According to the present invention, a process for producing a practical cholesterol-reduced substance by converting cholesterol contained in foods and feeds to coprostanol having very low intestinal tract absorbability by utilizing enzymatic action is provided.

The present invention relates to a method for producing a cholesterol-reduced substance, a cholesterol-reducing composition and a novel cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostane-3-one dehydrogenase for using the above-mentioned purpose.
It is widely known that excess intake of food having high cholesterol content increases the amount of cholesterol in serum and that high cholesterol content in serum is a significant factor in heart diseases. Therefore, processing techniques are required for selectively reducing the amount of cholesterol in food without deteriorating the quality of the food.
Among techniques for reducing the amount of cholesterol in food, a method is known that decomposes cholesterol with microorganisms (Japanese Laid-Open Patent Publication No. 267231/88) as a biochemical technique; however, this method produces by-products, therefore, it is not a safe method. Further, a method in which cholesterol is converted to epicholesterol by using an enzyme is known (WO93/25702).
Beitz, et al., U.S. Pat. No. 4,921,710 describes a method for converting cholesterol to coprostanol by using a cholesterol reductase derived from plants, and suggests a method for converting cholesterol to coprostanol by using a cholesterol reductase derived from bacteria such as Eubacterium species ATCC 21408. Also, Beitz, et al., U.S. Pat. No. 5,436,004 describes that the conversion ratio from cholesterol to coprostanol as 0.01% when a cream is treated using the above-mentioned enzyme derived from plants (see, column 5, table 1). However, such a low conversion ratio to coprostanol can not be admitted as practical level.
EYSSEN, British patent 1237483 describes that Eubacterium species bacterium separated from feces of rats reduces cholesterol to coprostanol, and also, in EYSSEN, Biochemica et Biophysica Acta, 348,279-284 (1974), it is estimated that the bacterium reduces cholesterol to coprostanol via 4-cholesten-3-one.
Beitz, et al., Applied Microbiology and Biotechnology 43, 887 (1995) describes that Eubacterium species bacterium (ATCC 51222) converts cholesterol in micelle to coprostanol, that 4-cholesten-3-one and trace amounts of coprostane have been detected in the conversion process, and that the reduction mechanism of cholesterol using the above-mentioned bacterium might be studied after pure preparations of the cholesterol reductase are obtained.
However, up to now, it has not been confirmed that cholesterol in a substance containing cholesterol is converted via 4-cholesten-3-one and coprostane-3-one to coprostanol by utilizing enzymatic action of cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase, coprostane-3-on dehydrogenase with using a coenzyme AND (P) and AND (P) H, and enzymes which convert cholesterol in a substance containing cholesterol to coprostanol via 4-cholesten-3-one and coprostane-3-one, respectively, have not been isolated from bacterium which reduces cholesterol, and in addition, no report have been proposed in which food is treated with these converting enzymes and microorganism containing the same.
It is known that cholesterol dehydrogenase derived from Nocardia, Alcaligenes, Proteus, which has optimum pH of around 9.0, requires AND (P) as a coenzyme, and is used for a quantitative determination of cholesterol (Japanese Post-Examined Patent Publication No.18064/90), however, this enzyme exhibits low activity at neutral pH, so that it can not be admitted as practical for food treatment.
Testing has been tried to obtain 4-cholesten-3-one dehydrogenase from the feces of a rat, however, the dehydrogenase is rapidly deactivated and can not be purified in this method, therefore, the method can not be admitted as practical (European J. of Biochemistry 37, 143 (1973)).
It is known that treatment of food with a phospholipase, protease and lipase accelerates conversion by a cholesterol oxidase (Japanese Laid-Open Patent publication No.76311/93), however, effect in enzymatic conversion of cholesterol to coprostanol is not known yet.
It is known that meat contains AND (H) (Journal of Food Science 37, 612 (1972)), and that nicotinamide inhibits decomposition of AND (Archives of Biochemistry and Biophisics 156, 143 (1973)). However, addition of nicotinamide in enzymatically converting cholesterol to coprostanol is not known.
It is known that an introduction of the cholesterol oxidase gene into lactic acid bacteria for decomposing food cholesterol (Applied Microbiology and Biotechnology 37, 330 (1992)).
As an applied example of these enzymes, the above-mentioned Beitz, et al., U.S. Pat. No. 5,436,004 suggests a treating method for reducing the amount of cholesterol in serum in which a cholesterol reductase derived from plants is orally administered. Further, it is reported that if a bacterium (ATCC 51222) which reduces cholesterol is orally administered to a rabbit suffering from hypercholesterolemia, the cholesterol level in serum decreases (Letters in Applied Microbiology 20, 137 (1995)).
Since absorbability of coprostanol through the intestinal tract is very low (American J. of Physiology 251, G495 (1986)), conversion of cholesterol to coprostanol is effective as a cholesterol reducing method. A practical method is not known for producing a cholesterol-reduced substance which reduces cholesterol by enzymatically converting cholesterol in food via 4-cholesten-3-one and coprostane-3-one to coprostanol. Also, a method for producing a practical enzyme which can be used in the above-mentioned production method is not known.
The object of the present invention is to provide a novel method for producing a practical cholesterol-reduced substance in food and feed. Another object of the present invention is to provide a novel cholesterol dehydrogenase having optimum pH in neutral pH range which is suitable for the above-mentioned practical method for producing a cholesterol-reduced substance, a novel 4-cholesten-3-one dehydrogenase and coprostane-3-one dehydrogenase having an optimum pH in a weak acidic pH range possessed by meat, and microbial cells and a treated material thereof containing these enzymes. A further object of the present invention is to provide a cholesterol-reducing composition which reduces cholesterol level in serum, comprising the above-mentioned three kinds of novel enzymes or comprising microbial cells containing these novel enzymes.
The present inventors have screened various microorganisms such as stock microorganisms mainly including 300 kinds (species categories) of actinomycetes, fungi and bacteria, aerobic bacteria separated from 100 kinds of various soils, and anaerobic bacteria from 7 feces samples of humans, 8 feces samples of mammals other than human and 9 feces samples of birds, to find a cholesterol reduction converting enzyme group having optimum pH in a neutral pH range or weak acidic range for solving the above-mentioned problems, and have intensively examined a culture medium for enzymatic activity detection and a method for recovering microbial cells, as a result, have accomplished the present invention.
Namely, the present invention relates to a process for producing a cholesterol-reduced substance and a method for reducing the amount of cholesterol, wherein cholesterol in a substance containing cholesterol such as meat, egg, milk, seafood and cooked processed food containing the same, or feed for animals, poultry and pisciculture, and the like, is treated with three kinds of enzymes consisting of cholesterol dehydrogenase having optimum pH in neutral pH range and 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase having optimum pH in a weak acidic range or microbial cells containing these enzymes, to convert cholesterol to coprostanol for reducing the amount of cholesterol.
Further, the present invention relates to the above-mentioned three kinds of enzymes consisting of cholesterol dehydrogenase having optimum pH in a neutral pH range and 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase having optimum pH in weak acidic range, produced by a strain belonging to Eubacterium, a cholesterol-reducing composition comprising these three kinds of enzymes, and a cholesterol-reducing composition comprising a strain belonging to Eubacterium which produce this enzyme group.
Further, the present invention relates to a process for producing a cholesterol-reduced substance and a method for reducing the amount of cholesterol wherein nicotinamide, phospholipase and nicotinamide, or a phosphate ion are added when cholesterol in a cholesterol-containing substance is converted to coprostanol by utilizing the above-mentioned enzyme group.
The process for producing a cholesterol-reduced substance of the present invention will be described in detail below.
In the present invention, examples of the cholesterol-containing substance include meat, egg, milk, seafood and cooked processed foods containing the same, or feeds for animals, poultry and pisciculture, and the like, but are not limited to them providing the substance containing cholesterol.
In the present invention, the cholesterol dehydrogenase is an enzyme conducting the following reaction.
Cholesterol+AND(P) xe2x86x924-cholesten-3-one+AND(P)H
Further, in the present invention, the 4-cholesten-3-one dehydrogenase is an enzyme conducting the following reaction.
4-Cholesten-3-one+AND(P)Hxe2x86x92coprostan-3-one+AND(P)
Similarly, in the present invention, the coprostan-3-one dehydrogenase is an enzyme conducting the following reaction.
Coprostan-3-one+AND(P)Hxe2x86x92coprostanol+AND(P)
In the present invention, the term xe2x80x9coptimum pHxe2x80x9d refers to a pH range in which relative active value is not less than 80% of the maximum active value, and the case in which pH 7.0 is included in such a pH range is referred to as xe2x80x9chaving optimum pH in a neutral pH rangexe2x80x9d in the present invention.
Similarly, the phrase xe2x80x9chaving optimum pH in a weak acidic rangexe2x80x9d in the present invention refers to a case in which pH 5.5 is included in the optimum pH range and the maximum active value exists in the acidic side.
For converting cholesterol in a cholesterol-containing substance to coprostanol via 4-cholesten-3-one and coprostan-3-one by utilizing the enzymatic actions of cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase sequentially in the present invention, any of an enzymatic conversion method using these enzymes and a microbial conversion method using microbial cells having these enzymatic activities can be used.
As the enzymatic conversion method, there is listed, for example, a method in which enzymes having activities of the above-mentioned cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase are added to a cholesterol-containing substance for converting cholesterol to coprostanol, and the enzyme which can be used in this enzymatic conversion method is not limited to a purified enzyme and may also be a crude enzyme which has not been purified but has enzymatic activity.
It is particularly desirable in this enzymatic conversion method to be sequentially treated with cholesterol dehydrogenase, the treatment with 4-cholesten-3-one dehydrogenase and the treatment with coprostan-3-one dehydrogenase continuously by successively-adding the enzymes and the like, from practical points of view such as simplification of the enzyme treatment process, improvement of conversion rate, and the like.
As the above-mentioned microbial conversion method, there is listed a method in which microbial cells having at least one enzymatic activity of cholesterol dehydrogenase activity, 4-cholesten-3-one dehydrogenase activity and coprostan-3-one dehydrogenase activity, or a treated material thereof are added to a cholesterol-containing substance for converting cholesterol to coprostanol, and it is desirable to use microbial cells having these enzymatic activities simultaneously or a treated material thereof. Further, a recombinant microbial cells having these enzymatic activities or a treated material thereof can also be used.
The material having the above-mentioned enzymatic activity (hereinafter, referred to as xe2x80x9cenzyme sourcexe2x80x9d) is usually added in the form of a powder or aqueous solution to a cholesterol-containing substance. Further, if necessary, coenzymes such as AND (P), AND (P) H and the like, enzymes such as phospholipase, lipase, protease and the like, or nicotinamide, phosphate ion and the like can be used alone or in combination together with the enzyme source.
In treatment for meat, for example, beef, pork, mutton or chicken, the enzyme source is mixed with minced meat, dispersed in sliced meat or injected in block meat. Further, the enzyme source can also be injected in blood vessels before and after slaughtering.
In treatment for milk, the enzyme source is added to milk, or milk is passed through a carrier to which the enzyme source is fixed. Further, in the case of fermented milk food, the enzyme source can also be added at milk fermentation.
In the treatment for eggs, the enzyme source is injected in the whole egg, or the enzyme source is mixed with yolk obtained by cracking the egg.
Further, when meat, milk, egg or seafood is cooked, the enzyme source may be added.
Regarding feeds for animals, poultry and pisciculture, it is possible to use a raw material for feed which has been treated with the enzyme source, or to mix the enzyme source in the process for preparing a feed.
In the above-mentioned enzymatic treatment, treatment conditions (temperature, time, pH) and the amount of enzyme to be added under which enzymatic conversion to coprostanol is possible are selected, and in general, the treatment is conducted at reaction temperature of 2 to 70xc2x0 C. and pH of 4 to 9 for 0.5 to 1xc3x97103 hours, and it is desirable to used a cholesterol reductase having optimum pH in the pH range possessed by the cholesterol-containing food itself to be treated in a practical point of view.
The amount used of each enzyme which is cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase or coprostan-3-one dehydrogenase to be used is from 1 to 1xc3x97105 units, preferably from 1xc3x97102 to 1xc3x97104 units per gram of cholesterol in food.
Optionally, AND(P) or AND(P)H which is a coenzyme of the above-mentioned enzyme can be added in an amount of 1xc3x9710xe2x88x924 to 2xc3x97102 g per gram of cholesterol.
Optionally, nicotinamide can also be added in an amount from 0.01 to 5% in terms of concentration in food. In meat which exhibits strong activity of decomposing these coenzymes it is preferable to added nicotinamide in an amount of 0.1% or more.
If required, a phosphate ion can also be added in an amount from 5 to 25 mM.
If occasion requires, it is also possible to add a phospholipase in an amount from 1 to 1xc3x97105 units per gram of a phospholipid and to add a lipase in an amount from 1 to 1xc3x97105 units per gram of a lipid.
Specific examples of the novel enzymes of the present invention include cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B. The method for production and the physical and chemical properties of these enzymes are described below.
The microorganism used in producing the novel cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A may be any microorganism having ability to produce the above-mentioned cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, and may also be a variant species or variant strain thereof. As a specific example of the microorganism having ability to produce the cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, for example, Eubacterium sp. CP 2 is listed.
The above-mentioned Eubacterium sp. CP 2 is a strain which has been newly separated from the feces of lions by the present inventors, and mycological properties thereof are as follows.
The present strain exhibits excellent growth by standing culture in a solution under anaerobic conditions at 37xc2x0 C. using the following basal medium. Tests regarding various properties were investigated under this condition.
Basal medium; 1% Casitone, 1% yeast extract, 0.5% soluble starch, 0.5% sodium pyruvate, 0.05% sodium thioglycolate, 0.05% calcium chloride, 0.0001% Resazurin, and 0.01% lecithin (pH 7.5)
(a) Morphological properties
{circle around (1)} Form of cell; short rod bacterium, and sometimes shown as almond figure.
{circle around (2)} Size of cell; 0.5 to 0.7 xcexcmxc3x970.7 to 1.0 xcexcm
{circle around (3)} Polymorphism; none
{circle around (4)} Mobility; none
{circle around (5)} spore; none
(b) Cultural properties
No growth was observed in bouillon plate agar-media and bouillon broth medium under aerobic or anaerobic conditions.
Cultural properties under a culturing condition and basal medium in which the present strain can grow are shown below.
{circle around (1)} Basal medium agar plate culture (culturing for 14 days)
i) Appearance of growth; forming weak small colony
ii) Color; transparent white
iii) Gloss; recognized
iv) Dispersible pigment; none
{circle around (2)} Basal medium solution culture (culturing for 3 days)
i) Growth on surface; none
ii) Turbidity; grown in the form of white emulsion at the bottom part
{circle around (3)} Gelatin stab culture in Basal medium
i) Condition of growth; excellent
ii) Liquefaction of gelatin; none
(c) Physiological properties in basal medium culture
{circle around (1)} Gram staining; positive
{circle around (2)} Reduction of nitrate; negative
{circle around (3)} Denitrification reaction; negative
{circle around (4)} MR test; negative
{circle around (5)} DVP test; negative
{circle around (6)} Generation of indole; negative
{circle around (7)} Generation of hydrogen sulfide; positive
{circle around (8)} Hydrolysis of starch; negative
{circle around (9)} Decomposition of esculin; positive
{circle around (10)} Utilization of an inorganic nitrogen source in a medium which has been obtained by removing 1% casitone and 1% yeast extract from the basal medium
i) Nitrate; negative
ii) Ammonium salt; negative
{circle around (11)} Generation of pigment; none
{circle around (12)} Urease; negative
{circle around (13)} Oxidase; negative
{circle around (14)} Catalase; negative
{circle around (15)} Growth range
i) Growth pH range; pH 6.0to pH 7.7 (optimum growth; around pH 7.3)
ii) Growth temperature range; 28 to 44xc2x0 C. (optimum growth temperature; around 35xc2x0 C.)
{circle around (16)} Attitude against oxygen; strictly anaerobic
{circle around (17)} O-F test (Hugh Leifson method); negative
{circle around (18)} Generation of acid and gas
i) L-arabinose; acid (none), gas (none)
ii) D-xylose; acid (none), gas (none)
iii) D-glucose; acid (observed), gas (none)
iv) D-mannose; acid (observed), gas (none)
v) D-fructose; acid (none), gas (none)
vi) D-galactose; acid (observed), gas (none)
vii) Maltose; acid (none), gas (none)
viii) Sucrose; acid (none), gas (none)
ix) Lactose; acid (none), gas (none)
x) Trehalose; acid (none), gas (none)
xi) D-sorbitol; acid (none), gas (none)
xii) D-mannitol; acid (none), gas (none)
xiii) Inositol; acid (none), gas (none)
xiv) Glycerin; acid (none), gas (none)
xv) Starch; acid (none), gas (none)
xvi) Metabolite from saccharides; butyric acid or acetic acid
(d) Other various properties; test according to An-IDENT
{circle around (1)} xcex1-glucosidase; positive
{circle around (2)} xcex2-glucosidase; positive
{circle around (3)} Alkali phosphatase; positive
{circle around (4)} xcex1-galactosidase; positive
{circle around (5)} Phenylalanineamino peptidase; negative
{circle around (6)} Decomposition of arginine; positive
{circle around (7)} Decomposition indoxyl acetate; negative
The present strain was of a gram positive strictly anaerobic short rod bacterium, did not form a spore, had no mobility, was negative against all of a catalase, oxidase and urease and formed acids from glucose and lactose, and main metabolite thereof was butyric acid or acetic acid. The classificational position of the strain having these mycological properties was compared with the description of Bergey""s Manual of Systematic Bacteriology vol. 2, 1986, as a result, the strain was identified as a bacterium belonging to Eubacterium, and the CP 2 strain was named as Eubacterium sp. CP 2. This strain was deposited in the name of FERM BP-5501 to National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, Japan) on Apr. 12, 1996.
As the medium used for culturing a microorganism which produces cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A of the present invention, any of a synthetic medium or natural medium containing a carbon source, nitrogen source, inorganic substance and the like can be used.
As the carbon source, for example, various carbohydrates can be used such as a soluble starch, lactose, pyruvic acid, glucose, molasses and the like, and the amount used thereof is preferably from 1 to 20 g/L.
As the nitrogen source, for example, ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium acetate, or nitrogen-containing organic compounds such as peptone, yeast extract, corn steep liquor, casein decomposed material, meat extract, and the like, can be used, and the amount used thereof is preferably from 1 to 20 g/L.
As the inorganic substance, for example, sodium chloride, calcium chloride, magnesium sulfate and the like are used, and the amount used thereof is preferably from 0.1 to 2 g/L. The amount used of a surfactant such as lecithin and the like is preferably from 0.01 to 1 g/L. The amount used of sodium thioglycolate is preferably from 0.1 to 1 g/L.
Culturing is conducted under an anaerobic condition by standing culture or stirring culture. Culturing temperature may advantageously be a temperature at which a microorganism grows and produces cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, and preferably from 35 to 40xc2x0 C. Culturing period varies depending on conditions, and culturing may advantageously be conducted until the maximum amount of cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A are produced, and usually from about 5 to 10 days.
Cholesterol dehydrogenase A that is produced by the CP 2 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction.
Cholesterol+NADPxe2x86x924-cholesten-3-one+NADPH
(b) Substrate specificity
The present enzyme reacts with steroid having a hydroxyl group at 3xcex2 position, and has relative activity of 38, 74, and 30 regarding xcex2 sitosterol, campesterol, stigmasterol when the activity of cholesterol is 100.
(c) Optimum pH: 6.5 to 7.8
(d) Stable pH: 5.3 to 9.0
An enzyme solution was allowed to stand still at 37xc2x0 C. for 15 minutes using various pH buffer solutions, and remaining activity was measured, and pH range in which activity was not less than 50% of the maximum active value was regarded as a stable pH range. (The following is the same.)
(e) Measurement of titer:
With 0.2 ml of 3 mM cholesterol micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM piperazine-N,Nxe2x80x2-bis-(2-ethane sulfuric acid) (hereinafter, referred to as xe2x80x9cPIPESxe2x80x9d) buffer solution (pH 7.5) containing 0.5% Triton X-100 and 1 mM dithiothreitol (hereinafter, referred to as xe2x80x9cDTTxe2x80x9d), and 0.1 ml of a 10 mM AND solution, the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of 4-cholesten-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of 4-cholesten-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of 4-cholesten-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing activity.
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM p-chloro mercury phenylsulfonate (PCMB), iodine acetamide and ethylenediamine tetraacetate (EDTA) are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 82 and 105 respectively, if the enzyme activity without adding inhibitor is defined as 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 95 and 3.2, respectively, in comparison with the case of no addition.
(i) Purification method:
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM MDTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10% glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the same buffer solution, then, adsorbed on Red Sepharose CL-6B (Pharmacia). Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADP) is used as a coenzyme.
(k) Molecular weight:
The molecular weight according to SDS polyacrylamide electrophoresis method is about 57,500.
The 4-cholesten-3-one dehydrogenase A of the present invention produced by the CP 2 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction.
4-cholesten-3-one+NADHxe2x86x92coprostan-3-one+AND
(b) Substrate specificity
The relative activities are 0, 41 and 0 for testosterone, progesterone and prognerone when the activity of 4-cholesten-3-one is defined as 100.
(c) Optimum pH: 5.4 to 6.5
(d) Stable pH: 5.5 to 7.2
(e) Measurement of titer:
With 0.2 ml of 3 mM 4-cholesten-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM PIPES buffer solution (pH 7.5) containing 0.5% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADH solution, the resulted mixture is added 0.05 ml of an enzyme solution, and reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of coprostan-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostan-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostan-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 6.0 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing the activity
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 47, 94 and 82 respectively, if the enzyme activity without adding inhibitor is defined as 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 110 and 0, respectively, in comparison to the case of no addition.
(i) Purification method:
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM MDTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10% glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the same buffer solution, then, adsorbed on Resource Column (Pharmacia). Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 1.0 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide (NADH) is used as a coenzyme.
(k) Molecular weight:
The molecular weight according to SDS polyacrylamide electrophoresis method is about 37,500.
The coprostan-3-one dehydrogenase A of the present invention produced by the CP 2 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction. coprostan-3-one+NADPHxe2x86x92coprostanol+NADP
(b) Substrate specificity
The relative activities are 13.2 for 5xcex1-cholesten-3-one when the activity of coprostan-3-one is defined as 100.
(c) Optimum pH: 5.2 to 5.7
(d) Stable pH: 4.0 to 7.5
(e) Measurement of titer:
With 0.2 ml of 3 mM coprostan-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 40 mM Britton-Robinson buffer solution (pH 6.0) containing 0.5% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADPH solution, the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction. The enzyme solution was mixed with equal amounts of activating fractions and left for 15 minutes at 5xc2x0 C., then used.
Then, the quantity of coprostanol produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostanol in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostanol per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing the activity.
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes in the presence of 0.5 mM NADPH, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 41, 82 and 101 respectively, if the enzyme activity without adding inhibitor is defined as 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 12 and 2.7, respectively, in comparison with the case of no addition.
(i) Purification method:
Method 1;
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0, containing 0.1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10% glycerol), then, passed through Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution, and fractions which have not been adsorbed on Blue Sepharose CL-6B are recovered, and dialyzed against a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0) (containing 0.5 mM NADPH, 1 mM DTT, 0.2% Triton X-100, 10% glycerol). The solution is adsorbed on DEAE-Cellulofine which has been equilibrated with the same buffer solution, then, the buffer solution flows with sodium chloride concentration being increased continuously from 0 to 1.0 M, and active fractions are collected and dialyzed against the buffer solution. The active fractions are adsorbed on Blue Sepharose CL-6B, and fractions which had not been adsorbed were recovered. The fractions which had not been adsorbed were heated at 95xc2x0 C. for 10 minutes to remove protein and to obtain activating fractions. Then, the active fractions which had been adsorbed on Blue Sepharose were eluted with the same buffer solution containing 1 mM NADPH with sodium chloride concentration being increased continuously from 0 to 1.0 M. To these active fractions, the above-mentioned activating fractions were added to prepare a purified sample.
Method 2;
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0, containing 0.1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0, containing 1 mM DTT, 0.2% Triton X-100, 30% glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against a 20 mM phosphoric acid-citric acid buffer solution B (pH7.5, containing 1mM DTT, 0.2% Triton X-100, 30% glycerol), then adsorbed on DEAE-Cellulofine which has been equilibrated with the same buffer solution B, then, the buffer solution flows with sodium chloride concentration being increased continuously from 0 to 1.0 M, and active fractions are collected and dialyzed against the buffer solution. The active fractions are dialyzed against the same buffer solution B, then adsorbed on Resource Column (Pharmasia). Then, the above-mentioned buffer solution B flows with sodium chloride concentration being increased continuously from 0 to 1.0 M and active fractions are collected to prepare a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADPH) is used as a coenzyme.
(k) Molecular weight:
The molecular weight according to SDS polyacrylamide electrophoresis method is about 29,000.
Then, the method for producing the novel cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B is described below.
The microorganism used in producing the present enzymes may be any microorganism having the ability to produce the above-mentioned cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B, and may also be a variant species or variant strain thereof. As a specific example of the microorganism having the ability to produce the cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B and belonging to Eubacterium, for example, Eubacterium sp. CP 1 is listed.
The above-mentioned Eubacterium sp. CP 1 is a strain which has been newly separated from the feces of humans by the present inventors, and mycological properties thereof are as follows.
The present strain exhibits excellent growth by standing culture in a solution under anaerobic conditions at 37xc2x0 C. using the following basal medium. Tests regarding various properties were investigated under this condition.
Basal medium; 1.2% a bovine brain extracted lipid, 1.8% Trypticase, 0.05% yeast extract, 0.13% dipotassium phosphate, 0.22% sodium chloride, 0.05% cholesterol, 0.04% L-cystine, 0.03% sodium thioglycolate, 0.05% agar, and a small amount of 0.05% Methylene Blue (pH 7.2)
(a) Morphological properties
{circle around (1)} Form of cell; long rod bacterium and grown in the form of chain
{circle around (2)} Size of cell; 0.3 to 0.5 xcexcmxc3x973.0 to 5.0 xcexcm
{circle around (3)} Polymorphism; none
{circle around (4)} Mobility; none
{circle around (5)} Spore; none
(b) Cultural properties
No growth was observed in the bouillon plate agar-media and bouillon broth media under aerobic or anaerobic conditions.
Cultural properties under culturing condition and basal medium in which the present strain can grow are shown below.
{circle around (1)} Basal medium agar plate culture (culturing for 21 days)
i) Appearance of growth; forming powdery colony
ii) Color; white
iii) Gloss; none
iv) Dispersible pigment; none
{circle around (2)} Basal medium solution culture (culturing for 5 days)
i) Growth on surface; none
ii) Turbidity; grows, but, discrimination from sediment in a medium by turbidity is difficult
{circle around (3)} Gelatin stab culture in basal medium
i) Condition of growth; excellent
ii) Liquefaction of gelatin; none
(C) Physiological properties in basal medium culture
{circle around (1)} Gram staining; positive
{circle around (2)} Reduction of nitrate; negative
{circle around (3)} Litmus milk; coagulation negative
{circle around (4)} Generation of indole; negative
{circle around (5)} Generation of hydrogen sulfide; negative
{circle around (6)} Hydrolysis of starch; positive
{circle around (7)} Decomposition of esculin; positive
{circle around (8)} Utilization of an inorganic nitrogen source in a medium which has been obtained by removing trypticase and yeast extract from a basal medium
i) Nitrate; negative
ii) Ammonium salt; negative
{circle around (9)} Generation of pigment; none
{circle around (10)} Urease; negative
{circle around (11)} Oxidase; negative
{circle around (12)} Catalase; negative
{circle around (13)} Growth range
i) Growth pH range; pH 5 to pH 8 (optimum growth; around pH 7)
ii) Growth temperature range; 33 to 40xc2x0 C. (optimum growth temperature; around 34xc2x0 C.)
{circle around (14)} Behavior against oxygen; strictly anaerobic
{circle around (15)} O-F test (Hugh Leifson method); negative
{circle around (16)} Generation of acid
i) L-arabinose; acid (small amount)
ii) D-xylose; acid (observed)
iii) D-glucose; acid (observed)
iv) D-mannose; acid (observed)
v) D-fructose; acid (small amount)
vi) D-galactose; acid (observed)
vii) maltose; acid (observed)
viii) Sucrose; acid (small amount)
ix) Lactose; acid (observed)
x) Trehalose; acid (none)
xi) D-sorbitol; acid (none)
xii) D-mannitol; acid (none)
xiii) Inositol; acid (none)
xiv) Glycerin; acid (none)
xv) Starch; acid (none)
xvi) Ribose; acid (observed)
xvii) Cellobiose; acid (observed)
xviii) Lactose; acid (observed)
xix) Melibiose; acid (observed)
xx) Raffinose; acid (observed)
xxi) Salicin; acid (observed)
xxii) Amygdalin; acid (observed)
xxiii) Melezitose; acid (none)
xxiv) Glycogen; acid (none)
xxv) Inulin; acid (none)
xxvi) Metabolite from saccharides; acetic acid
The present strain was gram positive strictly anaerobic long rod bacterium, did not form spores, had no mobility, was negative against all of a catalase, oxydase and urease and formed acids from glucose, and main metabolite thereof was acetic acid. The classificational position of the strain having these mycological properties was compared with description of Bergey""s Manual of Systematic Bacteriology vol. 2, 1986, as a result, the strain was identified as a bacterium belonging to Eubacterium, and the CP 1 strain was named as Eubacterium sp. CP 1. This strain was deposited in the name of FERM BP-5500 to National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, Japan) on Apr. 12, 1996.
As the medium used for culturing a microorganism which produces cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B of the present invention, any of the synthetic mediums or natural mediums containing a carbon source, nitrogen source, inorganic substance and the like can be used.
As the carbon source, for example, various carbohydrates can be used such as glucose, maltose, molasses and the like, and the amount used thereof is preferably from 1 to 20 g/L.
As a nitrogen source, for example, ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium acetate, or nitrogen-containing organic compounds such as peptone, yeast extract, corn steep liquor, casein decomposed material, meat extract, and the like, can be used, and the amount used thereof is preferably from 1 to 20 g/L. The amount of solvent-extract used (chloroform: methanol=2:1) of a bovine brain lipid is preferably from 1 to 20 g/L.
As an inorganic substance, for example, sodium chloride, calcium chloride, magnesium sulfate, potassium monohydrogen phosphate and the like are used, and the amount used thereof is preferably from 0.1 to 2 g/L. The amount used of cystine and sodium thioglycolate is preferably from 0.1 to 1 g/L.
Culturing is conducted under anaerobic conditions by standing culture or stirring culture. Culturing temperature may advantageously be a temperature at which a microorganism grows and produces cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B, and preferably from 35 to 40xc2x0 C. Culturing period varies depending on conditions, and culturing may advantageously be conducted until the maximum amount of these enzymes are produced, usually from about 3 to 10 days.
Cholesterol dehydrogenase B of the present invention that is produced by the CP 1 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction.
Cholesterol+NADPxe2x86x924-cholesten-3-one+NADPH
(b) Substrate specificity
The present enzyme reacts with steroid having a hydroxyl group at 3xcex2 position, and has relative activity of 67, 50 and 39 regarding xcex2 sitosterol, campesterol, stigmasterol when the activity of cholesterol is defined as 100.
(c) Optimum pH: 6.7 to 7.7
(d) Stable pH: 5.0 to 10.5
(e) Measurement of titer:
With 0.2 ml of 3 mM cholesterol micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 50 mM phosphoric acid buffer solution (pH 7.5) containing 1.0% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADP solution, to the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of 4-cholesten-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of 4-cholesten-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of 4-cholesten-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperatures:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing activity.
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes ,it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetic acid and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 98 and 102 respectively, if the enzyme activity without adding inhibitor is 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are both 0 in comparison to the case of no addition.
(i) Purification method:
Air is bubbled through the culture, and microbial cells adsorbed on the bubble are recovered and concentrated. The concentrated solution is subjected to centrifugal separation to collect the cells, and the cells are suspended in a 20 mM phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 1.0% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1 mM iodine acetic acid and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and the mixture is dialyzed against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic acid, and 0.5 mM PMSF). The crude enzyme solution is adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution containing 0.25% Triton X-100. Then, lipid components are eluted with the same buffer solution containing 1.0% Triton X-100, then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the above-mentioned buffer solution, then, adsorbed on DEAE-Sepharose Fast Flow which has been equilibrated with the same buffer solution containing 1.0% Triton X-100. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 0.5 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADP) is used as a coenzyme.
The 4-cholesten-3-one dehydrogenase B of the present invention produced by the CP 1 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction. 4-cholesten-3-one+NADHxe2x86x92coprostan-3-one+AND
(b) Substrate specificity
The relative activities are 0 for 5-cholestenon-3-one when the activity of 4-cholesten-3-one is defined as 100.
(c) Optimum pH: 5.3 to 7.0
(d) Stable pH: 5.2 to 8.0
(e) Measurement of titer:
With 0.2 ml of 3 mM 4-cholesten-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 1.0% Triton X-100and 1 mM DTT, and 0.1 ml of a10 mM NADH solution, the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of coprostan-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostan-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostan-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperatures:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing the activity.
(g) Range of temperature stability
After heating treatment at 37xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 99 and 125, respectively, if the enzyme activity without adding inhibitor is 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 107 and 0, respectively, in comparison with the case of no addition.
(i) Purification method:
Air is bubbled through the culture, and microbial cells adsorbed on the bubble are recovered and concentrated. The concentrated solution is subjected to centrifugal separation to collect the cells, and the cells are suspended in a 20 mM phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 1.0% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1 mM iodine acetic acid and 0.5 mM PMSF, and the mixture was dialyzed against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic acid, and 0.5 mM PMSF). The enzyme solution is adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution containing 0.25% Triton X-100. Then, lipid components are eluted with the same buffer solution containing 1.0% Triton X-100, then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the above-mentioned buffer solution, then, adsorbed on DEAE-Sepharose Fast Flow which has been equilibrated with the same buffer solution containing 1.0% Triton X-100. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 0.5M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide (NADH) is used as a coenzyme.
The coprostan-3-one dehydrogenase B of the present invention produced by the CP 1 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction. coprostan-3-one+NADPHxe2x86x92coprostanol+NADP
(b) Substrate specificity
The relative activities are 0 for 5xcex1-cholesten-3-one when the activity of coprostan-3-one is defined as 100.
(c) Optimum pH: 5.4 to 7.6
(d) Stable pH: 4.5 to 7.0
(e) Measurement of titer:
With 0.2 ml of 3 mM coprostan-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 1.0% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADPH solution, to the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of coprostanol produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostanol in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostanol per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 45xc2x0 C. is attended by increasing the activity.
(g) Range of temperature stability
After heating treatment at 45xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 95 and 112, respectively, if the enzyme activity without adding inhibitor is 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 105 and 0, respectively, in comparison to the case of no addition.
(i) Purification method:
Air is bubbled through the culture, and microbial cells adsorbed on the bubbles are recovered and concentrated. The concentrated solution is subjected to centrifugal separation to collect the cells, and the cells are suspended in a 20 mM phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 1.0% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1 mM iodine acetic acid and 0.5 mM PMSF, and the mixture is dialyzed against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic acid, and 0.5 mM PMSF). The crude enzyme solution is adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution containing 0.25% Triton X-100. Then, lipid components are eluted with the same buffer solution containing 1.0% Triton X-100, then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulting active fractions are dialyzed against the above-mentioned buffer solution, then, adsorbed on DEAE-Sepharose Fast Flow which has been equilibrated with the same buffer solution containing 1.0% Triton X-100. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 0.5 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADPH) is used as a coenzyme.
As the cholesterol-reduced composition of the present invention, microbial cells or treated materials thereof, crude purified enzymes, purified enzymes and the like containing these three enzymes may be used without any treatment provided they have activities of a cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase, and further, those in the form of tablets, powders, fine particles, granules, capsules, syrups and the like molded with vehicles which are acceptable for food or medicine may be used. The composition of the present invention may be added as a composition to be added into food or feed for reducing the amount of cholesterol in the food or feed, or may be orally administered via oral route for reducing the cholesterol level in serum. When a crude purified enzyme or purified enzyme is used as the composition of the present invention, the composition optionally may be advantageously prepared so that nicotinamide, phosphate ion or phospholipase is contained in the composition. Examples of the form of the oral composition of the present invention include tablets, powders, fine particles, granules, capsules, syrups, enteric agent, troches and the like. In the case of addition or administration, as the vehicle, any compound such as saccharides like sorbitol, lactose, glucose, lactose, dextrin, starch, crystalline cellulose and the like; inorganic compounds like calcium carbonate, calcium sulfate and the like; distilled water, sesame oil, corn oil, olive oil, cotton seed oil and the like, generally can be used. In preparing the composition, additives such as binder, lubricant, disperser, suspending agent, emulsifying agent, diluent, buffering agent, antioxidant, bacterium inhibiting agent and the like may be used.
The amount to be added may be advantageously controlled to be the above-mentioned value necessary for the enzymatic conversion or the microbial conversion in the method for producing a cholesterol-reduced substance of the present invention.
The dosage varies depending on age, sex, administration pattern, times of administration, form and the like, and regarding dosage for oral administration for adults, it is suitable that the amount of bacteria is 1xc3x97107 to 1xc3x971012 cell/day, preferably 1xc3x97108 to 1xc3x971011 cell/day and the amount of three enzymes is 10 to 1xc3x97105 unit/day, preferably 1xc3x97102 to 1xc3x97104 unit/day, and that the composition is divided into 1 to 4 portions for one day-administration. If necessary, dosage out of the above-mentioned restriction can also be adopted.

CHOLESTEROL DEHYDROGENASE, COPROSTAN-3-ONE DEHYDROGENASE AND 4-CHOLESTEN-3-ONE DEHYDROGENASE, COMPOSITIONS CONTAINING THE DEHYDROGENASES, AND METHOD FOR REDUCING AMOUNT OF CHOLESTEROL USING THE COMPOSITIONS

Abstract: Cholesterol in a cholesterol-containing substance such as meat, egg, milk, seafood and cooked processed foods containing the same, or feeds for animals, poultry and pisciculture, and the like, can be treated with three kinds of enzymes which are a cholesterol dehydrogenase having an optimum pH in a neutral pH range and 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase each having an optimum pH in a weak acidic range, or microbial cells containing the enzymes, for converting the cholesterol to coprostanol to reduce the amount of the cholesterol.


A collapsible frame is movable between a closed configuration and an open configuration. The frame includes a first side support assembly and a second side support assembly. Each side support assembly includes a collapsible first support member including a first platform and a collapsible second support member coupled to the first support member at a plurality of pivot points. The second support member includes a support joint movable with respect to the first platform for facilitating moving the collapsible frame between the closed configuration and the open configuration.

This invention relates generally to portable structures and, more particularly, to a collapsible frame for portable structures.
Conventional portable structures, such as portable chairs, beds and hammocks, are assembled to be movable between a closed configuration, for storing the structures in compact areas, and an opened configuration for supporting a person thereon.
However, such conventional structures may not be easily movable between the closed configuration and the open configuration due to complicated interconnections of the components. Further, several steps may be required to close the structure for storage or open the structure for use. Moreover, such conventional structures may require a large number of interconnected components, making the structure heavy and/or cumbersome and difficult to transport.

Portable structure having collapsible frame


A terminal device which can set a connection status of a communication tool line between terminal devices in various ways and an electronic conference system having such a terminal device are provided. The terminal device for communicating information between remote terminal devices comprises a connection status operation/control unit for setting a connection status of a line to be used by the communication tool, a connection modification unit for changing the line connection in accordance with the content set by the connection status operation/control unit, a connection status modification convey unit for outputting the content set by the connection status operation/control unit to other terminal device, and a connection status modification inform unit for receiving the content of the modification of the line connection from the other terminal.

1. Field of the Invention
The present invention relates to communication conference system and communication conference apparatus.
2. Related Background Art
Types of actual conferences are classified in the following major classes:
(1) Presentation Type (see 4-2 in FIG. 4)
One presentor is present and all others are audience. For example, a lesson in a school belongs to this class.
In the presentation type conference, the presenter may speak to the audience but the audience is not allowed to speak to the presenter. However, in questions and answers, the audience may speak to the presentor.
(2) Discussion Type (see 4-1 in FIG. 4)
There is no distinction between the presenter and the audience and all members participating the conference are allowed to speak. Brainstorming and discussion belong to this class. In the discussion type conference, all participants of the conference are allowed to speak and speeches by all other participants may be listened to.
(3) Combination Type (see 4-3 in FIG. 4)
A combination of the types 4-1 and 4-2 in FIG. 4. Several participants make discussion and other participants are the audience. A panel discussion belongs to this class.
As described above, there are various types of actual conferences.
However, in the prior art electronic conference system, means for realizing such variety of electronic conferences is not established and means for changing the connection of the line by the participants is not provided. As a result, the conferences other than the discussion type conference (2) in which all participants are interconnected cannot be realized.
In an actual conference of the type (2), a secret communication (by which conversation is made only between neighbors) is frequently conducted.
However, in order to achieve the secret communication, it is necessary to provide a separate communication line in addition to a main conference communication line to start a new conference. However, this method requires to set a new conference. In addition, tools used in the main conference may not be used for the secret communication. When a microphone is used in the main conference, a voice tool may not be used in the secret communication.
As described above, since the prior art system supports only a simple form of line connection, it cannot fully attain the inherent object of the electronic conference system of "to replace an actual form of conference".
The actual conferences may be classified into a formal conference such as a regularly held conference and an informal conference such as discussion by free participants.
In the formal conference, a chairman has been predetermined and the participants are normally predetermined persons, and means such as an electronic black board and an OHP are essential to smoothly conduct the conference.
On the other hand, in the informal conference, the participants are not fixed and a chairman is normally not elected and the electronic black board or OHP is not always required.
However, in the prior art electronic conference system, whatever type of electronic conference may be initiated, only one fixed user interface is provided to set an environment to start the conference.
As a result, even when the formal conference (which uses the same tools such as shared window tools, shared documents tools, voice tools and video tools by the same participants at every conference) is started, the same items must be set every time as the environment setting for the electronic conference and hence a big workload is required to start the conference. When the informal conference is to be started, the election of the chairman and the setting of the conference described above are in many cases not necessary, but such unnecessary items must be set. Thus, a big workload is required to start the conference.
It is considered that the electronic conference will become more and more versatile and the numbed of items to be set when the electronic conference is to be started will increase in the future. In such a case, if it is required to designate all necessary items to start the conference each time the electronic conference is to be started, a very troublesome work is required, which cannot be ignored.
In a common conference, a start time of the conference has been previously provided to the participants of the conference, and even if a participant is late to the conference, he may grasp how much time has elapsed since the start of the conference.
When a participant cannot attend the conference or will be late, it is common to provide notice to the chairman or participant of the conference a reason for absence or when he will come.
However, in the prior art electronic conference system, an invitee cannot immediately determine the elapsed time from the occurrence of the request to participate in the conference.
The participant (invitee) of the conference can convey only the information of participation or non-participation to the inviter (person who attempts to start the electronic conference) and the information which can be conveyed is extremely small.
Accordingly, when the invitee cannot participate in the conference, he cannot convey the reason for the non-participation or request a call in the course of conference because he is now busy on another work but may participate in the conference a few minutes later.

Communication conference system and communication conference apparatus


A method of sewing a decorative seam includes providing a material suitable for receiving stitches, folding a first portion of the material onto a second portion of the material to form a bend, sewing a first stitch to join the first portion to the second portion, the stitch being substantially parallel to the bend and spaced apart therefrom, the first stitch forming a seam, unfolding the material and sewing a second and third stitch into the material such that the second and third stitches are substantially parallel to the seam and arranged such that the second and third stitches are on opposite sides of the seam.

1. Field of the Invention
The field of this invention relates to a method of sewing a decorative seam into a material used in an automotive trim component and an automotive trim component having the decorative seam made using the method.
2. Background Art
A method of sewing a decorative seam and an automotive trim component having the decorative seam made by the method is disclosed herein. Examples of decorative seams are disclosed in U.S. Pat. Nos. 6,997,126 and 6,935,260 and in U.S. Publication Nos. 2007/0022931; 2005/0188907; 2005/0081771; and 2005/0081770.

Method of sewing a decorative seam and trim component made by that method

International Automotive Components Group North America, Inc.


A windshield wiper blade including a primary part having an elongated main body and an interior space formed in the main body, and at least two secondary parts is provided. Each secondary part has a first end and a second end, the first end being received into the interior space of the main body, and the second end being exposed, wherein the secondary parts are hingedly connected to the primary part to swivel relative to the primary part to accommodate a changing curvature of a surface of a windshield based on pressure applied thereto against the windshield.

1. Field of the Invention
Embodiments of the invention are directed generally to windshield wiper blades usable with various wiper arms. More particularly, the embodiments of the invention are directed to windshield wiper blades having segmented portions.
2. Description of the Related Art
Various types of windshield wiper blades are available which utilize respective adapters for connection of windshield wiper blades to various wiper arms of automobiles. Thus, a particular arm may be connectable to windshield wiper blades using the adapters. Efforts have been made in development of windshield wiper blades that provide consistent wiping regardless of the developing type of arm that the windshield wiper blade is attached to.


A method and system for automated intelligent electronic advertising. The method and system parse electronic information messages (e.g., e-mail messages, instant messages, etc.) for keywords and make search engine queries or database queries for electronic links based on the identified keywords. The electronic links returned from search engine queries or database queries are added to the electronic information messages creating a modified electronic information message. This allows additional electronic information to be accessed from the modified electronic information message based on information content of the original electronic information message.

The Internet and World-Wide-Web have changed the way organizations conduct business. Virtually every organization has a web-site that provides information about the organization and a description of the goods and/or services an organization may offer. As is known in the art, a "web-site" is group of related mark-up language documents and associated graphics and multi-media files, scripts, and databases, etc. that are served up by a server on the World-Wide-Web via the Internet. Business organizations also provide an electronic commerce (e-commerce) interface that allows users to purchase goods and/or services from such organizations.
There are many different type of web-sites on a spectrum ranging from very simple to very complex. Designing, implementing and deploying a web-site requires knowledge of markup languages such as Hyper Text Markup Language (HTML), eXtensible Markup Language (XML), programming languages such as JAVA, C++, C#, computer graphics functionality, multi-media functionality, etc. A knowledge of communications protocols such as Hyper Text Transfer Protocol (HTTP), Transmission Control Protocol (TCP), Internet Protocol (IP), e-mail protocols such as Simple Mail Transfer Protocol (SMTP), e-commerce protocols, encryption and other security protocols and many other types of protocols for various kinds of web functionalities.
Commercial web-sites offer many different types of electronic advertisements. The advertisements typically include electronic links to advertiser's web-sites. There are many problems associated with designing, implementing and deploying advertising on a web-site.
Another problem is that many types of online advertising are considered spam. Another problem is that pop-up advertising is being block by operating systems such as Windows XP and add-on software. This pop-up blocking is becoming a standard feature in many operating systems. Another problem is that banner ads and pay-per-click ads are no longer an effective means of advertising online.
Electronic mail (e-mail) is one of the most common types of electronic information sent and received. E-mail is sent and received over public networks such as the Internet, and many private networks such as intranets, local area networks (LAN), etc.
Advertisers and other information providers are always looking for new ways to use existing technologies such as e-mail for advertising. There are many free e-mail services that routinely add advertising to user's e-mail as a condition of providing free e-mail. However, there is no easy way to add advertising to e-mail. In addition, general e-mail advertising is often considered spam.
One attempt to solve these problems is "Gmail" provided by Google. Gmail includes a web-interface that displays e-mail. Electronic advertising is displayed on the web-interface that corresponds in part to content of the e-mail.
This it is desirable to use e-mail and other types of electronic information messages for providing advertising and other types of electronic information.

Method and system for automated intelligent electronic advertising


There is provided a test apparatus that tests a device under test. The test apparatus includes a main memory that stores a test instruction stream determining a test sequence for testing the device under test, a sequence cache memory that caches the test instruction stream, a transfer section that reads the test instruction stream stored on the main memory and writes the read stream into the sequence cache memory in accordance with a described sequence, a pattern generating section that sequentially reads and executes instructions from the test instruction stream cached on the sequence cache memory and outputs a test pattern corresponding to the executed instruction, and a test signal output section that generates a test signal according to the test pattern and supplies the generated signal to the device under test, in which the transfer section overwrites the instruction read from the main memory on a space area on the sequence cache memory or an area on which executed instructions are stored and prohibits overwriting the read instruction on an area on which instructions in a predetermined range is stored, the instructions being located in the predetermined range forward from a final instruction among the executed instructions according to the described sequence.

1. Field of the Invention
The present invention relates to a test apparatus and an electronic device. More particularly, the present invention relates to a test apparatus that tests a device under test and an electronic device including a test circuit that tests a circuit under test.
2. Related Art
A test apparatus for testing a device under test such as a semiconductor has been known. The test apparatus supplies a test signal with a predetermined logical pattern to the device under test, and detects a signal output from the device under test in accordance with this test signal. Then, the test apparatus compares the detected signal and an expected value to decide the good or bad of the device under test.
The test apparatus includes a main memory such as DRAM that stores sequence data (a test instruction stream), a cache memory that temporarily stores a test instruction stream, a transfer section that transfers sequence data from the main memory to the cache memory, a pattern generator that sequentially generates a test pattern, and a test signal output section that outputs a test signal with logic according to the test pattern. The pattern generator sequentially reads instructions from the sequence data stored on the cache memory, and executes the read instructions. Then, the pattern generator reads pattern data corresponding to the executed instructions from the memory, and sequentially outputs the read pattern data as test patterns. According to this, the test apparatus can supply a test signal with a predetermined logical pattern to the device under test.
Moreover, the transfer section sequentially reads sequence data from the main memory, and writes the read data into a space area on the cache memory. When the space area on the cache memory vanishes, the transfer section overwrites the read sequence data on an area on which an executed instruction is stored.
Meanwhile, the test apparatus can include a forward branch instruction in sequence data (for example, see Japanese Patent Application Publication No. 1998-78476). However, since an instruction to be executed next to the forward branch instruction is an already-executed instruction, the instruction may be overwritten by the transfer section. In this case, the pattern generator cannot read an instruction next to the forward branch instruction from the cache memory.
Moreover, in order to generate a test pattern every clock cycle, the pattern generator must perform a series of processes for reading an instruction from the cache memory, executing the read instruction, and then generating an address on the cache memory, in which the address is an address of an instruction to be next executed, for one clock cycle. Furthermore, it is desirable that the pattern generator can generate a test pattern in a shorter clock cycle, and thus it is desirable that a restriction of an executive operation for generating a test pattern is smaller.

Instruction address generation for test apparatus and electrical device


A method for forming channels within a dried chromonic layer is described. A coating composition is applied to a substrate, dried, and exposed to a hydrophilic organic solvent forming a channel pattern having a first set of channels, and a second set of channels that are substantially perpendicular to the first set of channels. A deposition material may be disposed within the channels to form a nanostructured pattern. An article having a channel pattern is further described.

Patterned layers of functional materials can be used in electric component manufacture as well as other applications. For example, multiple layers of differing layers of patterned materials may be used in the manufacture of flat panel displays, such as liquid crystal displays. Active matrix liquid crystal displays include a plurality of rows and columns of address lines that cross at an angle to one another, and form a plurality of crossover points. Techniques for applying patterned layers continue to evolve as the need increases for smaller structures in electric components.
Lithographic techniques can be used to generate small structures; however, as the size domain moves into the nanoscale range, significant technical challenges arise that can limit the use of lithographic techniques for nanostructures.
Self-assembly is another method that can be used to construct nanostructures. Molecular self-assembly is referred to as the assembly of molecules without guidance or management from an outside source. Many biological systems use self-assembly to assemble various molecules and structures such as the lipid bilayer membrane in cells.

Method for forming channel patterns with chromonic materials


Apparatus and method are disclosed for estimating direction of an arrival wave, such as radio or acoustic waves. A correlation matrix of an arrival wave is computed, and matrices for obtaining eigenvalues and eigenvectors are produced. Column norms of the matrix, which is subject to a Householder transformation, are computed. Whether a maximum value of the column norms is the same as or lower than a predetermined threshold is judged. A Householder transformation of the matrix is performed only if the maximum value of the column norms is determined to be greater than or equal to the threshold, and not when the maximum value of the column norms is less than the threshold. The eigenvectors and the eigenvalues of the correlation matrix are obtained by further implementing the Householder transformation. The number of arrival waves is judged from the number of times the Householder transformation is performed.

This invention relates to an apparatus for estimating direction of arrival wave, such as radio wave and acoustic wave, which arrives at a plurality of antenna elements.
A technique of estimating arrival directions of one or more arrival waves, such as radio waves and acoustic waves, of the same frequency which are caught by two or more antenna elements is well known. As one of such methods, MUSIC (Multiple Signal Classification) method is well known (see Japanese patent application the publication number of which is 2000-121716.
In MUSIC method, on the basis of a signal data vector of arrival waves, a correlation matrix thereof is obtained, and an eigenvalue decomposition operation for decomposing the obtained correlation matrix into eigenvectors and eigenvalues is implemented, and azimuths of the arrival waves are estimated from the thus obtained eigenvectors. In order to obtain the arrival azimuth, it is necessary to estimate the number of arrival waves in advance. Generally, eigenvalues are used in the estimation of the number of arrival waves. Such methods are AIC, MDL and threshold methods, for instance.
[Patent-related document 1] Japanese patent application the publication number of which is 2000-121716
In such an eigenvalue decomposition operation, methods with Householder's method or Jacobi method are known. If a Signal to Noise ratio of an arrival wave is high, that is, the arrival wave is very big in comparison with the noise, or a difference of arrival azimuths of two or more arrival waves is small, the ratio of a maximum value and a minimum value of the eigenvalues is high, so that underflow may occur in the operation of the smaller eigenvalue, and the computed eigenvalue may be a negative value or a value credibility on which is low. If a fixed decimal mode is used for a word size which is insufficient for an operation, the above-mentioned inconvenience is especially ready to appear.
If the credibility on the eigenvalues is low, the number of arrival waves which is estimated from the eigenvalues is likely to be incorrect. In addition, the vain operation is repeated in the eigenvalue decomposition operation in order to finally obtain the eigenvalue having low credibility, so that unnecessary operation is loaded on a processing unit.
The object of the invention is to provide an apparatus for estimating direction of arrival wave, for reliably estimating the number of arrival waves, extremely avoiding vain eigenvalue decomposition operations in order to overcome the above-mentioned inconvenience.

Apparatus for estimating direction of arrival wave


A seat back structure includes, a seat back frame which includes a panel material and a frame material joined with the panel material; a side frame which is included in the frame material and which is positioned in at least one edge side in a width direction of the panel material; and a first vulnerable portion which is formed in the side frame and which deforms when a load from an impact is applied. The first vulnerable portion is a slit formed by cutting out the side frame in a horizontal direction.

Conventionally, the Road Transport Vehicle Act determines the technical standard for structures and devices in an automobile in order to ensure safety and to protect the environment. As one standard, there is a safety standard for road transport vehicles. Such standard provides a method for testing the strength which a rear seat needs to protect passengers from luggage stored in a rear trunk when the automobile collides from the front and the luggage moves.
As a technique to satisfy such standard, there is a well-known technique described in patent literature 1, for example. The rear seat described in patent literature 1 includes a hinge mechanism which supports a seat back of a rear seat so that the seat back can be tilted to the front and the back with relation to a floor panel. Such hinge mechanism includes a portion which deforms according to a collision by luggage (block for testing) and which absorbs the impact when the luggage collides.


A universal fixture for holding printed circuit board assemblies during stencil printing, pick-and-place processing, and other PCB assembly processes. In one embodiment, a universal printed circuit board holder has a base and a plurality of support members movably attached to the base. The support members may project away from the base along support paths, and the support members are selectively positionable along the support paths when the support members engage a side of a printed circuit board assembly. Accordingly, the support members may be selectively positioned at heights corresponding to a topography of the side of the printed circuit board assembly to uniformly support the printed circuit board assembly.

Printed circuit board assemblies are used in computers, communications equipment, televisions, and many other products. In a typical printed circuit board assembly, many electrical components are attached to the top and bottom surfaces of a printed circuit board (PCB). Since the electronics manufacturing industry is highly competitive, it is important to maximize the through-put of processing PCB assemblies and to securely attach the electrical components to the PCBs.
The manufacturing of PCB assemblies involves many processes, one of which is surface mounting components to PCBs. To surface mount components to a first surface of a PCB, a dispenser deposits solder pads and/or adhesive pads onto the first surface of the PCB, and then a pick-and-place machine presses the components against the solder/adhesive pads. After the first side of the PCB has been populated with components, the PCB is inverted and the process is repeated to surface mount components to the second side of the PCB. The solder/adhesive dispenser is typically a stenciling machine, and the pick-and-place machine is typically a turret-type device.
Manufacturing issues related to supporting the populated sides of PCB assemblies are best understood in light of the structure and operation of conventional stencil printing machines. Conventional stenciling machines generally have a stencil plate, a movable platform under the stencil plate, and a PCB holder attached to the platform. Conventional stenciling machines also have two spaced-apart, parallel conveyor tracks to transport the PCB along a conveyor line between the stencil plate and the movable platform. In operation, the conveyor tracks engage opposing edges of the PCB and transport the PCB until it is over the PCB holder. The moveable platform then moves upwardly and engages the PCB holder with the downwardly facing side of the PCB to lift the PCB to the stencil plate. A wiper then moves across the stencil plate to press the solder paste or the adhesive compound onto the PCB. After the wiper stops, the work platform moves downwardly from the stencil plate to replace the PCB on the conveyor rails so that it can be transported to a pick-and-place machine.
In addition to maximizing the through-put of processing PCB assemblies, it is also becoming important to accurately mount a large number (e.g., 1000-1500) of very small components to one side of the PCB assemblies. An important aspect of surface mounting processes, therefore, is to accurately deposit very small solder pads and adhesive pads at precise locations on the PCB. Another important objective of surface mounting processes is to accurately press the electrical components onto the pads at the precise locations so that the electrical terminals of the components engage the desired solder pads. The PCB holders must accordingly hold the PCB assemblies flat with respect to the stencil plate so that a desired volume of the solder/adhesive compound is deposited in each pad across the surface of the PCB. Similarly, the PCB holders must hold the PCB assemblies flat in pick-and-place machines so that the terminals of the components engage the desired contacts on the surface of the PCB. Thus, it is generally desirable to uniformly support the underside of the PCB so that it does not bow or dip.
To uniformly support the underside of PCBs, one type of a conventional PCB holder has a platform and a number of pins removably attached to the platform to engage open areas or components on the underside of a PCB assembly. The pins of this type of a conventional PCB holder generally have a fixed height, and thus to engage a specific component, a pin having the correct height to support the specific component must be mounted to the platform at a location corresponding to that of the specific component. Another type of conventional PCB holder may have a platform, sidewalls projecting up from the platform to support the perimeter of a PCB, and spacers positioned within the sidewalls to support interior portions of the PCB. It will be appreciated that the pins and the spacers of conventional PCB holders generally prevent the PCBs from bowing to hold the PCBs flat for surface mounting processing.
Conventional PCB holders, however, encounter several manufacturing issues when components are surface mounted to both surfaces of a PCB assembly. For example, one problem with conventional PCB holders is that the pins or spacers may not be at the appropriate height or location to uniformly support the populated side of the PCB. The pins and spacers of conventional PCB holders are configured manually to engage vacant areas or to have a height that accommodates the components on the populated side of the PCB. However, it is extremely time-consuming to manually configure the individual supports on the platform to engage the vacant areas on a PCB. Moreover, it is even more difficult to select and position pins with the appropriate heights to engage components on the populated side of the PCB. Conventional PCB holders, therefore, may not uniformly support the PCB assemblies during surface mounting processes.
Conventional PCB holders are particularly difficult to operate efficiently in the manufacturing of contract PCB assemblies in which multiple runs of different PCB assemblies with different configurations of components are processed in a short period of time. Unlike dedicated PCB assembly manufacturing in which a large number of identical PCB assemblies are manufactured in each run, the down-time associated with configuring PCB holders in contract PCB manufacturing may significantly increase the cost of manufacturing the PCB assemblies. Therefore, there is a significant need to quickly and consistently support PCB assemblies with different configurations of components in the manufacturing of contract PCB assemblies.

Apparatus for holding printed circuit board assemblies in manufacturing processes


A lift truck with variable range with at least three wheels and a telescopic arm. The lift truck includes a steerable rear wheel and two front wheels, a driver station and a motor propulsion group secured to a chassis. The chassis has a compact configuration and is elongated in the longitudinal direction of advance of the truck, and has safety elements against the risk of loss of lateral stability. The safety elements are adapted to oppose turning over of the truck. The truck also includes a device to limit the maximum speed of advance based on a turning angle of the steerable wheel.

The invention relates to a motor driven lift truck, with variable range, in which the front end of the telescopic arm extends in front of the front wheels in the load carrying position.
The invention is particularly useful as a lift truck with variable range with three wheels.
There are known trucks with variable range with three wheels sold by the French company MANITOU BF under the name TMT: these trucks with variable range with three wheels have a U-shaped chassis, so as to be able to retract a load within the U-shaped chassis in the transport position. These trucks are particularly adapted to be loaded on the rear of a carrying vehicle, as is described in EP0 701 963.
Because of the particular structure of the TMT trucks adapted to be loaded on the rear of a carrying vehicle, these trucks have the drawback of a wide front track of the order of 2 meters and a short wheelbase of the order of 1.40 meter. This structure also has the drawback of a relatively short telescopic arm, permitting reaching a lifting height of the order of 3 meters.
Because of their particular structure permitting retracting the load within the U-shaped chassis in the transport position, these TMT trucks adapted to be loaded on the rear of a carrying vehicle have good lateral stability when turning.

Lift truck with variable range with at least three wheels


The subject invention relates to surfactant compositions of gemini surfactants and co-amphiphile(s), to a process for preparing such compositions, and to their uses, especially as emulsifiers and dispersants.

1. Field of the Invention
This invention relates to a surfactant composition of gemini surfactants and one or more co-amphiphile(s), to a process for preparing such composition, and to its uses, especially as emulsifiers and dispersants.
2. Description of the Prior Art
Mixtures of surfactants and co-amphiphiles and their use in emulsions or dispersions are well known. These mixtures have been designed for optimising the surfactant molecules pattern at the phase boundaries. When making emulsions, it is often intended to form liquid-crystalline, lamellar phases, which can be utilised to stabilise the emulsion.
Proportionate mixtures of special surfactants and co-amphiphiles are employed according to their applications. For those applications which require a high electrolyte content in aqueous solutions or emulsions/dispersions, ionic surfactants have up to now been considered to be largely unsuitable. In order to improve the selection of appropriate surfactants, efforts have been made to quantify or classify their polarities, thus enabling a more reliable assessment of the properties and application patterns. The HLB concept and its variations is mentioned here as an example of a simple approach.
Gemini surfactants, both as nonionic and cationic variants, have been known for more than twenty years, but it was not earlier than in the '90s that they stirred up a deeper interest. For a survey of the state of the art, see R. Zana, "Dimeric (Gemini) Surfactants" in Novel Surfactants Preparation, Applications and Biodegradability, C. Holmberg (ed.), Marcel Dekker (1998), p. 241.
Most of the recent publications deal with ionic gemini surfactants because these materials significantly enhance the interfacial activity, for instance expressed in terms of the critical micelle formation constant or the reduction in surface tension of water, in comparison with conventional surfactants.
At first gemini surfactants were mainly recommended for use in detergents and cleaning products.
JP-A 08/268 865 also describes the use of gemini surfactants in cosmetic preparations. In the formulations disclosed therein conventional anionic surfactants have been exchanged for anionic gemini surfactants, which are considerably less irritant to the skin, without laborious modification of the formulations.
EP-A-0 697 244 discloses amphoteric gemini surfactants, which can also be mixed with other anionic, nonionic, cationic, or amphoteric surfactants. Said surfactants are reported to be useful in detergents. The gemini surfactants (gemini amides) described in WO 95/19953 can be employed among others as components in customary cleaning preparations. In WO 95/19955 gemini polyethers have been disclosed as another class of gemini surfactants, which are useful for the same application mentioned hereinbefore. Mixtures of alkoxylated bisalkylphenol gemini surfactants and other surfactants are known from WO 97/23449.
In JP-A 11/60430 and JP-A 11/60437 the use of anionic gemini surfactants in cosmetics has been described. Reportedly, these surfactants can also be combined with other surfactants.
However, in contrast to what might be expected due to the substantial improvement in interfacial activity, no significant increase in efficiency or distinct improvement of application characteristics has been brought about by gemini surfactants as direct substitutes for conventional surfactants. The meagre advantage of such surfactant substitution cannot justify the effort of incorporating an additional building element in the basic structure.

Tenside composition containing gemini tensides and co-amphiphiles and production and use thereof


A biohazard-isolating bag is positioned in the interior space of a public mailbox. The biohazard-isolating bag receives mail that is deposited in the mailbox. A postal worker, upon retrieving mail from the mailbox, closes the biohazard-isolating bag. Contaminants that may be carried by letters or mailpieces are thereby contained within the bag and isolated from the environment exterior of the bag. The contents of the biohazard-isolating bag may be safely transported for further decontamination or processing.

1. Field of the Invention
The present invention relates to methods for depositing and retrieving mail into and from a mailing receptacle. More particularly, the present invention relates to equipment and procedures associated with the mailing process whereby handling mail is done in a secure method in order to guard against potential biohazards in contaminated mail.
2. Description of the Related Art
The United States Postal Service provides mail collection boxes in a multitude of public locations where members of the public may deposit mail items. Indeed this is true throughout the world; national postal authorities make public mailing receptacles open to the public. One of the chief design criteria for the public mailbox has been easy access and usability by the public.
In addition, the postal carriers and individuals who access public mailboxes typically do so in a hurried manner with little if any thought to potential hazards that may wait inside them. Thus the public mailbox makes an inviting target for those bent on anonymous and antisocial behavior.
These mailboxes have occasionally been the targets of pranks and vandalism. Recently there was a spate of more serious incidents involving contaminated mail. It is believed that mail was deposited and delivered through typical mail channels where the mailpieces had been deliberately contaminated with biological hazard material. Investigation has confirmed that some mailpieces deposited in mailboxes were contaminated with anthrax spores. Some members of the postal service, as a result of their handling contaminated mail, suffered exposure to the biological materials.
In response to these incidents, procedures have been developed to treat mail that is believed to be contaminated with biohazard or infectious materials. Decontamination methods have been developed that will render contaminated mail safe or harmless. However, application of these decontamination methods typically requires that suspected mail be transported from a collection location to a decontamination site. Thus, even with present decontamination procedures, there exists a risk of exposure to harmful agents within the mail while the mail is in transport to the decontamination site. Thus, there is a need to develop equipment and methods to minimize the chance of infection or exposure to biohazard material in the mail during the period between mail collection and decontamination.
The present mailbox design provides little protection against the threat posed from biohazards in contaminated mail. Accordingly, there is a need to improve the mailbox design. In addition, there is a need to develop practices and methods whereby letter carriers may perform their duties in a safe and secure manner.
It would be desirable to provide a means whereby contaminated mail in a mailbox can be collected with minimal risk of human exposure to infectious material potentially found in the mail.
It would also be desirable to retrofit the existing mailbox design such that mailboxes need not be replaced in their entirety. It would be advantageous to provide a means for secure handling of mail that can be applied to the mailbox design that is now in use.
It would also be desirable to provide a method to improve security in postal collection that can be enacted quickly and easily.