mushrooms produce spectacular diversity of biologically active biomolecules encompassing laccase , lectins , nucleases , proteases , antifungal proteins , ribosome inactivating proteins , and polysaccharides , polysaccharide  peptides and polysaccharide  protein complexes . many of these proteinaceous and nonproteinceous molecules manifest potentially worthwhile activities including antitumor , hiv-1 reverse transcriptase inhibitory , and immunomodulatory activities [ 1 , 2 ] . a variety of mushroom constituents are known to have health promoting activities and therapeutic potential . many mushrooms have a good taste and are highly nutritious . thus mushrooms are popular in the diet of many people all over the world . proteases catalyze degradation with high ( e.g. , trypsine ) or low ( e.g. , subtilisin ) specificity . [ 5 , 6 ] . the requirements of brewing , dairy , meat , leather , detergent , and photographic industries make proteases useful and commercial . the sale of industrial enzymes , a significant percentage of which is used for detergents , is a business involving millions of dollars . proteases have been reported from a number of mushrooms , including agaricus bisporus , armillariella mellea , cordyceps sinensis , flammulina velutipes , grifola frondosa , helvella lacunosa , lyophyllum cinerascens , pleurotus eryngii , pleurotus ostreatus , pleurotus citrinopileatus , and tricholoma saponaceum . xylaria hypoxylon , commonly known as candlestick fungus , carbon antlers , or stag 's horn fungus , is an inedible mushroom belonging to the genus xylaria . in china , x. hypoxylon is a kind of competitor fungus found in the cultivation of straw mushroom ( volvariella volvacea ) . two tetralone derivatives xylariol a and b , and two -pyrone derivatives xylarone and 8,9-dehydroxylarone possessing cytotoxic activities were isolated from the culture broth of x. hypoxylon [ 21 , 22 ] . sex cytochalasins binding to actin in muscle tissue , a xylose - specific lectin with antiproliferative , and antimitogenic activities have also been found in the fungus [ 23 , 24 ] . in view of the importance of proteases and the differences in characteristics of proteases from different sources and the dearth of information on protease from wild mushrooms , the present study was undertaken to purify and fruiting bodies of x. hypoxylon were purchased from a company specializing on straw mushroom in beijing and identified by institute of microbiology , chinese academy of sciences . the sources of other biochemical and chemical reagents used in this work are as follows : deae - cellulose , cm - cellulose , coomassie brilliant blue r-250 , glycine , casein , trypsin , and yeast trna , were obtained from sigma . q - sepharose , superdex 75 , molecular mass standards , and akta purifier were purchased from ge healthcare ( usa ) . a water extract of the fruiting bodies of x. hypoxylon ( 500  g ) was prepared by homogenization in distilled water ( 4  ml / g ) . following centrifugation of the homogenate at 12000  g for 20 minutes , tris - hcl buffer ( ph 7.2 ) was added to the supernatant obtained until the concentration of tris was 10  mm . ion exchange chromatography of the supernatant on a 5  20  cm column of deae - cellulose was then carried out in 10  mm tris - hcl buffer ( ph 7.2 ) . after removal of the flow - through fraction ( d1 ) , the column was eluted stepwise with 0.2  m nacl and then with 1  m nacl in the starting buffer to yield fractions d2 and d3 , respectively . fraction d3 was dialyzed , lyophilized , and chromatographed on a q - sepharose column ( 2.5  20  cm ) in 10  mm tris - hcl buffer ( ph 7.0 ) . when all the unadsorbed proteins ( collected as fraction q1 ) had been eluted , the column was eluted with a linear concentration ( 0 - 1  m ) gradient of nacl added to 10  mm tris - hcl buffer ( ph 7.2 ) . the second and most strongly adsorbed fraction , q3 , was dialyzed , lyophilized , and then applied to a 2.5  20  cm column of cm - cellulose . the column was eluted with 10  mm nh4oac buffer ( ph 4.5 ) until all the unadsorbed proteins had been eluted and collected as fraction cm1 . adsorbed proteins were desorbed with a linear concentration ( 0 - 1  m ) gradient of nacl in 10  mm nh4oac buffer ( ph 4.5 ) to yield fractions cm2 and cm3 . final purification was conducted by fplc - gel filtration of fraction cm2 on a superdex 75 hr 10/30 column in 0.2  m nh4hco3 buffer ( ph 8.5 ) using an akta purifier . sds - page was assayed using the protocol of laemmli and favre , using a 12% resolving gel and a 5% stacking gel . at the end of electrophoresis , fplc - gel filtration was carried out using a superdex 75 hr 10/30 column which had been calibrated with the undermentioned molecular mass standards . the molecular mass of the protein was determined by comparison of the elution volume with those of molecular mass standards including blue dextran ( to determine void volume ) , phosphorylase b ( 94  kda ) , bovine serum albumin ( 67  kda ) , ovalbumin ( 43  kda ) , soybean trypsin inhibitor ( 20  kda ) , and bovine -lactalbumin ( 14.4  kda ) . amino acid sequence analysis was carried out using an hp g1000a edman degradation unit and an hp1000 hplc system .   in this assay , an improved method of satake et al .   was used . in brief , a casein solution , which was used as substrate , was freshly prepared as follows : 0.1  g casein was added into 10  ml mes buffer ( 200  mm , ph 7.0 ) . following centrifugation at 12000  g for 20 minutes , the precipitate was removed and the resulting solution could be used as the protease substrate . the test sample or trypsin solution ( as positive control ) ( 20  l ) was mixed with the above casein solution ( 180  l ) and then incubated at 37c for 15  min . subsequently , 400  l of 5% trichloroacetic acid ( tca ) was added for ending the enzymatic reaction . the reaction mixture was then cooled to the room temperature before centrifugation at 10000  g for 15  min . the absorbance of the supernatant was read at 280  nm against water as blank using a uv - spectrophotometer . protease activity was calculated based on the activity of trypsin ( 7900 baee units / mg according to sigma ) in the protease assay using casein as substrate . protease activity was tested over the ph range ( ph 39 ) and temperature range ( 20c100c ) . casein in different buffers including 0.1  m naoac ( ph 35 ) , 0.1  m mes ( ph 57 ) , and 0.1  m hepes ( ph 79 ) were used to determine the optimal ph value . to determine the optimal temperature , the reaction mixture was incubated at 20c , 30c , 37c , 45c , 50c , 60c , 70c , 80c , and 100c in 0.1  m mes buffer ( ph 7.0 ) for 30  min . in this assay , the purified protease was exposed to the following inhibitors : phenylmethylsulfonyl fluoride ( pmsf , 1.0  mm ) , ethylenediaminetetraacetic acid disodium salt ( edta , 1.0  mm ) , pepstatin a ( 0.2  mm ) , and lima bean trypsin inhibitor ( 0.25  mm ) for 30  min at room temperature . control , in which inhibitors were substituted by mes buffer ( ph 7.0 ) , was taken as 100% . the assay for hiv-1 reverse transcriptase inhibitory activity was carried out using the assay kit from boehringer mannheim ( germany ) and the reaction protocol of zhao et al . . the assay for antifungal activity toward fusarium oxysporum , rhizoctonia cerealis , rhizoctonia solani , and sclerotinia sclerotiorum was carried out using the method of lam and ng . the ribonuclease activity of the purified protease was assayed following the method of mock et al .   and using yeast trna as substrate . the assay for hemagglutinating activity was measured with a 2% suspension of rabbit red cells in phosphate - buffered saline ( ph 7.2 ) at 20c with the method of zhang et al . . after ion exchange chromatography of the fruiting body extract on deae - cellulose , protease activity resided only in fraction d3 which was most strongly adsorbed on the column . protease activity was again enriched in the most strongly adsorbed fraction , q3 , derived by ion exchange chromatography of fraction d3 on q - sepharose . the unadsorbed fraction q1 lacked protease activity and the adsorbed fraction q2 exhibited very low protease activity ( table 1 ) . further purification of the unadsorbed fraction q3 on cm - cellulose allowed separation of a small unadsorbed peak from two adsorbed peaks ( figure 1 ) . the first adsorbed fraction ( cm2 ) had much higher protease activity than the following adsorbed fraction ( cm3 ) ( table 1 ) . cm2 was resolved into two peaks of similar sizes upon gel filtration on superdex 75 ( figure 2 ) . su2 appeared as a single band with a molecular mass of 43  kda in sds - page ( figure 3 ) and as a single peak with the same molecular mass upon rechromatography on superdex 75 ( data not shown ) . the purified protease exhibited an activity of 459.3  u / mg ( table 1 ) . a comparison of characteristics of x. hypoxylon and other fungal proteases is listed in table 2 . the protease was strongly inhibited by pepstatin a , but not significantly affected by pmsf , edta , and trypsin inhibitor ( table 3 ) . the protease activity increased steadily as the ph was raised from 3.0 to 6.0 and then remained high when the ph was further raised to 8.0 . there was an approximately 12% decrease in activity as the ph reached 9.0 ( figure 4 ) . as the temperature was raised to 100c , there was a quick fall in activity . however , about 30% of the maximal activity was remained at 100c ( figure 5 ) . at a protease concentration of 1  m , 5  m , 15  m , and 25  m , 17.5% , 42.3% , 65.6% , and 79.8% inhibition , respectively of hiv-1 reverse transcriptase were observed ( figure 6 ) . it did not exhibit antifungal , ribonuclease , and hemagglutinating activities ( data not shown ) . the present study constitutes the first report on the purification of a protease from xylaria hypoxylon . a comparison of chromatography behavior of x. hypoxylon and other fungal proteases is listed in table 2 . x. hypoxylon protease , just like h. lacunose protease , adsorbs on deae- , cm - cellulose , and q - sepharose . a protease from p. citrinopileatus unadsorbs on deae - cellulose and q - sepharose , but adsorbs on cm - cellulose . so far a few ascomycetous proteases from genus aspergillus , cordyceps , helvella , and xylaria have been reported , and only c. sinensis , h. lacunose proteases , and the present one are from mushroom species . compared with each other x. hypoxylon and h. lacunose proteases manifests a close temperature optima ( 60c and 65c , resp . ) , while c. sinensis protease possesses a quite low optimal temperature of 30c . x. hypoxylon and c. sinensis proteases exhibit a same molecular mass of 43  kda which is higher than that of h. lacunose ( 33.5  kda ) . c. sinensis and h. lacunose proteases are both alkaline proteases ( with optimal ph of 9.5 and 11 , resp . ) , while x. hypoxylon proteases has a fairly stable optimal activity in a ph range of 68 . microbial proteases display a range of molecular masses from 18  kda to 126  kda . x. hypoxylon protease exhibits an intermediate molecular mass ( 43  kda ) , which is the same as c. sinensis protease , but higher than those of proteases from p. eryngii ( 11.5  kda ) , t. saponaceum ( about 18  kda ) , a. mellea ( 18.5  kda ) , g. frondosa ( 20  kda ) , p. citrinopileatus ( 28  kda ) , and h. lacunose ( 33.5  kda )   and lower than p. ostreatus ( 75  kda ) . its n - terminal sequence is largely different from previously reported fungal protease sequences , suggesting that it is a new protein .   among the protease inhibitors tested , pmst , edta , and trypsin inhibitor have no significant effect on the protease activity , suggesting that it is neither a serine protease , nor a metalloprotease . the activity of the protease was adversely affected by pepstatin a , indicating that it is an aspartic protease . to date , many serine proteases have been isolated from mushrooms , such as a. bisporus , cordyceps sobolifera , h. lacunose , and h. marmoreus . other mushroom proteases are metalloproteases , including a. mellea , g. frondosa , and t. saponaceum . only a few mushroom proteases are belonging to aspartic proteases , such as p. eryngii protease . x. hypoxylon protease is relatively thermostable because its activity remains within the range of 65%100% of its maximal activity at 60c as the temperature varies from 20c to 70c . at a high temperature of 80c , a significant portion ( about 50% ) of enzyme activity remains and at 100c a. mellea protease is stable up to 55c , but its activity rapidly declines at higher temperatures . the optimal temperature of x. hypoxylon protease is very close to that of h. lacunose protease ( 65c ) , and higher than that of p. eryngii ( 45c ) , a. clavatus ( 50c )   h. marmoreus ( 50c ) , and t. saponaceum ( 55c ) . the protease activity remains essentially unaltered in the ph range 68 and 60%100% of the maximal activity is present even of the ph is changed from 39 . eryngii protease demonstrates an acidic ph optimum of 5.0 , while several proteases of other mushrooms manifest optimal ph in the alkaline range , such as proteases from c. sinensis ( ph 9.5 ) , p. citrinopileatus ( ph 10 ) , and h. lacunose ( ph 11 ) . rapid decline in activity is noted , when the ph value is lower or higher than ph 7.5 . hence , the isolated protease from x. hypoxylon appears to be relatively stable with regard to variations in ambient ph and temperature , compared with h. marmoreus protease . x. hypoxylon protease demonstrates hiv-1 reverse transcriptase inhibitory activity , with an ic50 value of 8.3  m . many mushroom proteins , such as lectins , ribonucleases , and proteases , manifest inhibitory activity toward hiv-1 reverse transcriptase . the ic50 values of i. umbrinella lectin , l. shimeji ribonuclease , p. eryngii protease , h. erinaceum lectin , are 4.7  m , 7.2  m , 30  m , and 31.7  m , respectively [ 16 , 30 , 42 , 43 ] . x. hypoxylon protease is the first ascomycetous protease reported manifesting hiv-1 rt inhibitory activity . up till now , only a basidiomycetous protease from p. eryngii has been reported to inhibit hiv-1 rt . inhibitory activity of the purified protease is higher than those of p. eryngii protease and h. erinaceum lectin , but lower than those of i. umbrinella lectin and l. shimeji ribonuclease . the inactivity of x. hypoxylon protease in hydrolyzing trna , inhibiting fungal growth and agglutinating rabbit red blood cells indicates that it is free of contamination with ribonucleases , antifungal proteins , and lectins that are commonly produced by mushrooms .   in summary , x. hypoxylon protease is a unique enzyme with a distinctive n - terminal sequence , relatively high thermostability and the first report of hiv-1 reverse transcriptase inhibitory activity from ascomycota mushrooms .
a novel aspartic protease with hiv-1 rt inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom xylaria hypoxylon . 
 the purification protocol comprised distilled water homogenization and extraction step , three ion exchange chromatographic steps ( on deae - cellulose , q - sepharose , and cm - cellulose in succession ) , and final purification was by fplc on superdex 75 . 
 the protease was adsorbed on all the three ion exchangers . 
 it was a monomeric protein with a molecular mass of 43  kda as estimated by sds - page and fplc . 
 its n - terminal amino acid sequence was hytellsqvv , which exhibited no sequence homology to other proteases reported . 
 the activity of the protease was adversely affected by pepstatin a , indicating that it is an aspartic protease . 
 the protease activity was maximal or nearly so in the ph range 68 and in the temperature range 3560c . 
 the purified enzyme exhibited hiv-1 rt inhibitory activity with an ic50 value of 8.3  m , but was devoid of antifungal , ribonuclease , and hemagglutinating activities .