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1 A rush job that doesnt work (out)oodaks | 06/01/2011 | See all oodaks's reviews (8)First up, credit where credit is due - the Heart Rate Monitor is great, works perfectly, and provides realtime feedback, which in turn makes exercising more interesting. All good so far.However, it is an excercise tool, and it just doesnt work anywhere like it should. The devil here is in the detail - all Kinnect games are all Kinnect "game/title", this one is merely in brackets like an after thought - thats because it is.This title was not built for the Kinect, it was built for the Wii and PS3, and this is just a hack of that for the Kinnect - a straight up way for EA to get more money out of something already done.Does that matter? Massively. Heres why. The tracking is poor. However, that actually doesnt matter, as long as you can get on with your Workout. Well, you can't. It just randomly pauses, for no reason at all, and you constantly have to yell at it to try and activate the voice command, and when that fails, and it often does, you then have to reach for a controller to hit the A button.It often pauses when you are just holding a stretch - you havent gone anywhere or moved or turned the lights off - it saw you perfectly fine you for the first 15 seconds of holding the stretch, why then does it decide it cant see you and pause!!Its random to, and it just disrupts your workout by breaking your rhythm while you stop and get it going again - the whole point of this title is an exercise tool yet it stops you all the time from exercising! No one would tolerate a First Person Shooter if you couldnt do any First Person Shooting!To qualify my review - I have plenty of clear space (move the coffe table and couch out of the way), plain carpet and wall, good lighting, a healthy heart(the monitor is not registering a heart attack or flatline), and a very mild accent. I have other Kinnect titles that perform amazingly perfect and dont randomly pause all the time.One more thing - EA couldnt even be bothered to license some decent tunes to work out to - music being widely used by most people to aid exercise. You can add your own tunes, but again, its just another point of how little EA done to get this"working" on the Kinect.I have used a friends "Your Shape : Fitness Evolved" that is a proper Kinect built title, and it shows as it is mint - 1,000,000,000 x better.EA, this is a disgrace. I will stick with it only becasue I have already handed over my cash, though will move to something better as soon as I can.We can only hope EA do it properly if a Sports Active 3 ever gets made.If you havent brought this, please dont - go for "Your Shape : Fitness Evolved" or wait until EA do a proper version.There will be no "Update" to fix these problems, as its at the core of the games engine - no chance some patch could fix it.I give it 1 Star for the Heart Rate Monitor - hopefully when SA3 arrives I will be able to use it with that and it wont be a complete waste of my cash.EA, "its NOT in the game" this time.
1 Avoid.Blank2k | 28/12/2010 | See all Blank2k's reviews (11)I've had this since launch, and with Kinect a few exercises don't register, and it random loses you so it pauses the game & waits for you to return.... even though you've not moved from the cameras view, and video tutorials have a nasty habbit of popping up mid-workout & voice control usually means your shouting "Skip" "Skip!" "Skiiipp" "SKIP!!" "SKIPPPP!!!" "SKIP!!!!!!" in hopes it'll pick you up resulting in a very tedious workout.Also on every other menu it likes to "Connecting To EA Servers" and EA servers seem to be very poor, some times this message will be on screen for 5 seconds other times 5 minutes until it decides the servers aren't there or you cancel it (until you goto another menu)The game itself is very good, but it was clearly designed for Wii/PS3 and then EA decided they'd bodge some Kinect into it. (And i gather PS3 version has a few issues aswell)I'd of reviewed this sooner, but ive been awaiting an update for EA Sports Active 2 to fix these problems, and well Finally! EA have released an update that fixes, well, pretty much nothing of relevance.If i had kept my receipt it'd of certainly rammed it back, but i didn't so EA screwed me out of another 60 quid, I'd recommend buying Yourshape instead as its motion detection is infinitely superior.Edit: I'd love to know if people rating this 5/5 are actual using the X360 version or just PS3/Wii - There's 3 of us with this game in a variation of room sizes & lighting situations, 1 has returned it, the other is baring his teeth hoping for an update, and i'm using it as a coffee coaster.
5 Really goodmonkeywrench7 | 19/11/2010 | See all monkeywrench7's reviews (1)I have found this to be really good! It makes you warm up and cool down between exercises and really can get you sweating! The sensors work very well and feels like a quality product.Similar to wii fit but without the scales!
1 just activeAmbitiousTerror | 01/01/2011 | See all AmbitiousTerror's reviews (3)I bought this for xmas and have had lots of problems with it..first the box states a minimum space 2.1 meters i have 2.5 and cant use a lot of the exercises as you need to face the sensor and need to be low to the floor on many exercises a minimum of 3 metres (9+ feet) is probably best... the movment pauses and does not track movement very well
5 Good workoutSymonep | 04/01/2011 | See all Symonep's reviews (2)I have Ea sports and EA sports more workouts on the Wii but loved the idea of not having to strap the controler to my leg. After reading reviews i was in 2 minds about buying it, I did and have had no problems except sometimes with the floor workouts as i need to move the kinect to a different level but it isnt an issue to do this. Much prefer this to the wii version where i had many more problems with it recognising movements.


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Our Company
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Our gunsmithing shop is open from 10am to 7pm Monday through Saturday and closed on Sundays
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capable of handling virtually any job. Replace old sights, restore your great grandfather's hunting rifle, or personalize your firearm with a custom paint job. Our gunsmiths have over 50 years of combined experience and no job is too big or too small. If you can dream it we can make it a reality.
We charge in fifteen minute increments and have a minimum charge of $30
We charge a diagnostic fee of $45 which includes a full dis assembly and cleaning on all firearms.
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Sweet Estrella is an affectionate, family-oriented Pittie mix who loves to love!
Adorable and uber social Sophie is a fun-loving, quirky kitty with a face like Grumpy Cat!
If you're looking to heal a sweet kitty's heart, please read more about sweet Bunny.
We have some darling new small dogs looking for loving homes!
Join us Sunday, March 22nd for the always-popular egg and dog-bone hunt – The Hunt is On!
Looking to give back to homeless dogs and cats this year? We have many great volunteer opportunities.
Shop to your heart's content while supporting CCHS through Cafe Press!
Can you guess… how many different vocal sounds dogs & cats have?
Spring is kitten season – learn what to do if you've found kittens in your area.
CCHS endeavors to educate the public to foster compassion, responsibility and respect toward all animals by providing information and resources on a variety of animal and people related topics.


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Bronze Portrait of Tiberius
Date: Daily through September 22, 2014,
Location: Museum, Floor 2, Getty Villa
Admission is free. An advanced timed-entry ticket is required.
Following a yearlong conservation project and subsequent exhibition, this over-life size bronze portrait of Tiberius from Herculaneum remains on view at the Getty Villa. Marking the two thousandth anniversary of Tiberius's accession as Emperor, the statue is displayed in a gallery devoted to Men in Antiquity (209). There he stands in the company of busts of other Roman emperors, including his predecessor Augustus and successor Caligula.


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Quote: Your attitude, almost always determines your altitude in life.
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We presented the framed motivational poster with the signatures of our management team to the general manager on boss's day. His comment was that it was the coolest, best boss's day gift ever! I was proud to present the gift as it was of high quality and being able to add our signatures and a personalized plaque made it even more special. The other managers were very impressed with the gift I had picked out to give to our GM from all of us.
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Delivery was 4 days earlier than expected. We ordered late and I was hoping it would arrive the morning of the day we were supposed to present it. The item arrived early which allowed us to get all the signatures and wrap the gift ahead of time.
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5.5" Kubaton Self Defense Jabber w/ Keyring- Black. 5.5" overall. Pointed tip for self defense purposes. Has a convenient keyring so you can carry it with you anywhere. Easy grip surface.
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October 10, 2012
September 21, 2012
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Leonardo has yet to fill out a bio.
So you quited.. :(
I used to LOVE Zoobilee Zoo!
Oh and also, check out this writer. He needs the support :/
That's pretty BS, you are a great writer. Its probably because you haven't written in a while that's why. They always do that.
I have been better, LOL. Its nice to hear from you. I miss reading your work.
:D so when are you planning on writing again? :)
MAZUSA!!!!!! Thats it, How are you?
I am starting to not recognize what writer this is? The name has me confused.
I love the name :)


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News Feature
Travel to Croatia for unique healthy eating fair
16 February 2011Travelling to Croatia next month may be a popular action among Britons who love to eat healthy and delicious food.
That is because the Mediterranean Health Food and Medicinal Herbs fair is coming to the ancient city of Dubrovnik between March 24th and 27th.
Locals and tourists alike converge on the Valamar Lacroma resort for the event, which attracts numerous exhibitors showing off their tasty wares.
In addition to the chance to try and buy top-class cuisine, guests can sit in on educational presentations and lectures at tables around the venue.
The fair is said to be the most important date in the calendar in terms of organic agriculture in Croatia.
Dubrovnik restaurants and bakeries also come out in force to present their delicacies and perhaps inspire people to dine out while on holiday in Croatia.
Other special events taking place in Dubrovnik include the city's carnival, which is already underway and runs until March 6th.
Croatian Villas offering villas, apartments and waterside properties from the people who really know Croatia.
Latest News
- Bikers show motoring holiday possibilities in Croatia - 29 June 2011
- Dubrovnik holidaymakers can enjoy magical musical performance - 29 June 2011
- Croatia 'ideal for a sailing holiday' - 29 June 2011


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Colonel John - 2009 Wickerr Stakes
increased his total earnings to $1539012. After winning the Santa Anita Derby, the colt was one of betting choices in the Kentucky Derby Presented by Yum! Brands (gr. I) but wound up sixth behind Big Brown after encountering traffic troubles in the 20-horse field. Becrux, winner of the Wickerr Stakes in 2006 and '07, ran well to be runner-up and paid $6.60 and $4.20, while Ferneley returned $3.80 to show. ... Colonel John horse racing del mar trevor denman Wickerr Stakes garret gomez Harty Eoin ...
Tags: Colonel, John, horse, racing, del, mar, trevor, denman, Wickerr, Stakes, garret, gomez, Harty, Eoin
Jesse W. Hammock racing on Attainable
Jesse W. Hammock racing on Attainable. Delaware Park, Sept. 2, 1979. Race 1 for the day. Length of the race was 6 Furlongs. The horse Attainable was trained by Anthony Dutrow, brother or trainer Richard Dutrow jr. trainer of Preakness winner Big Brown. ... "Jesse W. Hammock" "Richard Dutrow" "Anthony Dutrow" "Big Brown" Preakness "Horse racing" "Delaware Park"
Tags: Jesse W. Hammock, Richard Dutrow, Anthony Dutrow, Big Brown, Preakness, Horse racing, Delaware Park
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1. What inspired you to become a director?
I tried many different things to begin with before trying my hand at direction. I think of myself as a very good liar. When I was a kid, I realized I could get people's attention easily when I told interesting lies, but white ones. This made me a storyteller. My main trigger was a workshop with Anisul Haque and Mostafa Sarwar Farooki at Chittagong Film Centre in 2002. After that, I felt like I wanted to be a storyteller by profession.
2. What was your first production as a director?
My first production, titled '2 in 1' was in 2006. This was almost based on a real story, told from two different perspectives by two directors, I also starred in it.
3. What is your most favourite production of all time?
In the world of Bangladeshi television, my favourite one is 'Spartacus '71', produced in 2008, by Mostafa Sarwar Farooki.To me, amongst all the different works on Bangladesh's Liberation War, be in on TV or celluloid, this is the best of all. In the world of films, 'Elite Squad', both the first and its sequel (directed by José Padilha), are my favourites. The movies show how a security force called the Elite Squad was put together to protect the Pope during his visit to Brazil, which was necessary since Brazil was overrun with crime and violence. The first movie shows the squad's successes and popularity. But, the sequel shows how the Elite Squad abuses their power and becomes the problem themselves. I could not help but appreciate how similar the story is to our very own Rapid Action Battalion (RAB).
4. Who have you enjoyed working with the most?
It is very difficult to mention one person! There are so many people I enjoy working with. But if I have to choose one, then its Zahidul Haque Apu, he is a Radio Foorti RJ by profession, but also an actor and has worked with me on several television fictions.
5. What is your most embarrassing memory as a director? Did you ever make a mess of things?
In 2007, I really liked an external shooting location in Uttara, Dhaka. I thought it was ideal, and I made all the arrangements for the scene to be shot there. On the day of the shooting, I found out that the house did not have the required power line! We needed a 440 KV electricity connection, but the house had a 220 KV one (which common houses have). The embarrassing part was that I was trying to convince my crew that we have to manage with this. They informed me that this was not enough to even turn on the shooting lights! I was so ignorant, that the crew had to actually connect the equipment and turn on the switches to show me that the lights would not turn on. I learnt a big lesson that day, and obviously had to pack up and change the location.
6. What are some of the common challenges you face as a director in Bangladesh?
Television and film, as industries in Bangladesh, do not have all its support functions. They are incomplete as industries. As a director, I have to be in charge of every component imaginable, starting from fundraising, casting, marketing, getting the output signed with a television network. If there were specialists who could take care of these others facets, then I could better focus on my work as a director.
7. In your opinion, what is missing from the overall media today?
As a nation, I think Bangladeshis have become very black and white in terms of what they want on television, in movies or in the media in general. And this was not always the case; you can't talk as much about reality today as you could before. I think what's missing is the appetite to look at the negative sides of society, the stomach to take all the dark corners along with what's good. This means, as a director, my options are very limited, and I know that I have to restrict my thinking in order to make sure my productions see the light of day.
8. Television directors are seen to try their hand at making movies. Do you think television direction is a starting point to get to movies?
Yes, true. Bangladesh does not have any formal process or institute to groom these talents, rather, people start by assisting someone who is already established. For example, I started working with Mostafa Sarwar Farooki, and learnt step-by-step working on the job. So, yes, often people will start with television productions before trying their hand at movies.
9. What would be your word of advice for those who want to enter the world of making TV fiction?
I would say you have to make sure you actually have the passion for it, and have foresight, because you cannot expect to make good money in the first few years. You have to be very hard working, because you have to micro-manage every part of the production you are in charge of.
10. Where do you see yourself ten years from now?
In ten years, I hope to have visited twenty countries, and hope to be making movies!
Interviewed by Zakir Mushtaque


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Mario Batali's Chestnut-Apple Ciambella
From Delish
This moist and delicious Bundt cake has an almost pudding-meets-a-cloud-in-heaven texture, thanks to the chestnut flour and the shredded raw apple.
For more great recipes from Mario Batali, pick up his book Molto Batali: Simple Family Meals from My Home to Yours and catch him on ABC's newest talkshow The Chew.
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Nutritional Information
(per serving)
Calories | -- |
Total Fat | -- |
Saturated Fat | -- |
Cholesterol | -- |
Sodium | -- |
Total Carbohydrate | -- |
Dietary Fiber | -- |
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Quentin Bacon
Serves: 8
Yields: 8 to 10
U.S. | Metric | Conversion chart |
- 1/2 cup(s) dried currents
- 1/2 cup(s) grappa
- 1 cup(s) light brown sugar, packed
- 1/2 cup(s) granulated sugar
- 1.5 cup(s) cake flour
- 1/2 cup(s) chestnut flour
- 1/2 teaspoon(s) ground cloves
- 1 teaspoon(s) baking soda
- 1 teaspoon(s) baking powder
- 1 teaspoon(s) salt
- 6 (eggs) separated, at room temperature
- 1/2 cup(s) extra virgin olive oil
- 1/2 cup(s) buttermilk
- 1/2 cup(s) fresh chestnuts, chopped
- 2 Rome apples (or any soft tart cooking apple), peeled, seeded, and shredded
- 2 cup(s) confectioners sugar
- 4 teaspoon(s) light corn syrup, or more if needed
- 1 cup(s) mascarpone, at room temperature
- Place the currants in a small bowl, add the grappa, and set aside to soak for 1 hour.
- Preheat the oven to 350 degrees F. Lightly butter and flour a Bundt pan.
- In a large bowl, combine the brown sugar, granulated sugar, cake flour, chestnut flour, cloves, baking soda, baking powder, and salt. Mix until well blended. Add the egg yolks, the oil, and the buttermilk, and, using an electric mixer, beat until smooth.
- Drain the currants, reserving the soaking grappa. Fold the currants, chestnuts, and apples into the batter.
- In a separate bowl, beat the egg whites until stiff. Fold the egg whites into the batter, and then pour the batter into the Bundt pan. Place the pan in the oven and bake for 1 hour, or until a toothpick inserted into the cake comes out clean.
- Remove the pan from the oven and set it aside to cool for 10 minutes. Then invert the cake onto a plate.
- In a small bowl, stir together the confectioners sugar, 4 teaspoons of the reserved soaking grappa, and the corn syrup until smooth and glossy. (If it is too thick to spread,add another teaspoon of corn syrup.) Pour the icing over the peak of the cake all around the ring, and allow it to set for 10 minutes.
- Drink a shot of the remaining grappa in the kitchen before serving the cake with a bowl of the mascarpone on the side.
From Molto Batali: Simple Family Meals from My Home to Yours ©2011 HarperCollins Publishers - Buy the book
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We are particularly in need of dog and/or cat food and Large Dog Houses right now to help these animals survive the hard weather seasons! They do not need to be new but they do need to be in good usable condition. There are many dogs that we come across while doing outreach who have dog houses that are in horrible condition or do not have a dog house at all. Many of these dogs may not survive without an adequate shelter. You can drop off dog houses or any other donations at our drop-off locations listed below or contact us on Facebook or through email if you need someone to pick up your donation. We have several drop off locations around the city. Following is a list of addresses where you can drop off your donations closer to home. Thank you!
Lee's Summit, MO: 409 SW Lakeview Blvd., LS, MO 64063 (leave items on screened back porch)
Overland Park, KS: 5208 W. 104th St., OP. KS 66207 (left side of the front porch)-PLEASE NO HAY OR STRAW BALES AND NO LARGE DOG HOUSES!
North of the River (Liberty area) 8641 NE 74th Terr, KCMO 64158 (front porch)
Independence: 12900 E. 49th street, Independence, MO 64055 (right side of the house)
Monetary Donations or items small enough to be sent through the mail can also go directly to Chain of Hope at P.O. Box 16926, Kansas city, MO 64133
Other Items We Are In Need Of:
Large rawhides/pig ears
Medium-Large collars, preferably the buckle type
Metal bowls (can be old pots, etc.)
Super weight tie-out cables
Dog and cat food
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Advantage/Frontline products for dogs and cats
Fly-catching bags
Gas card
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OLED or Otherwise (ORGANIC LIGHT-EMITTING DIODE)
is a LIGHT EMITTING DIODE (LED) in which the Electroluminescent layer is a film of Compounds that are Organic and release light energy when electricity is passed through them. The layer of the Semiconducting materials is situated between two electrodes. OLED belong to either one of two families: Those based on Polymers and those based on small molecules.
Their usage is spread over a wide area of devices and gadgets eg.Television screens, Computer Monitors, Mobile phones,PDA's ,watches and for advertising to mention but a few. The need for the technology especially in lighting is brought about by the fact that some of the traditional sources for lighting e.g. Incandescent lamps, light bulbs, consume about 45-50 % of all the lighting energy and yet produce only about 14% of light which is measured in Lumens. About 90% of the energy goes to the production of heat which is really inefficient. Fluorescent lamps are three to four times more energy efficient, but still, a sizable fraction of the used energy is wasted. Therefore a more efficient lighting method is required. This is where OLED comes into play.
OLEDs are energy converting devices, in that they convert electric power to Light energy based on the process of electroluminescence. Whenever a voltage is applied on to the electrodes, charges move in the device under theinfluence of the electric field. Holes are moved from the anode and Electrons from the cathode.The Recombination of the two charges lead to the formation of a photon which is light energy. The frequency given because of the energy gap is given by(E=hv) Researchers had their eyes on OLEDs during the 1960s because of the Potentially high quantum efficiency of luminescence and the ability to generate a wide variety ofcolors. Unfortunately, due to high operating voltagesof about 1000V it became clear that they were not going to be practical. Not for long though, because in 1987, Dr. Ching Tang and Mr.Van Slyke from Eastman Kodak developed a heterostructure double layered device containing active "small molecules" that combined a low operating voltage of less than 10Volts with good brightnessof greater than 1000 cd/m2 and a respectable luminous efficiency of 1.5lm/W, research once again gained the momentum. In 1990, electroluminescence from polymers was achieved by Burroughes.From then on, OLED research has gained acceleration and as far as device efficiency levels are concerned it has surpassed incandescent lighting.
The advantages of OLEDs include:
- They have a high efficiency in terms of power usage and large area sources.
- They produce a great deal of brightness and the angle of view is wide.
- They are usually thin Flat and FlexibleLow voltage and fast switching technology.
- They allow for form freedom and tunable emissions.
- They are used in flexible displays.
Disadvantages include:
- Water can damage the organic materials of the displays.
- Outdoor performance is poor because the Metallic cathodeusually acts as a mirror leading to poor readability in bright litareas.
- OLED displays can be damaged by overexposure to Ultraviolet Light.
- One of the most discouraging aspect of OLEDs is theirlifespan which can around 14,000 hours to half the original brightness when used for flat-panel displays.
Research is on going to on ways of extending the life spans of the OLEDs and also on lowering down the cost of production. Currently there is a steady market for the technology and the trend looks encouraging for the next foreseeable future.


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Chuck Inglish - Swervin Feat. Sir Michael Rocks
Swervin Chuck Inglish Feat. Sir Michael Rocks
Although it's a Chuck x Mikey collabo it's not officially 'The Cool Kids' but it's still nice to hear. This is off Chuck's upcoming solo album, "Convertibles," of course with the production from Chuck.
Listen to Swervin, the new track by Chuck Inglish featuring Sir Michael Rocks which was dropped on Tuesday, July 30th, 2013. Chuck Inglish's odds keep getting better with each new leak, and Swervin will undoubtedly improve his chances of winning. It's a worthy addition to a respectable catalogue that has seen a lot of growth since Chuck Inglish debuted in this game. We're looking forward to the follow-up.
Check out Chuck Inglish on Came Thru/Easily , featuring Mac Miller & Ab-Soul . Check out Chuck Inglish's profile page for the most recent info, news, songs, and mixtapes. Check out Chuck Inglish's Twitter.
How's the chemistry here? How do you feel about the production here?


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Lady Gaga Shows Off Engagement Ring for Terry Richardson [PHOTOS]
It looks like pop diva Lady Gaga is on quite a ride on her Born This Way Ball tour, which features 110 shows around the world. Her good friend and famed photographer Terry Richardson followed her on her visit to Tokyo where she performed her second album Born This Way.
Lady Gaga To Wear Giorgio Armani For Born This Way Ball Tour [PHOTOS]
The 26-year-old strikes silly poses for Terry as she gets dolled up backstage and although she had just gotten out of a relationship with ex Taylor Kinney, she flashes a rock on her ring finger as Terry joked and captioned in his blog, "Gaga showing off her new engagement ring!" Good one, guys. Terry joined Lady Gaga and her crew in Disneyland Tokyo and both sported their Mickey and Minnie Mouse ears together.
Watch 50 Of Lady Gaga's Most Outrageous Fashion Statements [VIDEO]
Checkout Gaga looking flashier than ever in yet another meat dress. She takes it down a notch compared to her controversial meat dress she wore at the 2010 MTV awards.
Don't forget to Pin-It!
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* One bedroom 4th floor condo. Located downtown and adjacent to marina and gulf bay. Walking distance to amenities. Superb accommodation offered fully updated and fully furnished. Tiled throughout. All new appliances, internet, TV, stereo, piano. Linen provided. Laundry room on each floor. Air conditioning.Parking space for one car.Exercirse roome with all new equipment. Security entrance. Concierge between 8.30 - 4:30 Mon-Fri.
* Clubhouse for entertainment and pool.
(AVAILABE July 01 -2015 ,TILL Oct -01-2015 )
Two hour drive to Disney theme parks. One hour drive to Bush gardens. 45 minute drive from Tampa International airport and ten minute drive to local Sarasota airport.
Rate Period | Nightly | Weekend Night | Weekly | Monthly * | Event | ||||
My Standard Rate
May 1 2015 -
Oct 27 2016
7 night minimum stay
* Approximate monthly rate. Actual rate will depend on the days of the month you stay.
Payment is usually accepted in the quoted currency (USD) unless the currency and the amount is specifically agreed in advance with the owner / advertiser.
One month reservation required. With a security deposit of $500.00 and first / last rent.
Winter retes $2,200.- NOV, DEC, JAN, FEB, MARCH, APRIL-2015
Includes electric water internet association & cable.
A refundable deposit of $500 is required against delapidations and key loss.
And $100. for cleaning.


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People in treatment for multiple myeloma may be eligible to receive grants to help with treatment-related transportation costs through CancerCare's "Door to Door" program. The grants can be used for expenses such as gasoline, parking and tolls, and taxi, bus, or train fare to and from medical care.
To apply for a grant, download a financial assistance application from our website or call 800-813-HOPE (4673).
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BioMed Research International
Volume 2013 (2013), Article ID 175012, 14 pages
Production and Characterization of Keratinolytic Protease from New Wool-Degrading Bacillus Species Isolated from Egyptian Ecosystem
1Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, New Borg Al-Arab, P.O. Box. 21934, Alexandria, Egypt
2Botany and Microbiology Department, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt
3King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia
Received 2 December 2012; Revised 7 March 2013; Accepted 28 March 2013
Academic Editor: Periasamy Anbu
Copyright © 2013 Mohamed A. Hassan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry.
1. Introduction
Keratins are classified as fibrous proteins called scleroproteins that occur abundantly in epithelial cells. These proteins are insoluble in water, weak acid and alkali, and organic solvents and are insensitive to the attack of common proteolytic enzymes such as trypsin or pepsin [1]. The animal remains rich in α-keratin such as animal skin, hair, claws, horns, and wools.
The important property of these proteins is the presence of high cystine content that differentiates keratins from other structural proteins such as collagen and elastin. Both a high cystine content as well as a high content of glycine, proline, serine, and acidic amino acids and a low content of lysine, histidine, and methionine (or their lack) as well as the absence of tryptophan are also characteristic of keratins [2, 3]. Numerous disulfide cystine bonds present in keratin to bind peptide chains and packed as α-helices as in hair and wool or in β-sheet arrangements as in case of feathers. The disulfide linkage and the tight secondary structure of keratins make them difficult to be hydrolysed by common proteolytic enzymes [4].
The major problem of α-keratin hydrolysis is the presence a high numbers of disulfide bonds that make it insoluble in nature and resistant to proteases hydrolysis [5]. Keratinolytic protease enzymes are spread in nature and elaborated by different groups of microorganisms that can be isolated from polluted area with keratin wastes [6]. A vast variety of Gram-positive bacteria including Bacillus, Lysobacter, Nesternokia, Kocuria, and Microbacterium as well as a few strains of Gram-negative bacteria such as Xanthomonas, Stenotrophomonas, and Chryseobacterium are confined as keratin degraders [7–10]. Most of keratin degrading bacteria belong to the genus of Bacillus [11].
The keratinous substrate such as feather and wool can be degraded in basal medium by microorganisms which are capable of utilize keratin as sole carbon and nitrogen source [12]. Keratinolytic proteases have broad substrate specificity where they have the ability to hydrolyze soluble protein such as casein, gelatin, and bovine serum albumin. Additionally, they can hydrolyze the insoluble protein including feather, silk, and wool [13]. Keratinolytic proteases mostly belong to serine or metalloproteases showing sequence similarity with subtilisin group of proteases [14, 15]. In recent years, more demands to keratinolytic proteases are increasing due to their multitude in industrial applications such as the feed, fertilizer, detergent, and textile industries. The present study describes the isolation and identification of new Bacillus amyloliquefaciens MA20 and B. subtilis MA21 strains from Egyptian ecosystem that grow well on wool as sole carbon source. Moreover the two strains are able to degrade wool and their enzymes can be used to improve the wool quality. This paper includes full characterization of the keratinolytic protease which explains that the best environmental conditions can be used to improve the wool quality in industry.
2. Materials and Methods
2.1. Sample Collection
Different types of samples were collected from different Governorates of Egyptian ecosystem included Alexandria, Behera, Qaliubia, Mania, and Asiut. These fresh samples were varied such as soil, sand soil, humus, waste wool, and rhizosphere under olive trees. These samples were collected in sterile falcon tubes and transported to the microbiological lab in (City of Scientific Research and Technological Applications).
2.2. Strains Isolation
The bacterial strains were isolated by suspending 1 g of soil samples in a 10 mL sterile 0.85% (w/v) saline solution and then treated for 20 min at 80°C. This will enable the isolation of the spore-forming bacteria. Luria-Bertani (LB) agar medium with 1% (w/v) skim milk was used for their cultivation by spreading 0.1 mL of each 10−5 and 10−6 dilutions. The plates then were incubated for 24 hours at different temperatures [4, 16]. The colonies which give clear zones formed by hydrolysis of skim milk were picked. Pure bacterial isolates were obtained by reculturing individual colonies several times on fresh LB agar medium to produce single colony from each.
2.3. Strains Selection
Twelve selected strains isolated according to the diameter of clear zone were cultured on medium containing 0.5 g NaCl, 0.3 g K2HPO4, 0.4 g KH2PO4, and 10 g wool per liter; pH 7 and incubated for 5 days at 37°C. The wool was used as sole carbon and nitrogen source for detecting potent strains that have the ability to degrade the wool completely. Three strains degraded the wool and the supernatants of their culture were assayed on plate containing 1% gelatin powder which is soluble in phosphate buffer pH 7. After determining the existence of the activity (by the clear zone of the supernatants), Bacillus sp. MA20 and Bacillus sp. MA21 were selected and preserved for further investigation.
2.4. Bacterial Identification
While the phenotypic characteristics and isolation method of the two selected isolates indicate that they are related to Bacillus group but further identification was conducted.
The strains identification are included the spore morphology, Gram stain, and motility. The morphological and physiological characteristics of the bacterial isolates were compared with the data from Bergey's Manual of Determinative Bacteriology [17].
2.5. Scanning of Bacillus sp. MA20 and Bacillus sp. MA21 by Scanning Electron Microscope
The bacterial smear was prepared by centrifuging the bacterial cultures at 12,000 rpm for 20 min. The pellets were washed 2 times by saline solution. The pellet was suspended in sterilized distilled H2O. The bacterial film was prepared and fixed on glass slides till complete drying. The smear was coated with gold using sputter coater. The golden coated sample was scanned at 20 KV acceleration voltages at room temperature.
2.6. Genetic Identification and Differentiation
2.6.1. DNA Extraction
The genomic DNA of Bacillus strains was isolated using modification method from Sambrook et al. [18].
2.6.2. Identification by 16S Ribosomal RNA (rRNA)
PCR Amplification according to Sambrook et al. [18]. The PCR amplification reactions were performed in a total volume of 50 μL. Each reaction mixture contained the following solutions: 2 μL of DNA (40 ng), 1 μL of 10 pmol forwarded 16S-rRNA primer (5′-AAATGGAGGAAGGTGGGGAT-3′); 1 μL of 10 pmol reverse 16S rRNA primer (5′-AGGAGGTGATCCAACCGCA-3′); 0.8 μL of 12.5 mM (dNTP's); 5 μL of PCR buffer included MgCl2, and 0.2 μL Taq polymerase (1 Unit) and water-free DNAse and RNAse were added up to 50 μL. The PCR apparatus was programmed as follows: 3 min denaturation at 95°C, followed by 35 cycles that consisted of 1 min at 95°C, 1 min at 58°C, and 1 min at 72°C, and a final extension was 10 min at 72°C. The products of the PCR amplification were analyzed by agarose gel electrophoresis (2%).
2.6.3. PCR Cleanup and 16S rRNA Sequencing
The PCR products were cleaned up for DNA sequencing following the method described by Sambrook et al. [18]. Automated DNA sequencing based on enzymatic chain terminator technique, developed by Sanger et al. [19], was carried out using 3130X DNA Sequencer (Genetic Analyzer, Applied Biosystems, Hitachi, Japan).
2.6.4. Phylogenetic Analysis
Similarity analysis of the nucleotides was performed by BLAST searches against sequences available in GenBank. For phylogenetic tree construction, multiple sequences were obtained from GenBank and the alignments were performed using MEGA 5 software version 5.1 [20].
2.7. Keratinolytic Protease Production
Bacillus sp. MA20 and Bacillus sp. MA21 were first inoculated in liquid LB medium to produce large amount of cells. After 18 hrs, the colony forming units (CFU)/mL culture were , and 2% volume of the liquid medium was transferred to the production medium using 250 mL flask containing 100 mL of the production medium. The production medium containing (w/v) NaCl, 0.5 g/L; K2HPO4, 0.3 g/L; KH2PO4, 0.4 g/L; wool, 10 g/L; and the pH was adjusted 7.0–7.2 using 2N of NaOH and HCl [16]. The cultivated media were incubated at 37°C and 200 rpm for 5 days. Culture supernatants were obtained by centrifugation at 12,000 rpm and 4°C for 30 min. The different supernatants which contain the crude enzymes were used in assay and analysis of enzymes.
2.8. Enzymes Assay
2.8.1. Detection of the Proteolytic Activity on Plates
The crude enzyme of Bacillus sp. MA20 and Bacillus sp. MA21 was screened for their proteolytic activity using agar well diffusion plate method described by Amara et al., after modification [21]. One gram of gelatin powder was suspended in 100 mL phosphate buffer pH 7 and autoclaved. After sterilization, the soluble component was added to sterile water containing agar (18 gm agar/L). The suspension then stirred gently and distributed in Petri dishes (25 mL/plate). After complete solidification of the agar on plates, wells were punched out of the agar, by using a clean sterile cork borer. The base of each hole was sealed with a drop of melted sterile water agar (15 g agar per liter H2O) using sterile Pasteur pipette. Fifty μL of each bacterial supernatant was added to each well and preincubated at 4°C for 2 hrs and then overnight incubated at different temperatures.
2.8.2. Visualization of the Enzyme Clear Zone
Coomassie blue (0.25%, w/v) in methanol-acetic acid-water 5 : 1 : 4 (v/v/v) was used in plates staining to visualize the gelatin hydrolysis where 10 mL was added to each plate and incubated in room temperature for 15 min followed by removing the staining solution from the plates surfaces and washing gently by distilled water. Then the plates were destained using destining solution (66 mL methanol, 20 mL acetic acid, and 114 mL H2Obidest) for a suitable time [22]. Also extracellular protease detection was determined according to Vermelho et al., [23] with modification. Staining was performed with 0.1% amido black in methanol-acetic acid-water 30 : 10: 60 (v/v/v) for 5–20 min (according to the quality of amido black stain) at room temperature. Regions of enzyme activity were detected as clear areas, indicating that hydrolysis of the substrates has been occurred.
2.8.3. Enzyme Assay Spectrophotometrically
Preparation of Casein in Different Buffers with Different pH. Casein Hammarstein was weighted in a quantity of 0.325 g and dissolved in 50 mL of different buffers at different pH. The mixture was dissolved by heating gently to 80–90°C without boiling in water bath according to Amara and Serour [24] or using hot plate with stirring for more accurate because casein is highly stick with the walls of the container. The following buffers were used:(i)sodium phosphate, pH 6-7-8 (0.1 M)(ii)glycine/NaOH pH 9-10 (0.1 M)(iii)sodium phosphate dibasic/NaOH pH 11 (0.05 M)(iv)KCl/NaOH pH 12 (0.2 M).
2.8.4. Optimum Temperature of the Enzymes
Ten μL of each supernatant which contains the crude enzyme was added to 490 μL of the casein Hammarsten soluble in buffer pH 7. The enzymes-substrate mixture was incubated at different temperatures of 20, 30, 40, 50, 60, 70, and 80°C using water bath for 15 min. After the incubation period the enzyme reaction stopped by adding 500 μL of 10% trichloroacetic acid (TCA). The mixture was allowed to stand in ice for 10 min and then centrifuged at 13,000 rpm for 10 min. The absorbance of each sample was determined spectrophotometrically at 280 nm and their tyrosine content derived from the tyrosine standard curve which was carried out according to Amara and Serour [24] and the enzyme activity was determined as Unit/mL.
2.8.5. Optimum pH of the Enzymes
Ten μL of each supernatant which contains the crude enzyme was added to the 490 μL of the casein Hammarsten soluble in different pH values. The enzymes-substrate mixture was incubated at 60°C which acts as optimum temperature in a water bath for 15 min. At the end of incubation period the enzyme reaction stopped by adding 500 μL of 10% trichloroacetic acid (TCA). The mixture was allowed to stand in ice for 10 min and then centrifuged at 13,000 rpm for 10 min. The absorbance of each sample was determined spectrophotometrically at 280 nm and their tyrosine content derived from the tyrosine standard curve and the enzyme activity determined as Unit/mL.
2.8.6. Confirmation of Optimum pH and Temperature
The previous reaction was performed using casein Hammarsten soluble in Glycine/NaOH pH 9. The enzyme substrate mixture was incubated at various temperature of 20, 30, 40, 50, 60, 70 and 80°C in water bath for 15 min. The enzyme activity was calculated as mentioned previously.
2.8.7. The Optimized Enzymes Reaction
The optimized reaction is a mixing between 10 uL of crude enzyme and 490 μL of the casein Hammarsten soluble in Glycine/NaOH pH 9. The mixture was incubated at 60°C in water bath for 15 min. The reaction was stopped with 500 μL of 10% TCA. The mixture was allowed to stand in ice for 10 min and then was centrifuged at 13,000 rpm for 10 min. The enzyme activity was determined as mentioned above using tyrosine standard curve.
2.8.8. Thermal Stability
The thermostability was carried out by preincubating the crude enzyme solution at a temperature range of 4°C to 80°C for 0.0 to 24 hrs. The residual activity was measured with standard enzyme reaction. The control is enzyme reacted at zero time (consider as 100%) [16].
2.8.9. pH Stability
The pH stability was determined by preincubating the enzyme solution in buffers with different pH values (3–12) at room temperature from 0.0 to 48 hrs. The residual activity was measured with standard enzyme reaction. The control is enzyme reacted at zero time and considered as 100% [16]. The following buffers were used:(i)citrate buffer pH 3-4-5-6 (0.1 M)(ii)sodium phosphate pH 7-8 (0.1 M)(iii)glycine/NaOH pH 9-10 (0.1 M)(iv)sodium phosphate dibasic/NaOH pH 11 (0.05 M)(v)KCl/NaOH pH 12 (0.2 M).
2.8.10. Effect of Inhibitors
The inhibitors were added to supernatants which contain the produced enzyme and were incubated for 30 min at 30°C before being tested for proteolytic activity. Protease inhibitors phenylmethanesulphonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), β-mercaptoethanol, and the detergent sodium dodecyl sulfate (SDS) were used. The inhibitors stocks were prepared in distilled water except that PMSF was prepared by using isopropanol. The final concentrations of PMSF, EDTA, and β-Mercaptoethanol are 5 mM and 1 mM while SDS is 0.5% and 0.1% (w/v). The control was enzyme mixed with distilled water instead of inhibitors. Control activity was considered to be 100% [25].
2.8.11. Effect of Metal Ions
The effect of metal ions on protease activity was investigated using two concentrations of 5 mM and 10 mM (final concentration). The metal stock solutions were prepared in distilled water and diluted to the appropriate concentrations. The enzyme solution was mixed with the different metal solutions and incubated for 30 min at 30°C before assay. A control was also included where the enzyme was mixed with distilled water instead of metal solution. Control activity was considered to be 100% [25]. ZnCl2, MgCl2, CuSO4, Urea, HgCl2, CaCl2, BaCl2, Guanidin HCl, and MnCl2 were used.
2.8.12. Effect of Solvents
The effect of the different solvents (Methanol, Ethanol, DMSO, Isopropanol, Tween 20, and Triton X100) on protease activity was investigated using a concentration of 1% and 0.5% (final concentration) [25].
2.9. First-Dimension Protein Electrophoresis
2.9.1. Sodium Dodecyl Sulfate Polyacrylamid Gel Electrophoresis (SDS-PAGE)
Characterization of proteins and evaluation of the protein enrichment process SDS-PAGE was performed in a discontinuous SDS-PAGE vertical slab gel electrophoresis apparatus as described by Laemmli [26]. Discontinuous SDS-PAGE consisted of a stacking gel (5%, w/v, pH 6.8) and a separating gel (12%, w/v, pH 8.8). The separating gel was prepared in a 1 mm slab gel (10 × 10 cm).
2.10. Gel Staining Using Silver Stain
The gels were stained using silver nitrate staining methods as described by Blum et al. [27].
2.11. Zymogram for the Detection of Protease Activity Using SDS-PAGE
As above in the case of protease zymography using SDS PAGE and the separating gel concentration was 12%. The samples were mixed with 5X sample buffer without β-mercaptoethanol and without boiling.
After running the gel at 80 V the SDS-PAGE gel was stripped off from the gel plate and soaked in 1% triton X100 for 2 hrs to change the solution every 1 hour or in 2.5% triton X100 for 30 min for removing SDS and renaturating the enzymes.
The gel was washed three times with tape water and soaked in 1% gelatin powder solubilized in Glycine/NaOH buffer pH 9 for 90 min at 60°C. The gel was washed with buffer for 10 min before staining.
Then the gel was stained with Coomassie blue stain for 2 hrs. After that the gel was destained till the active bands appear.
2.12. Zymogram for Detecting Protease Activity Using Native PAGE
The gel was carried out using all components of the SDS-PAGE and the same conditions but without SDS as well as the buffers described above. The samples were prepared by mixing with 5X sample buffer for native PAGE and without boiling. The gel was washed with tap water and soaked into 1% gelatin powder as mentioned previously. Then the gel was stained with Coomassie blue stain for 2 hrs. After that, the gel was destained till the active bands appear.
2.13. Two-Dimension Polyacrylamide Gel Electrophoresis (2D-PAGE)
First-dimension isoelectric focusing (IEF) was performed using Ettan IPGphor3 and the second-dimension SDS-PAGE was applied using vertical electrophoresis of Ettan DALTtwelve System. The experiment was carried out using the operation instruction of manufacturer (GE healthcare company). The gels were stained with Coomassie stain where Coomassie blue R-250 (0.02 g) in methanol-glacial acetic acid-distilled water (10 mL, 5 mL, and 100 mL resp.) was used.
3. Results and Discussion
3.1. Strain Isolation
Microbial keratinolytic protease has been described for various biotechnological applications in food, detergent, textiles, and leather industries, and yet the growing demand for these enzymes necessitates the screening for novel keratinolytic microorganisms with potential applications [28, 29]. Keratinolytic protease has been described for several species of Bacillus [30, 31] due to the broad distribution of keratinase among these genera, and this study focused on keratinolytic protease production from them.
A total of 48 pure cultures of spore-forming bacteria were isolated and purified which obtained from different samples collected from Governorates of Egypt. All isolates were screened using selective method for Bacillus isolation. The proteolysis activities of all the isolates were detected using the plate test method containing LB agar medium with 1% (w/v) skim milk. Among the isolates analyzed, 12 isolates exhibited proteolytic activity in which they had a halo diameter of fivefold longer than the colony diameter. All isolates have the proteolytic activity but do not have the ability to degrade wool, for that the twelve selected isolates were grown using a medium which contain (w/v) NaCl, 0.5 g/L; K2HPO4, 0.3 g/L; KH2PO4, 0.4 g/L; wool, 10 g/L; and the pH was adjusted at 7.0–7.2 using 2N NaOH and HCl. The flasks were incubated for 5 days until the wool has been completely degraded by some isolates. Three Bacillus isolates show the ability to degrade the wool and gained the names Bacillus sp. MA20, Bacillus sp. MA21, and Bacillus sp. MA10. In the last screening step, the obtained supernatants from the cultivation of three above Bacillus strains culture media were assayed using agar well diffusion in Petri dishes including gelatin powder plate which suspended in phosphate buffer pH 7 as in Figure 1. Bacillus sp. MA20 and Bacillus sp. MA21 were selected for studying the keratinolytic protease enzyme based on the diameter of clear zone (Figure 1). By refering to their isolation source, the two selected strains were found to be isolated from Minia Governorates. This method is simple but proves to be efficient for determining the best enzyme-producing isolates. Out of the three strains, the two which were given the best result have been subjected to further identification.
3.2. Strains Identification
3.2.1. Identification by Morphological and Biochemical Tests
The morphological and physiological characteristics of the isolated strains were compared with the data from Bergey's Manual of Determinative Bacteriology [17] who revealed that Bacillus sp. MA20 and Bacillus sp. MA21 are matching with those of B. subtilis group. In previous articles on taxonomy, species included in the B. subtilis group are the following: B. velezensis, B. atrophaeus, B. mojavensis, B. malacitensis, B. axarquiensis, B. nematocida, B. vallismortis, B. subtilis, and B. amyloliquefaciens [32–34].
The two strains are aerobic, motile, and Gram-positive rods. The smears of Bacillus sp. MA20 and Bacillus sp. MA21 were scanned by scanning electron microscope which indicates the bacterial size of the two Bacillus strains which was measured by slime view program software.
The results of biochemical tests of the Bacillus sp. MA20 and Bacillus sp. MA21 indicated that they are related to B. subtilis, and B. amyloliquefaciens which summarized in Table 1.
3.3. Identification Based on Genetic Materials (DNA)
3.3.1. DNA Extraction
The isolated DNA was analyzed by gel electrophoresis and the quality of the DNA for each sample has been identified for further investigation.
3.3.2. Identification by 16S Ribosomal RNA (rRNA)
The amplified 16S rRNA gene from the DNA of Bacillus sp. MA20 and Bacillus sp. MA21 was determined using 2% agarose gel. The size of the amplified fragments was determined by using size standard (Gene ruler 50 bp–1031 bp DNA ladder). The PCR products were visualized under UV light and photographed using gel documentation system. Approximately 380 bp of 16S rRNA gene was amplified.
The PCR products were purified and sequenced using 16S forward primer. The sequences of Bacillus sp. MA20 and Bacillus sp. MA21 were deposited in national center for biotechnology information (NCBI GenBank) under the Accession numbers (HQ115599.1–HQ115600.1), respectively. The basic local alignment search tool (BLAST) algorithm was used to retrieve for homologous sequences in GenBank.
The Bacillus sp. MA20 revealed 98% identity to Bacillus amyloliquefaciens and Bacillus subtilis while Bacillus sp. MA21 revealed 97% identity to Bacillus subtilis and Bacillus amyloliquefaciens. Based on the morphological, biochemical, and molecular characteristics, the Bacillus sp. MA20 and Bacillus sp. MA21 were designated as B. amyloliquefaciens MA20 and B. subtilis MA21, respectively.
A phylogenetic tree based on the comparison of 16S rRNA sequences of reference strains was constructed. The phylogenetic analysis was performed with 341 bp sequences using the software MEGA 5 [20], using the neighbour-joining method and based on Jukes-Cantor distances. The branching pattern was checked by 500 bootstrap replicates (Figures 2 and 3).
3.4. Media Screening for Keratinolytic Protease Production
Keratinolytic proteases are largely produced in a basal medium with keratinous substrates, and most of the organisms could utilize keratin sources such as feather and wool as the sole source of carbon and nitrogen [35, 36]. B. amyloliquefaciens MA20 and B. subtilis MA21 were tested on eight nutrient media. The selected media is medium (1) which is containing (w/v) NaCl, 0.5 g/L; K2HPO4, 0.3 g/L; KH2PO4, 0.4 g/L; wool, 10 g/L, and the pH was adjusted 7.0–7.2 using 2N of NaOH and HCl.
Korniłłowicz-Kowalska, (1997) and Amara and Serour (2008) reported that the mass loss of the keratin substrate is the most reliable indicator of microbial keratinolytic abilities. B. amyloliquefaciens MA20 and B. subtilis MA21 have the ability to degrade wool completely after incubation for 5 days and remain it as powders in the bottom of flasks [24, 37].
3.5. Enzyme Production and Characterization
The keratinolytic protease enzyme was produced using production medium mentioned above for each of B. amyloliquefaciens MA20 and B. subtilis MA21, and the crude enzymes were characterized.
3.6. Detection of the Proteolytic Activity on Plates
The crude enzymes of B. amyloliquefaciens MA20 and B. subtilis MA21 were assayed by agar well diffusion methods on plates containing gelatin soluble in glycine/NaOH buffer of pH 9. The Coomassie blue and amido black bind to gelatin and whole plate giving the colour of dye except the hydrolysis areas which appear as transparent without dye (Figure 4).
3.7. Characterization of Keratinolytic Protease Enzyme
3.7.1. Influence of pH and Temperature
The effect of temperature and pH on enzymes activity and stability was determined. The optimum temperature and pH for protease activity of enzymes produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was found to be 60°C, 9.0, respectively.
The enzymes activity was investigated at pH 7 and different temperatures (Figure 5). The proteolytic activities were determined at optimized temperature 60°C and different pH (Figure 6). For confirmation from the optimized reaction, the enzymes activity was assayed at pH 9 with different temperatures (Figure 7).
Results revealed that the keratinolytic protease from B. amyloliquefaciens MA20 and B. subtilis MA21 is similar to those produced using bacteria, actinomycetes, and fungi and has a pH optimum in a neutral-to-alkaline range [38, 39]. The optimal temperature for activity was also found in the usual range for keratinolytic protease (30–80°C).
The maximum activity of protease enzyme produced by B. amyloliquefaciens MA20 was 922 U/mL at pH 9 and 60°C while the maximum activity of protease enzyme produced by B. subtilis MA21 was determined as 814 U/mL at the same conditions. The alkaline pH of the keratinolytic protease enzyme from B. amyloliquefaciens MA20 and B. subtilis MA21 suggests a positive biotechnological potential.
The enzymes were stable at temperatures between 4 and 70°C. The enzyme produced by B. amyloliquefaciens MA20 is more thermostable than enzyme produced by B. subtilis MA21 (Figures 8 and 9). The results of thermal stability indicated that the keratinolytic protease from B. amyloliquefaciens MA20 is more thermostable than the enzyme produced by B. subtilis MA21.
The thermal stability studies give the enzyme from B. amyloliquefaciens MA20 the advantage for using in industrial applications which are the main objective of this study.
The crude enzyme from B. amyloliquefaciens MA20 is described as stable over a broad pH range of 4.0–12.0, but the best stable pH is 9 and 10 (Figure 10). Figure 11 indicates that the enzyme from B. subtilis MA21 was stable in pH range (5.0–12.0) with high stability at pH 9. The stability of keratinolytic proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 has been suggested to offer great advantages for industrial purposes such as wastewater treatment and leather tanning [40].
3.8. Influence of Protease Inhibitors, Solvents, and Metal Ions on Enzymes Activity
Mostly keratinolytic proteases belong to the subtilisin family of serine proteases with cysteine proteases, which have higher activity on casein [41]. The keratinolytic proteases produced by Bacillus sp. are often serineproteases, such as the enzymes produced by B. licheniformis [42], B. pseudofirmus [43], and B. subtilis [44, 45]. Protease activity of enzymes prepared from B. amyloliquefaciens MA20 and B. subtilis MA21 was completely inhibited by serine protease inhibitor (PMSF). The result indicated the presence of the serine group in the enzyme active site. The enzymes activity was partially inhibited by EDTA (Tables 2 and 3). This suggests that the keratinolytic protease from the Bacillus strain belongs to keratinolytic serine protease family.
The stability of keratinolytic proteases in presence of SDS acts as a positive advantage of enzymes feature because it indicated the possibility of using them in different industrial purposes as detergent industry, leather industry, and wool improvement. The stability toward SDS is important because a few authors reported that SDS-stable enzymes are also not generally available except for a few strains such as Bacillus clausii I-52 [46] and Bacillus sp. RGR-14 [47]. The effects of various solvents and metal ions on enzyme activity were examined in order to find which ions are stimulators and which are inhibitors of the catalytic process.
The metal ions were used with two final concentrations (5.0 mM and 10 mM) while the used solvents with final concentrations (1% and 0.5%). The effects of metal ions and solvents on enzyme activities are summarized in Tables 2 and 3.
3.9. First-Dimension Protein Electrophoresis
The production of extracellular keratinolytic proteases by B. amyloliquefaciens MA20 and B. subtilis MA21 was evaluated by SDS-PAGE and zymogram analysis. Giongo et al. found multiple bands after zymogram of keratinolytic protease produced by three strains of Bacillus sp. P6, P7, and P11 using feather degrading medium [25]. Growth of the two strains in presence 10 g/L of wool resulted in the production of multiple proteases, as observed by a zymogram on gelatin (Figures 12 and 13). Multiple clear zones were observed and detected using (PAGE Ruler Prestained Protein Ladder) which indicated that the proteolytic activity was not due to a single protein. The native-PAGE zymogram of enzymes from the 2 strains was performed and compared with protein ladder (Figures 14 and 15). The zymogram using native page was carried out for detecting the bands of protein where the purification performs using native protein. Multiple bands could be detected on native-PAGE zymogrm.
3.10. Two-Dimension Protein Electrophoresis
The two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is an advanced technique that depends on protein separation firstly according to the pH and secondly based on the molecular weight. This technique was carried out to differentiate between the crude keratinolytic proteases obtained from B. amyloliquefaciens MA20 and B. subtilis MA21. After the separation based on the pH, the strips were applied on gel for separating according to molecular weight. The gels were stained with Coomassie blue for detecting the protein spots.
In order to classify the keratinolytic protease enzyme, a systemic comparison of 2D maps of proteases was conducted with image master 2D Platinum 6 software. The two gels were photographed by high quality scanner image (Figure 16).
The results were analyzed using image master 2D Platinum 6 software. The spots were detected before matching between the two gels and the report was obtained (Table 4). Every spot refers to the presence of one protein. The gel which was loaded with keratinolytic protease from B. amyloliquefaciens MA20 had 237 spots while the gel applied with B. subtilis MA21 had 291 spots.
The 2 gels were matched and consider gel resulted from crude enzymes of B. amyloliquefaciens MA20 as reference and the final report that summarizes the difference between 2 gels was obtained (Table 5). The 2 gels were matched and the percent of matching between them was 13.25%.
4. Conclusion
This paper described in details different methods that lead to the production of keratinolytic protease from two Bacillus sp. strains. Different methods and assays ranging from simple to advanced were carried out to prove that the enzymes have the ability to degrade wool. The two used strains have been selected based on that they belong to Bacillus sp. and show the best keratinolytic activities. The study included molecular and bioinformatic tools to identify the two Bacillus sp. strains. 16S rRNA, phylogenetic tree, Blast search for nucleotide similarity, scanning electron microscope, SDS-PAGE, and 2D-PAGE have been used to differentiate the both strains and their produced enzymes. The study succeeded in characterization of Bacillus sp. strains which were named Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21. The enzyme activities either on agar diffusion plates or through the enzyme activity bioassays under different experimental conditions proved that the both strains are able to produce different keratinolytic protease enzymes. B. amyloliquefaciens MA20 and B. subtilis MA21 could be used in large-scale production of keratinolytic protease enzymes where these enzymes were stable at temperature range between 4 and 70°C, and over a wide range of pH values (4–12), as well as, stable against organic solvents and detergents. The characters of keratinolytic enzymes produced by B. amyloliquefaciens MA20 and B. subtilis MA21 could play an important role especially in industrial applications; therefore this research acts as preliminary studies for applying the keratinolytic proteases in wool quality improvement.
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Later in 1992 an advertising executive, captivated by the kooky musicians, urged them to audition for the Kit Kat television commercial. They did and got the job. Then a talent scout who had seen them suggested the band try out for the popular TV show Ed McMahon's International Star Search.
"I had no idea what Star Search was," Khramov explains. "The closest thing we had in Russia was a show called Hello, We Are Looking for Talent. But we tried out anyway." They competed and won first prize, copping $5000, which they promptly spent on a Dodge Ram 350 van. The vehicle, which toted equipment and a mattress, logged hundreds of thousands of miles on trips all over North America as the band began doing shows at festivals and colleges.
By 1994 Limpopo had made its way to Miami, thanks to the local band I Don't Know (reborn -- sans accordionist -- in the summer of 1997 as the more serious and rockier Humbert), whose members discovered Limpopo in 1993 at the South by Southwest music conference in Austin. "We loved what they were doing and were dying to play with them," explains Ferny Coipel, guitarist for I Don't Know/Humbert. "A year later they came to Miami to play at FIU and we arranged a gig with them at the Talkhouse. Everyone was amazed by their instruments, their style, their vocals, and of course their energy."
"Their show is nuts," adds Humbert bassist Tony Landa, another I Don't Know alumnus. "They use jingle bells and accordions, and the kind of dancing they do is indescribable. You just have to see it. On top of all that, they're great musicians -- they can play any kind of music."
Still performing at festivals and in small clubs, Limpopo returned to Miami a year later for another show with I Don't Know, this time at Churchill's Hideaway in Little Haiti. According to that club's owner, Dave Daniels, "This was probably the best show we ever had here. Limpopo was funny, fresh, and different. The crowd wasn't dancing as much as jumping up and down. They loved it."
The van finally gave out a few years ago, but Limpopo continues on. Since 1994 they've recorded three CDs -- Crazy Russian Folk 'n' Roll, Give Us a Break, and Traffic Jam in Moscow -- on their own Folk 'n' Roll label. Distributed at Tower Records and available at shows or directly from the band's Website (www.limpopo.com, which also offers videos, coffee mugs, T-shirts, and memberships to the group's Klub Komrade fan club), the records feature upbeat original songs, many of them written by Khramov and sung in Russian. Last year's Traffic Jam contains a couple of Khramov-written bonus tracks sung in English. Traditional Western instruments (trombone, guitar, keyboards) mix with unconventional ones such as various balalaikas and a wood-slat rattle known as a tresheotki.
The band's lineup changed significantly in 1996 when Bernov and Yuzov split to form the rock-and-rollier Red Elvises. They were replaced by keyboardist Boris Bolshakov, percussionist Andrei Baranov, and bassist Dimitri Mamokhin, plus dancer Sasha Kalinin from the Moiseyev Dance Company. (Shades of the Happy Mondays!) Last year Kalinin and Mamokhin departed to pursue independent projects, so Limpopo is once again a quartet, back where it was in 1992.
Well, not quite. They're not exactly playing for handouts any more, although they occasionally pick up their instruments and perform at the Third Street Promenade just for kicks. "Good things still happen to us because of playing on the street," Khramov explains. "Recently we got a lot of great bookings from doing it -- one was at the Beverly Hills Country Club."
They've also expanded their audience, taking their spirited shows into Los Angeles public schools. "Kids are another interesting market for us," Khramov points out. "They really enjoy our show. They just laugh all the time." The band has contributed songs to a pair of movie soundtracks -- the low-budget The Russian Godfather, and Somewhere in the City, a racy Sandra Bernhard film released only in Europe -- and they're shopping their tunes to a major world-music label. Finally, they're working to enhance the Limpopo experience. Their current road show consists of the quartet playing its bouncy Russian folktunes and the occasional rock or jazz standard, with accordionist Fedorko providing his signature eye-popping acrobatics. But eventually the band hopes to mount a more elaborate stage show that incorporates several dancers and musicians.
"It's a new step for us, a new energy," Khramov says. "We are trying to do something more crazy, more creative -- to connect dancers and musicians not just with Russian music but with modern American music. We want to create wonder for people, to excite them with something they've never ever seen before."
Limpopo performs with Humbert Friday, February 27, at 10:00 p.m. at Churchill's Hideaway, 5501 NE 2nd Ave; 757-1807. Admission is $7.


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JIB: Claim Your Favorite People, Places,
Products and Events Around the World.
Change City
Reviewed by: Toni Kervina
Opening Date: Monday, May 14, 2012
Alexander Wang has opened a second flagship boutique in Beijing, and the look of the store is just as cool as the New York designer's eponymous clothing line. The two-level, 5,000-square-foot space was designed by Paris-based architect Joseph Dirand in collab with Wang, and is appropriately situated in Sanlitun Village, the center for all things luxe in Beijing (neighbors include Alexander McQueen and Maison Martin Margiela.) The design follows in line with the NYC flagship, with sleek, strong lines that emphasize raw materials. Upon entry, guests are greeted by a large marble sculpture meant to look like an inkblot test. A bronze prism sculpture cuts across the top floor, and even the shelving is bronze. Nearly all the furniture and fixtures were custom created for the store, including a concrete sofa with goat hair and infinity tables. Once you've managed to peel your eyes away from the design, you'll be able to shop the brand's complete men and women's collections, as well as the recently launched Objects collection.