Hi Mr Kada!

Yes it is possible. Depending on the enzyme you want to detect you can design peptides with labels to measure cleavage activity. I always use the MEROPS database for that purpose (http://merops.sanger.ac.uk/). Addition of mirrored (D-) amino acids will increase the specificity of the assay. At the current moment I have 2 article regarding this topic (pubmed Kaman WE) maybe these can help? Contact me in case you`re unable to view the full text!

Good luck!

Wendy

Dear Narayana

Normally, in cultural based bacterial enzyme detection methods, the substrate enzyme is added to the medium, if the enzyme be present, its effects will be detected by various methods, e.g. changing in opacity of the medium (amylase, lipase), changing the pH and its effect on the pH indicator (beta-galactosidae), liberating of chromophore from the substrate and many various mechanisms. I think it is better to define your enzyme and your bacteria, too. Then it is easy to find or propose suitable method for the enzyme and special method for the strain. Please consider there are a lot of enzymes and a lot of bacterial strains. Which one do you need?

Dear Sir,

Thank you for your kind reply. Actually my research area is diversity of Vibrios associated with seaweeds and mangrove rhizosphere. apart from the diversity i am also interested to explore the enzymes from my strains. In that context I would like to know if there is any common protocol (mostly culture based which i can afford in my lab) by which I can detect various enzymes.

i guess this may require probably extracting the enzymes followed by partial purification.

There are two enzyme (oxidase & catalase) you can directly determine by using culture. Rest of enzyme is media specific means growing in specific substrate and having different protocol for quantitative. For qualitative specific media will be require to determine enzyme production. Probably every microbiology lab have all these kind of media and if not asked to colleague. A little (50 gm) amount of media is more then enough to screen 100 bacterial culture by girding in plate. You can also differentiate culture enzyme production based on intensity/zone of production in plus minus scale (None –; weak +; moderate ++; strong +++).

Good luck

Your work seems to be very interesting. However, before I extend any help to you, my main question is what is the precise objective in trying to identify enzymes, because every organisms has a wide variety of enzymes for different metabolic purposes. It may not be possible to isolate and identify all the enzymes without an objective.

If you are interested in a large number of enzymes, try commercial assay systems like API50 or the Biolog system.

Your topic is nice. Do you choose your enzyme(s) or no? If yes, please write. If no, there are two approaches to choose them. 1) applied approach, to find suitable enzyme for biotechnology. 2) basic approach, to find specific enzyme in a certain metabolic route. I am ready to help you, for fast communication, you can send email to [email protected]

It is probably much better to use APIZYM. Good luck

Indeed you can determine many enzyme activities. Enzymes acting on carbohydrates can be analyzed with the test APIS or Biolog. The tryptophanase through the indole test. Oxidase by adding the specific reagent. Catalase with hydrogen peroxide. They are also described in the literature determination of dehydrogenases (due to reduced TMF red coloration occurs is determined by espectrofotmetria) and quantification of acid and alkaline phosphatases it is also possibleetc.

My objective is to explore enzymes which has significance industrially and biotechnological application. The suggestions and questions you have given are valid and I should be precise in selecting what kind of enzymes I am interested. I had just started my work on the diversity and with respect to bacterial enzymes I am new to this area of research. As all the above suggested I will try to make myself precise to select the enzymes. Thank you all for your valid suggestions.

It seems APIZYM or Biolog are very costly which I cannot afford in my lab. Can you suggest protocols for culture based methods to identify enzymes.

Dear Kada,

What I understand from above discussion and from your problem, is actually you want to check first qualitative detection of enzymes production by your culture and after then you want quantify specific enzymes of Industrial / biotechnological importance. For this consult any experimental microbiology book for qualitative/ quantitative determination of enzymes or by research paper. Though, I think book will be better option for you as you are beginner. For your connivance here I can provide you protocol of some enzymes. Contact me [email protected]

Virtually all bacterial enzyme can be detected by culture mehod as long as it has a known substrate, so you have to be specific about the exact enzyme you want to detect.

Are you going to detect specifc enzymes? Also, are they extracellular or intracellular?

SCREENING OF ACTINOMYCETES FROM EARTHWORM CASTINGS FOR THEIR ANTIMICROBIAL ACTIVITY AND INDUSTRIAL ENZYMES

Vijay Kumar1; Alpana Bharti 1; Yogesh Kumar Negi 1; Omprakash Gusain2 ; Piyush Pandey1; Gajraj Singh Bisht1

Brazilian Journal of Microbiology (2012): 205-214

The methods recommended by Concepcion Calvo are suitable for any laboratory, both well financial sustained and modest ones. The catalase, oxydase, indol, TSI, MILF and the rest of the enzymes tests are easy to carry on in any laboratory and with good results. You may also add in your Petri dish a certain substance (known for the searched emzyme) and a color indicator in order to identify a certain enzyme's action.

Is better to use more tests in order to confirm your bacterial enzymatic production.

In addition to all answers below you can use gelatin to a basal medium to detect gelatinase. Add 4% of gelatin in basal medium and inoculate the bacterium. Incubate for 24h at the appropriate temperature and store at 4 °C for an hour and then check the mobility of the fluid. If it is gelatin positive then the medium will be acting like water when you tilt the beaker, if not it is gelatin negative.

Another test that has not been mentioned below is caseine test. Add 2% low fat milk in a petri dish supplemented with nutrient agar and place a fresh clean column of the bacterium as a spot on the dish. Incubate for 24h at the appropriate temperature and measure the zone of hydrolysis around the bacterium. A caseine positive bacterium forms an very district clear zone, a caseine negative bacterium does not form a zone. I can support you with more tests but you can find many and more elaborate at Bergey's Manual.

what kinds enzyme do you want to detect? There are many kind of bacterial enzyme that need different detection..

You should know what kind of interested enzyme? Many methods used to determine the activity are available on the internet including journals .

detection of enzymes can be done like streptolysin/fibrinolysin from bacteria streptococcus which can be used for incase of myocardial infarction: I think you must be interested in extraction of such enzymes

Which enzyme you want to identify? I will provide you analytical protocol for specific enzyme.

Hi Mr.Kada

you dont say what kind enzyme you want detect. If you have enzymes are secreted, You can remove it from the culture with percipitation and santrifuge, but if your bacterial enzymes are internal you should use of bacterial lysis, for example with Lysosime.

I request a modified question to be posted for a healthy discussion

I think Mr. KADA is looking for a general methodology....and some articles

Please specify what type of resistance enzymes that you looking for...ESBL?AmpC?Carbapenemase; KPC,MBL,OXA?

Dear razali we are discussing out of a nonspecific question: i feel really difficult to comment unless the question is modified: you were right in asking if he was looking for any resistance enzymes

let me paraphrase this question for him: I need to detect hydrolytic enzymes for some research topics, i.e: Chitinase, protease, cellulases, legninases, lipases .. etc

or oxidoreductases ??!! let us try to open this discussion on the quantitative and qualitative test levels available in what u read in published literature. thanks alland happy new year

We can keep guessing but what exactly our mate (narayamurthy) wants only he can make it clear. I am looking forward for a response

Yes, Of course you can detect bacterial enzymes by culture based methods. but let me know which type of enzyme you are interested? depending on the type of enzyme method may vary. so be specific about your question.

Hi Mr Kada!

Yes it is possible. Depending on the enzyme you want to detect you can design peptides with labels to measure cleavage activity. I always use the MEROPS database for that purpose (http://merops.sanger.ac.uk/). Addition of mirrored (D-) amino acids will increase the specificity of the assay. At the current moment I have 2 article regarding this topic (pubmed Kaman WE) maybe these can help? Contact me in case you`re unable to view the full text!

Good luck!

Wendy

The API ZYM system is a rapid system for detection of bacterial enzymes- alkaline and acid phosphatases, butyrate esterase,caprylate esterase lipase, myristate lipase, leucine,valine and cystine aminopeptidases, trypsin, chymotrypsin,

phosphoamidase, ot-galactosidase, f-galactosidase, fl-glucuronidase, ax-glucosidase, fl-glucosidase, ,B-glucosaminidase, (x-mannosidase, and cx-fucosidase.

Simply add the substrate of the enzyme you want to detect in the culture medium, then detect the enzyme substrate reaction after the end of incubation by formation of clear zones or addition of reagent that can react with the end product.

Dr Kada,

Have you check in the review from Orenga et al. (J. Microbiol. Methods 2009 - 79: 139-55) if you find the answer to your technical issue?

Good luck

Sylvain