Wednesday, November 19, 2008

Laccase Enzyme Secreting Bacteria Isolation

1.Laccase Enzyme

Laccase enzyme typically contains 15–30% carbohydrate. It has an acidic isoelectric point and has a molecule mass of 60–90 kDa. Laccases are the model enzymes for multi-copper oxidases and participate in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. For catalyzing the oxidation of non-phenolic substrates, laccase requires the presence of a mediator in the medium. A mediator is a small molecule that behaves like an ‘electron shuttle’ between laccase and substrate and these small molecular-mass compounds are converted into stable radicals by means of enzymatic oxidation.

1.1 Methods used for screening of laccase producing bacteria

Discovery of novel laccases with different substrate specificities and improved stabilities is important for industrial applications. Microbes that produce laccases have been screened for either on solid media containing coloured indicator compounds that enable the visual detection of laccase production.

Material and method: Extracellular laccase test were performed according to modification of method proposed by Rayner and Boddy [Rayner ADM,Boddy L.Fungal Decomposition of wood.Its biology and ecology.NY:Wiley;1988.]

A) Plate Test :
Requirement: 2 to 5 days old culture on NA plates
Chemical: Guaiacol,96% ethanol
Procedure: Add 2-3 drops of guaicol (1.24g in 100 ml of 96% ethanol) and incubate for 4 hours,it give purple color.

B) Plate test :
Laccase activity visualize on plates containing 0.02% guaiacol as a reddish brown Zone, due to oxidative polymerization of guaicol by the action of laccase.

C)Laccase activity also visualize by native PAGE by soaking gel in 50 mM sodium acetate buffer (pH 4.5),containing 2mM guaicol.After several minutes enzymatic band with a brown color appeared because of the polymerization of guaicol.[Purification and characterization of a Phenoloxidase (laccase) from Lignin Degrading Basidiomycete, PEDRO M . COLL 11 may 1993]

D) Laccase activity confirmed by spectrophotometrically at 525 nm, through the oxidation of syringaldazine to its quinone form, using a molar absorptivity of 65,000 M−1 cm−1 for the product. The reaction mixture contained 1.5 ml phosphate buffer (100 mM, pH 6.5), 0.2 ml syringaldazine (0.5 mM in ethanol solution), and 0.1 ml of culture filtrates.
Alternatively, laccase activity was determined by the oxidation of 2,2′-azinobis(3-ethylbenthiazoline)-6-sulfonato (ABTS) The reaction mixture contained 0.3 mM ABTS in malonate buffer (50 mM, pH 4.5), and the oxidation was followed at 420 nm (420=36,000 M−1 cm−1).

E)Plate Test : Syringaldazin Ager Plates
Inoculate bacteria on Syringaldazin Ager Plates.If laccase like enzyme present gives purple color within 72 hours.

87 comments:

Anita said...

nice info.

haleema ayub said...

can u pl help me...
my work is on laccase producing bacteria...it s reported tat laccase is secreted by halotolerant organism..so m trying to isolate a strain from sea water tat produce laccase...do u have any idea of wat media shud be used to screen???/

Yogendra Singh and Anita Singh said...

Hi thanks for showing your interest in www.ligninblack.blogspot.com. As u said that u r working over halotolerant bacteria for laccase activity. Before going onwards try these suggestions:
1. halotolerant means not only sea water.
2. halotolerant can be acidophilic or alkaliphilic.
3. Start ur experiment with pulp and paper mills black liquor/soil/solid waste separated during filtration of black liquor. I am confident that there u will get halotolerant alkaliphilic bacterial strains.

If these content help u any how then follow my site. I am happy to see ur name follower list.

warm regard from ligninblack.

haleema ayub said...

thanks fa d info....but to work on tat source wat kinda medium can be used?????
now m using MH media with guaiacol to screen but m nt getting any +ve results...very difficult to sort out d problem...can u help me pl??????

Yogendra Singh and Anita Singh said...

Mueller-Hinton (MH) is gud for isolation of halotolerant microorganism. I need little details to solve your problem:
1. tell me about the samples location from which u want to isolate the concern strain.
2. Will u go for enrich ur sample before starting isolation.

Try this suggestions:
1. Samples (soil/water) enrich with
Guaiacol or ABTS or Tannic acid and incubate it for 24 to 72 hrs.
2. Star isolation by serial dilution on on plates containing 0.02% guaiacol or 0.5% Tannic acid agar plate. The colony appears redis to dark brown is showing laccase activity.
3. The 0.02% guaiacol or 0.5% Tannic acid agar plate may contain any media (like Nutrient Agar with series of lower to higher salt concentration) for screening.

haleema ayub said...

isolating from sea water....just wanna screen d bacteria capable of good laccase activity...hav got some good colonies but colour change is not observed even media with guaiacol....thanks fa d info...helped me a lot....

Yogendra Singh and Anita Singh said...

use 0.5% tannic acid agar plates instead of guaiacol.

haleema ayub said...

hi...
it has been a long time i scrapped u...atlast i have isolated a strain that produce laccase from samples of thrissur using guaiacol as substrate..and also optimised the ph and temp for the activity of laccase...
coming to the next step i have been advised to characterise the strain (biochemical methods) what are ways to do the characterisation?????....thanks in advance...
your ideas have helped me a lot:)

Yogendra Singh and Anita Singh said...

Hi Haleema nice to see u again. Your query about characterize the strain (biochemical methods).
These are the simple test like:
A. Carbon Source Utilized by ur Strain:
For Ex.
1.Glucose (Dextrose) test
2.Lactose test
3.Sucrose test etc

B.MRVP (methyl red-Vogues Proskauer Test and IMViC Test
C. Test for Acid/gas production.
D. The Indole Test
E. The Citrate Test

U may add more in the sequence as per your requirement.

afreen said...

need your help.i am trying to isolate laccase enzyme from cyanobacteria. can u suggest me the screening method for the identification of laccase enzyme?
?Also i need the formula and protocol for the determination of laccase enzyme activity by using ABST as a substrate.
hope to get a response.

Yogendra and Anita Singh said...

Dear, afreen thanks for showing your interest in ligninblack.
Your quary 1: can u suggest me the screening method for the identification of laccase enzyme?
Answere: I already suggested several methods to test Laccase activity including ABTS.
{laccase activity was determined by the oxidation of 2,2′-azinobis(3-ethylbenthiazoline)-6-sulfonato (ABTS) The reaction mixture contained 0.3 mM ABTS in malonate buffer (50 mM, pH 4.5), and the oxidation was followed at 420 nm (420=36,000 M−1 cm−1).}

Feel free to ask anything else

afreen said...

First of all,thanks alot for your resonse.
I wouldlike to know th formula for calculating laccase enzyme activity using ABTS or syringaldazine as substrate.

unnamalai said...

Hai sir, I'm B.tech final year student. I'm interested in Laccase enzyme.. Problem is guaiacol is not dispersed in buffer and in media too. its just floating as an oil drop. i tried wit Sodium actate buffer, citrate phosphate buffer, KH2PO4-NaOH buffer.. So i'm unable to carry out my assay.. I dont have ABTS also...kindly suggest me wat to do else ...

unnamalai said...

Hai sir, I'm B.tech final year student. I'm interested in Laccase enzyme.. Problem is guaiacol is not dispersed in buffer and in media too. its just floating as an oil drop. i tried wit Sodium actate buffer, citrate phosphate buffer, KH2PO4-NaOH buffer.. So i'm unable to carry out my assay.. I dont have ABTS also...kindly suggest me wat to do else ...

Yogendra and Anita Singh said...

Dear unnamalai, thanks for showing your interest in ligninblack. Your query: Problem is guaiacol is not dispersed in buffer and in media too ? Answerer: Dissolve 1st in distilled water. Example: dissolve 1gm guaiacol in 60 to 70 ml of water. Then go further for your experiment

Yogendra and Anita Singh said...

Dear, afreen please go through the given below link:
http://www.plasma.uaic.ro/COMB/analele%20stintifice/2008/3_manole%208pag.pdf

Hcam said...

thank you so much for all of these interesting ideas, hope from you to help me
I am working on some Natural products microbial transformation and I am interesting in laccase producing microorganisms
could you help me to know the interesting habitats that I should target to get the best variety of laccase microorganisms. at this time I am working on isolating micros from tree stem.

Yogendra and Anita Singh said...

Dear Hcam thank for this pleasant comments for ligninblack. Your query: habitats that I should target to get the best variety of laccase microorganisms?
Answer: Various natural habitats has been reported for isolation of Laccase producing microorganisms like:
• White rot fungus : isolated from such as decaying tamarind wood
• organisms living in digestive tracts probably use other enzymes for the breakdown of plant (poly)phenolics, such as diverse peroxidases.
• pulp and paper mills black liquor/soil/solid waste separated during filtration of black liquor
• Wood chip heaps soils etc

Karuna Dhiman said...

hello sir, I am karuna dhiman and presently I am working on laccase producing bacteria bioprospecting from paddy fields and paper mill samples. I got many isolates which shows red colour on guaiacol media but further screening on syringaldazine give no purple colour even they grow on syringaldazine containing media. so can u suggest me any other method which i could use for screening.I shall be very thankful to you.

Yogendra and Anita Singh said...

Dear, Karuna Dhiman:

Go for 0.5% tannic acid agar plate. isolate brown-black producing colony and then go for

ABTS assay.laccase activity was determined by the oxidation of 2,2′-azinobis(3-ethylbenthiazoline)-6-sulfonato (ABTS) The reaction mixture contained 0.3 mM ABTS in malonate buffer (50 mM, pH 4.5), and the oxidation was followed at 420 nm (420=36,000 M−1 cm−1).

or

mystic said...

Dear all,
I am planning to isolate hemicellulase, laccase, lipase, protease and rennin enzymes from soil. Can anyone help me about which selective media I can use for these enzymes? And do you know how can I identify the bacteria or fungi that is producing these enzymes, is API test is useful for this purpose?
I would appreciate if you could give a comment about this.
Thank you.

prabinshr4u said...

sir, i am a student doing thesis in isolation of lignin degrdading organisms. i used o-tolidine to screen laccse producing organism but i am not getting any result do u know how can i use o-tolidene for obtaining result. i am also plannin to use alpha naphthol how good is it..??
thank you.

Yogendra and Anita Singh said...

Hi prabin, thanks for showing your interest in www.ligninblack.blogspot.com. To screen-out laccase enzyme go for ABTS or tannic acid or guaiacol tests. Fro o-tolidine, alpha naphtho you have to work on certain fungal species, they can possibly breakdown these compounds. However, for bacterial species, ABTS or tannic acid or guaiacol tests are good. regards

prabinshr4u said...

thank you sir.
sir i am also trying to study dye decolorizing ability of fungal and bacterial species. but while using dyes like congo red malachite green or other dyes, they become faint when i mix them with pda. and so dye decolorizing ability is being difficult to study.
can u plz help in this also.
thanking you.

Yogendra and Anita Singh said...

Hi prabin nice to see you again.
I found a good article on this matter.
Hope it will help you.
Plz go through this link:

http://www.ajol.info/index.php/ajb/article/viewFile/56232/44676

Yogendra and Anita Singh said...

Prabin also see this link may be useful for you:

http://www.omicsonline.org/2155-6199/2155-6199-2-128.pdf

Karuna Dhiman said...

thanku very much sir.......ur suggestions help me a lot in my research work....sorry for the late reply actually i was busy somewhere else in last few months...........but t5hanks a lot sir

Karuna Dhiman said...

thanku very much sir.......ur suggestions help me a lot in my research work....sorry for the late reply actually i was busy somewhere else in last few months...........but t5hanks a lot sir

Karuna Dhiman said...

thanku very much sir.......ur suggestions help me a lot in my research work....sorry for the late reply actually i was busy somewhere else in last few months...........but t5hanks a lot sir

Karuna Dhiman said...

thanku very much sir.......ur suggestions help me a lot in my research work....sorry for the late reply actually i was busy somewhere else in last few months...........but t5hanks a lot sir

Yogendra and Anita Singh said...

Thanks Karuna for this nice gesture.

monalinikam said...

I want to know composition of sodium tartarate buffer for lacasse assay.

Yogendra and Anita Singh said...

To prepare 10 mM tartaric acid (sodium) buffer solution (pH=2.9)
Composition
Tartaric acid (M.W.=150.09): 7.5 mmol (1.13 g)
Sodium tartrate dihydrate (M.W.=230.08): 2.5 mmol (0.58 g)
Add water to make up to 1 L.

OR

You may also use following for Laccase Assay

Laccase was assayed by monitoring the oxidation of 2,2 azinobis (3-ethylbenz-
thiazoline)-6 sulphonate (ABTS) by the enzyme extract (Wolfendon and Wilson 1982) at
pH 4.5 and 35 oC temperature. Laccase activity assay was performed in 2.1 mL reaction
mixture containing 1 mL of 50 mM malonate buffer (pH 4.5), 1 mL of 1mM ABTS and
0.1 mL of enzyme solution, and ABTS oxidation was followed at 420 nm (ε420 36000 M
cm-1). The absorbance of each sample was taken after a 10 min. interval. Control samples
contained 0.1 mL of distilled water instead of enzyme solution. One unit laccase activity
was defined as change in absorbance of the assay mixture 420 nm in 10 min. The change
in absorbance in 10 min. was equivalent to µM of ABTS oxidized in 10min. That result
was then converted to uM of ABTS oxidized per min to calculate the IU.

abhibhawan said...

Hello sir and madam
I want to know that for plate assay using guaiacol, how much is to be added since Im having guaiacol in liquid form? Secondly whether it has to be added before or after autoclaving? Actually Im preparing B&K media for using guaiacol

monalinikam said...

Its quite late to reply but Sorry and Thank you very much sir.
Monali.

Yogendra and Anita Singh said...

Dear abhibhawana:

Go for this link : It discussed a lot about using Guaiacol supplemented agar.

http://scihub.org/ABJNA/PDF/2010/4/1-4-591-599.pdf

Also, Dont autoclave the liquid guaiacol if required use siring filter to sterilize it. Add it in last.

The liquid guaiacol you have has certain concentration which is mentioned on the bottle.

Yogendra and Anita Singh said...

Dear monalinikam, I replied you on the same day. I am sorry that you will not get it on time. Nice to see you again.

monalinikam said...

Sir, for purification of ligninolytic enzyme what should be the cut off of dialysis membrane and from where i can ordered this?and also which column is suitable for purification?

Yogendra and Anita Singh said...

Hi monalinikam nice to see you again..Hope this will help you..
Try this for laccase purification: Ammonium Sulphate fractional precipitation, and Sephadex G-100 gel filtration chromatography.

Purification of Laccase
Ammonium sulfate precipitation:
Crude laccase extract from 5-d-old cultures was centrifuged (3,000×g) for 15min
o
at 4 C. The supernatant was placed in an ice bath, crystals of ammonium sulfate were
added to attain 50% saturation, and the mixture was kept overnight at 4 oC. The resulting
precipitate was collected by centrifugation at 3000×g for 20 min at 4 oC. The pellets of
precipitated proteins were discarded. In the supernatant, more crystals of ammonium
sulfate were added to attain 90% saturation, and the mixture was again kept overnight at
4oC and centrifuged as previous. The pellets were dissolved in minimal volume of 50
mM sodium malonate buffer at pH 4.5 and dialyzed against distilled water with 4 equal
changes of water every 6 h to remove residual ammonium sulfate.

Gel filtration chromatography:
Laccase was further purified by gel filtration chromatography using a Sephadex
G-100 (Sigma, USA) column (120×2cm). Phosphate buffer (100 mM) with 0.15 M NaCl
was used as elution buffer at a flow rate of 0.5 mLmin-1. All the active fractions were
pooled.

Extraction and Purification of Extracellular Laccase:
After filtration of the fermention broth, the filtrate was centrifuged at 3,000×g for
20 minutes at 4 oC to obtain clear supernatant. The supernatant with a laccase activity of
202400 U/200mL and specific activity of 234.26 U/mg was used as crude enzyme
extract. The enzyme was precipitated at 90% ammonium sulfate saturation to obtain a
specific activity of 271.16 U/mg, which was equivalent to 1.16 purification fold. The
pellets (precipitate) were dissolved in minimal volume of 50 mM sodium malonate buffer
at pH 4.5 and dialyzed against distilled water to remove the extra salt.

You may get these things easily from your local vendor. May also go for Sigma products also.

monalinikam said...

Thanks a lot sir.
your blog is helping lot of students working in this field and i am one of them.

monalinikam said...

Sir, i want to know about cut off of dialysis membrane for ligninolytic enzyme purification.so that i can purchase this.

Yogendra and Anita Singh said...

Dear Monali, the laccase molecular wt. is coming between 60-90 kda, So, you may go for 10,000 MW cutoff dialysis tubing which is easily available. But must sure that your raw concentrate purified by by earlier said methodology.

* Kindly add your name in Followers list.

regards

abhibhawan said...

thank u sir and maam for replying. The paper which u have mentioned. im following that only.I prepared the B&K media by filter sterilising guaiacol. but i dissolved it in water before sterilising, was this alright? secondly, can i use the first method which u have mentioned in ur list that is using 2-5 days old culture on NA plates?

Yogendra and Anita Singh said...

Dear Abhibhawan,
You may use it directly.....
Follow these Instructions
Preparation Instructions:
One gram of guaiacol dissolves in 60-70 ml of water or
1 g of glycerol. It is miscible with alcohol, chloroform,
ether, oils, glacial acetic acid, and slightly soluble in
petrolium ether. Although it is soluble in a sodium
hydroxide solution, guaiacol forms a sparingly soluble
compound in a moderately concentrated potassium
hydroxide solution.

You may also use the above said method of NA plate test. Also, Try 0.5% tannic acid agar plate/NA plate. But here autoclave 0.5% tannic acid and agar plates separably and then mix it finally and made your plates under laminar hood. Strik the culture on these plates and isolate the brown-black colour producing colony.

regards

plz list your name in follower list.

Yogendra and Anita Singh said...

Dear abhibhawan,
In this article the guaiacol is added to media before autoclave.

Plz go through this also
http://www.ijest.info/docs/IJEST10-02-06-102.pdf

But i suggest go for syringe sterilization of guaiacol (mix 1 gm guaiacol in 60-70 ml of sterilize water and go for final syringe sterilization and add it to the media)

abhibhawan said...

Hello sir
Im using the methods you have mentioned but still not able to isoalte a single laccase producing bacteria.Is the same method applicable for alkaliphiles?Is it because iam using liquid guaiacol that not getting the results?I have not tried the tannic acid procedure

Yogendra and Anita Singh said...

Dear, abhibhawan
Try the tannic acid plates also.
Before going onward,enrich your sample (select the good samples):
Try this suggestions:
1. Samples (soil/water) enrich with
Guaiacol or ABTS or Tannic acid and incubate it for 24 to 72 hrs.
2. Star isolation by serial dilution on on plates containing 0.02% guaiacol or 0.5% Tannic acid agar plate. The colony appears redis to dark brown is showing laccase activity.
3. The 0.02% guaiacol or 0.5% Tannic acid agar plate may contain any media (like Nutrient Agar with series of lower to higher salt concentration) for screening.

Kashif Roy said...

Hi
i want to screen laccase producing bacteria using tannic acid plate test. please guide me how to do it. should i add tannic acid before or after autoclaving LB media? how much old bacterial culture should be used to streak plates? should i add copper to bacterial culture? what will b +ve result?
Thanks

Yogendra and Anita Singh said...


Hi kashif, Nice to see you.
Use 0.5% tannic acid agar plate/NA plate/LB plate. But here autoclave 0.5% tannic acid (in distilled water) and media (LB/NA) separately and then mix it finally and made your plates under laminar hood. Strike/spread the culture on these plates and isolate the brown-black colour producing colony.
Use Enriched media: Sample (soil/water etc) + 0.5% tannic acid, incubate for 24hr to 72 hr at 37 degree centigrade. This sample should be serially diluted and finally spread over tannic acid plates (LB/NA).
Purify the black brown color producing bacteria by strike again on tannic acid plates.

Prabin said...

hello sir.. im very thankful to your suggestions and advices. i again had a problem sir. i also have guaiacol in liquid form and it is not soluble in water. i knew through this blog that i can use organic solvents so i want to try with ethanol but what percent of ethano to use.. i am confused and how to make 10 mM of the solution because only molecular wt is given.
thank you sir

Yogendra and Anita Singh said...

It is very simple dude:
Guaiacol Preparation
1 000 ML = 124.14 g/lit = 1 Mole
Take 100 micro liter of Guaiacol liquid and dilute in 10 ml of distilled water/Glycerol/absolute alcohol. It is your 10 millimole Guaiacol .

Prabin said...

oh..thank you sir. thank you very much. but sir when i use alcohol or glycerol will it not have any effect in growing bacterias. i had studied that absolute alchohol denatures protein by penetrating cell of bacteria thus killin it.
please guide me in this case also.

thanking you

Yogendra and Anita Singh said...

Hi prabin, There is lots of use of Guaiacol. Like for Enzyme assay or for screening of laccase producing bacteria. its on you where you want to use it.
Tips
For screening of bacteria: Use circular very small filter disk (autoclave) and pour it in Guaiacol and put on the bacterial colony. OR you may also use the method mention in the article.

monalinikam said...

how to make malonate buffer of pH 4.5.

abhibhawan said...

Hello sir and madam,
Now i have identified some laccase producing bacteria .Now i want to know how to find out laccase producing alklaiphiles?

Yogendra and Anita Singh said...

Collect sample from alkaline environment. Go for same procedure by which you have isolate the laccase bacteria. Only you have to take care is pH of your culturable media (that must be more than 7).

Karuna Dhiman said...

hello sir ur suggestion to use tannic acid help me in screaning laccase producing bacteria. Now I have to do laccase activity u suggested me a method but I donot get any result with that. so can u please suggest me some other method and formula to calculate activity.

tinku rajasekhar said...

sir can i isolate laccase producing bacteria from seawater is it possible

Yogendra and Anita Singh said...

Yes you can, Kindly go through the comments of Haleema Ayub. regard

tinku rajasekhar said...

sir can u plz tel how can i screen laccase producing bacteria from seawater any special medium is required for it

Yogendra and Anita Singh said...

Mueller-Hinton (MH) is gud for isolation of halotolerant microorganism. Try this

tinku rajasekhar said...

thanku for ur suggestion sir

tinku rajasekhar said...

thanks for ue suggestions sir

tinku rajasekhar said...

sir can u plz tel the formula to calcuate grams to liter and liter to percentage

Prabin said...

hello again dear sir and madam..first of all sorry to be late to wish u both happy dasami n dipawali..wish u enjoyed them..i isolated many bacteria and among them 7 showed positive in tannic acid but none showed in guaiacol..i chose one and tried to see laccase activity with ABTS but in 90 seconds absorbance increased only by 5-9 units..i would like to know if bacterial laccase donot degrade ABTS..and would like to know if there is any protocol for determining laccase activity using tannic acid...plus i measured lignin degradation ability and i observed increase in abosrbance at 280 nm each day...is it possible sir..??

thanking you very much for your help..
prabin

abhibhawan said...

Hello sir and madam
I want to know why to use sodium acetate buffer only for determination of enzyme activity and that too of acidic pH?

Basit said...

Hello Sir i found the conversation very interesting.......it was really very knowledege gaining comments....thnx u alll....

abhibhawan said...

Hello sir,
I want to know after finding out laccase positive bacteria how to find its enzymatic activity. Im not asking for the purified enzyme. Just want to know how much enzyme is being produced by the specific bacteria

Yogendra and Anita Singh said...

Dear,

Use this do determine the crude extract of laccase.

Laccase activity confirmed by spectrophotometrically at 525 nm, through the oxidation of syringaldazine to its quinone form, using a molar absorptivity of 65,000 M−1 cm−1 for the product. The reaction mixture contained 1.5 ml phosphate buffer (100 mM, pH 6.5), 0.2 ml syringaldazine (0.5 mM in ethanol solution), and 0.1 ml of culture filtrates.
Alternatively, laccase activity was determined by the oxidation of 2,2′-azinobis(3-ethylbenthiazoline)-6-sulfonato (ABTS) The reaction mixture contained 0.3 mM ABTS in malonate buffer (50 mM, pH 4.5), and the oxidation was followed at 420 nm (420=36,000 M−1 cm−1).
or
you may also try by plotting standard curve with purified laccase enzyme activity and crude extract of laccase activity

Basit said...

thanx a tonne...dea Yogendra Singh and Anita Singh ,,,,remain blessed

abhibhawan said...

Sir
Thank you for replying. I want to know how to calculate activity using guaiacol as a substrate since this is available in my lab. Secondly what is meant by culture filtrate. Which media is to be used for growing the bacteria? Either nutrient broth or the one conatining CuSO4 which was used for screening.

saumya singh said...

sir i am trying to isolate laccase producing bacteria from rice straw... can u please guide me with this.

Vaanavil Regia said...

very useful info! can u plz tell me of simple plate screen assays for lignin peroxidase and manganese peroxidase enzymes too? thank u :)

prolab seo said...

Hello,
This is a really good read for me, Must admit that you are one of the best bloggers I ever saw.Thanks for posting this informative article.

Recombinant proteins

Nareshkumar said...

hello sir, thanks for your novel advoices. I am trying to isolate Laccase secreting bacteria. I am still in screening. I have tried with agriculture soil sample, compost sample but I am unable to get positive colonies. Can you please help me which source sample shall I go for getting novel bacteria?. I have liquid Guaiacol, what volume shall I add in NA media?. It will be a great favour if you help me out. Thank you...

anjan mahi said...

hello sir
I m Mahtab,and working on production of laccase enzyme from bacteria & i have problem for the assay i dont have ABTS chemical, suggest me some assay process using other then ABTS plz

Pratibha Baral said...

Can you help me
i want to know how can we isolate laccase from fungi

Nigar Siddiqui said...

Hi, nigar here sir.....
M interested in isolation of actinomycets n bacterial laccase. ... need your suggestion for sample collection n screening methods.

Pavani said...

Hi Sir, let me know the easy protocol for Laccase assay using ABTS

priyanka said...

Hi sir, can u help me how to calculate laccase activity by using abts. i need the formula.

pranjoti said...

Hello sir,
I am Pranoti Joshi, I am student of Bachelor of Engineering in Biotechnology Engineering.

I went through most of the discussion above. This is very great that you help to people facing problems related to the topic.

I am working on immobilization of laccase. Can you please suggest me the cheapest substrate for laccase..?? We have purchased laccase from Sigma Aldrich. Also we have 1- naphthol available in our lab. Can we use it for assay..?? If yes, Can I get protocol ..??

Thanking you in anticipation.

lalita limaye said...

how do I isolate laccase producing Actinomycete? I have isolated and tested many actinomycete strains but none has given positive results. What's going wrong?>

Parvathy G said...

Can you suggest the best medium for both qualitative and quantitative screening for bacterial laccase enzyme. My work is based on stain removal by bacterial laccase enzyme.

Pranita Roy said...

Sir,i'm involve in screening of laccase producing bacteria........just wanted to know about how bacteria appears on ABTS plates ?

Roshan Agricos said...

PLEASE TELL ME THE DETAIL PROTOCOL TO CARRY OUT LACCASE ASSAY USING ABTS /SYRINGALDEHYDE.. ETC

Roshan Agricos said...

I AM WORKING ON MICROBIAL DECONSTRUCTION OF RICE STRAW DEGRADATION .PLEASE TELL ME THE HOW TO CARRY OUT LACCASE ASSSAY USING SUBSTRATES LIKE ABTS

Memona Saeed said...

hi....i am working on laccase enzyme producing bacteria tell me is it produced by bacillus subtilis??