Isolation and screening of cellulase producers

/Isolation and screening of cellulase producers
Isolation and screening of cellulase producers2018-02-23T10:29:14+00:00

Isolation and screening of cellulase producers

Introduction

Cellulases (3.2.1.4) also known as 1,4-(1,3;1,4)-β-D-Glucan-4-glucanohydrolases are enzymes that hydrolyse cellulose. Cellulose is a linear polysaccharide of glucose and is connected by β-1,4 linkages. The enzymatic mechanism involved in the breaking down of cellulose involves two steps: first, a prehydrolytic step wherein anhydroglucose chains are swollen or hydrated and secondly, hydrolytic cleavage of the now susceptible polymers either randomly or endwise. The first step would involve an enzyme designated C1 and the second, hydrolytic enzymes termed Cc. A third type of enzyme is β-glucosidase (cellobiase).The C1 component attacks highly ordered (crystalline) cellulose, i.e., cotton fibres or Avicel, but have little effect on soluble derivatives such as carboxymethyl cellulose (CMC). C1 “decrystallizes” or hydrates cellulose chains whereas Cc consists of exo and endo β-1,4 glucanases that attack soluble derivatives or cellulose that has been acid or alkali swollen. In simple words, the cellulosic enzyme system is composed of 3 major components: endo-ß-glucanase (EC 3.2.1.4), exo-ß-glucanase (EC 3.2.1.91) and ß-glucosidase (EC 3.2.1.21). The mode of action of each of these being:

1. Endo-p-glucanase, 1,4-ß-D-glucan glucanohydrolase, CMCase, Cx: “random” scission of cellulose chains yielding glucose and cello-oligo saccharides.

2. Exo-P-glucanase, 1,4-ß – D-glucan cellobiohydrolase, Avicelase, C1: exo-attack on the non-reducing end of cellulase with cellobiose as the primary structure.

3. ß-glucosidase, cellobiase: hydrolysis of cellobiose to glucose.

Although a large number of microorganisms are capable of degrading cellulose, only a few of these microorganisms produce significant quantities of cell-free enzymes capable of completely hydrolysing crystalline cellulose in vitro. Fungi are the main cellulase-producing microorganisms, though a few bacteria and actinomycetes have also been reported to yield cellulase activity. Commercially, cellulases are produced by the members of the family Trichoderma and Aspergillus.

Cellulases find a wide range of application in agricultural and industrial areas. These enzymes are used in the detergent industry since it aids in re-sizing of the fibres. Besides that, cellulases are used in fruit juice processing industry and in the paper and pulp industry.

In this experiment, congo red dye is used to check the cellulose activity. Congo red shows a strong interaction with polysaccharides containing contiguous β (1,4) linked D-glucopyranosyl units and β (1,3) D glucans. This helps in enumerating and characterising cellulolytic microorganisms by the intense colour formation of the dye-glucan complex. The NaCl creates ions which helps in leeching of the dyes from the area where the dye doesn’t bind.

Requirements

  1. Nutrient agar
  2. CMC (carboxy methyl cellulose)
  3. 0.5% congo red dye
  4. 1M NaCl
  5. Toothpicks

Procedure

  1. Obtain soil samples and make serial dilutions.
  2. Make a solution on 0.5% CMC by dissolving it separately in water.
  3. Spread 0.1ml of an appropriate dilution on a nutrient agar plate. Incubate overnight at 37°C.
  4. Now separate the colonies and streak them on nutrient agar platesto obtain pure cultures. Perform gram staining to confirm the purity of the cultures. Incubate overnight.
  5. Take a fresh plate of nutrient agar enriched with 0.5% CMC. Perform picking of the colonies, from the nutrient agar plate and make sure there is one colony on each square. Incubate overnight.
  6. Check for growth. Flood the plate with congo red dye and leave for 5-10 minutes.
  7. Wash with 1M NaCl solution. Check for zone of clearance and measure the diameter.
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