Enzymes cellulases

Enzymes cellulases

1. Industrial fungal metabolites Enzymes Cellulases . V.S.Patil Professor, Department of Botany Shri Shivaji College of Arts, Commerce,& Science Akola

2. Introduction- Cellulase  is any of several enzymes produced chiefly by  fungi,  bacteria, and   protozoans  that  catalyze  cellulolysis , the decomposition of cellulose and of some related polysaccharides . It is the enzyme, that act serially or synergistically to decompose cellulosic material . Cellulases break down the cellulose molecule into  monosaccharides  ("simple sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides .   It makes a major constituent of plants available for consumption and use in chemical reactions .  In many herbivorous animals  like cattle and sheep horses , cellulases are produced by symbiotic bacteria .   Endogenous cellulases are produced by a few types of metazoan animals, such as some   termites, snails,   and   earthworms .

3. It include   endo-1,4-beta-D-glucanase (beta-1,4-glucanase, beta-1,4-endoglucan hydrolase , endoglucanase D, 1,4-(1,3,1,4)-beta-D- glucan 4-glucanohydrolase),  carboxymethyl cellulase  ( CMCase ), avicelase ,  celludextrinase ,  cellulase A,  cellulosin AP, alkali cellulase ,  cellulase A 3, 9.5 cellulase , and  pancellase SS.  M ost animals (including humans) do not produce cellulase in their bodies and can only partially break down cellulose through fermentation, limiting their ability to use energy in fibrous plant material. It is also found in green microalgae ( Chlamydomonas reinhardtii ,  Gonium pectorale  and  Volvox carteri ) and their catalytic domains (CD) belonging to GH9 Family.   cellulose, is a water-insoluble polymer .

4. Fungal species known to degrade cellulose encompass members of the Ascomycota , Basidiomycota , and chytrids. Aerobic fungi are used for industry to synthesize multi-enzyme complexes, cellulosome . Cellulases have been produced and characterized from different aerobic fungi such as Aspergillus , Trichoderma , Penicillium .

5. Structure- Most fungal cellulases have a two-domain structure , with one catalytic domain and one cellulose binding domain, that are connected by a flexible linker.  This structure is adapted for working on an insoluble substrate, and it allows the enzyme to diffuse two-dimensionally on a surface in a caterpillar-like fashion. Both binding of substrates and catalysis depend on the three-dimensional structure of the enzyme which arises as a consequence of the level of protein folding. The amino acid sequence and arrangement of their residues that occur within the active site, the position where the substrate binds, may influence factors like binding affinity of ligands , stabilization of substrates within the active site and catalysis. Temperature, pH and metal ions influence the non-covalent interactions between enzyme structure. Multimodular cellulases are more efficient than free enzyme (with only CD) due to synergism because of the close proximity between the enzyme and the cellulosic substrate.

6. Types and action- 1.Endocellulases   - Cellulose 1,4-beta-cellobiosidase (reducing end ).It is an enzyme with systematic name  4-beta-D-glucan cellobiohydrolase (reducing end ) .  This enzyme catalyses the following chemical reaction. Hydrolysis of (1->4)-beta-D- glucosidic linkages in  cellulose  and similar substrates, releasing  cellobiose  from the reducing ends of the chains. The CelS enzyme from  Clostridium thermocellum  is the most abundant subunit of the  cellulosome  formed by the organism.

7. 2.Exocellulases  or cellobiohydrolases - Glucan 1,4-beta-glucosidase  (or  4-beta-D-glucan glucohydrolase ) is an enzyme that catalyses the hydrolysis of (1->4)-linkages in 1,4-beta-D-glucans and related oligosaccharides, removing successive  glucose  units . This is one of the  cellulases , enzymes involved in the hydrolysis of cellulose and related polysaccharides; more specifically, an  exocellulase , that acts at the end of the polysaccharide chain. Other names for this enzyme are  exo-1,4-beta-glucosidase ,  exocellulase ,  exo-beta-1,4-glucosidase ,  exo-beta-1,4-glucanase ,  beta-1,4-beta-glucanase ,  exo-1,4-beta-glucanase , and  1,4-beta-D-glucan glucohydrolase . Exocellulases are further classified into type I , that work processively from the reducing end of the cellulose chain, and type II , that work processively from the nonreducing end.

8. 3.Cellobiases or  beta- glucosidases -   It is an enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in beta-D- glucosides and oligosaccharides, with release of glucose.

9. 4.Oxidative cellulases -   depolymerize cellulose by radical reactions, as for instance  cellobiose dehydrogenase (acceptor).

10. 5.Cellulose- phosphorylases   depolymerize cellulose using phosphates instead of water . Cellulose is the most abundant biomaterial derived from the living organisms on the earth. Plant is the major contributor to the cellulose pool in the biosphere being synthesized through the process of photosynthesis. It consists of β-D- glucopyranoside units that are linked together via β-D- glucosyl bonds.

11. 1.Progressive (also known as processive ) type- It will continue to interact with a single polysaccharide strand . 2.Nonprogressive cellulase will interact once then disengage and engage another polysaccharide strand.

12. Production of Cellulases - It has been produced from fungal species such as Aspergillus ornatus , Penicillium sp., Aspergillus terreus , Aspergillus niger , Rhizopus sp, Trichoderma longibrachiatum , Beauveria Bassiana , lichtheimia romosa , Phaffomycetaceae , Dipodascaceae , Trichoderma citrinoviride , Humicola insolens etc. These fungi produce extracellular cellulase enzymes when they are grown on media containing plant polymers, or short oligosaccharides as an energy source, and when cultivated on media containing easily metabolizable sugar such as glucose, the expression of these enzymes is repressed.

13. Factor Affecting Cellulase Production- 1.Fermentation Method- Produced through solid state fermentation (SSF) and submerged fermentation ( SmF ) In solid state fermentation (SSF) the fungal species is grown on one or more solid substrate such as rice straw, wheat bran, corn husk, cassava cake, or sugar cane bagasse without or very low water content. The microorganism can be grown for long period of time. The enzymes including cellulase and metabolic byproducts are secreted into fermentation medium and medium supplements or nutrients are rapidly utilized and a continuous supply with is needed. SMF has several advantages such as simplicity of sterilization, heat and mass transfer, process monitoring (pH, temperature, and soluble molecules) and automation, and extraction and recovery of enzymes and bioactives .

14. 2. Carbon Source- Sarbose , maltose, sucrose, lactose, dextrose, galactose , cellobiose , and CMC ( Carboxymethylcellulose ). 3. Nitrogen Source- organic nitrogen source are peptone, yeast or beef extract, tryptone or soybean meal. Inorganic nitrogen source like ammonium sulphate, ammonium chloride, ammonium hydrogen phosphate. 4. pH and Temperature- pH 4 or pH 5.0 and temperature mostly 30˚C . 5. Incubation Time- from 96 h to 192 hours. 6.culture media containing avicel (10 g/l) as carbon source, urea (1.2 g/l), yeast extract (1.0 g/l), KH2PO4 (6.0 g/l), and MgSO4⋅7H2O (1.2 g/l) with an agitation speed of 220 rpm and aeration rate of 0.6 vvm .

15. Mechanism of cellulolysis - The three types of reaction catalyzed by cellulases:1. Breakage of the noncovalent interactions present in the amorphous structure of cellulose ( endocellulase ) 2. Hydrolysis of chain ends to break the polymer into smaller sugars ( exocellulase ) 3. Hydrolysis of disaccharides and tetrasaccharides into glucose (beta- glucosidase ).

16. Uses- 1.For commercial food processing in  coffee . It performs   hydrolysis of cellulose during drying of beans . 2.In textile industry and in laundry detergents . 3. I n the pulp and pape industry  for various purposes. 4. For pharmaceutical applications . 5. in the fermentation of biomass into  biofuels . 6. as a treatment for phytobezoars , a form of cellulose   bezoar  found in the human stomach, and it has exhibited efficacy in degrading polymicrobial bacterial  biofilms  by hydrolyzing the β(1-4) glycosidic linkages within the structural, matrix exopolysaccharides of the extracellular polymeric substance (EPS ). 7. Amino acids synthesis. 8. animal feeds. 9. Waste Management  

17. 10. Wine and Beverage Industry. 11. in agriculture where they are used to hydrolyze the cell wall of plant pathogens thus controlling the plant infection and diseases. 12. improvement of the soil quality. 13. in food processing during fruit and vegetable juices manufacturing to improve extraction. 14. increase extraction of olive oil under cold processing conditions and to improve its antioxidants and vitamin E contents. 15. cellulases like Digestin help to relieve digestive problems such as malabsorption .