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.
•Most 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.

16. 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.

17. 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.

18. 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.

19. 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).

20. 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. In 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

21. 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.