CHAPTER 4ENZYME CATALYSIS

ENZYME CATALYSIS

Application of Hydrolytic Enzymes. Enzyme Immobilization Technology:- Methods of enzyme immobilization.- Immobilized enzyme kinetics.

SUBCHAPTER

INTRODUCTION

OBJECTIVE OF THIS CHAPTER: To study some of the applications of enzymes. To examine immobilized enzyme catalyst formulations (allow sustained, continuous use of the enzyme).

Some enzymes of industrial importanceName Source Application Notes

Diastase MaltDigestive aid, supplement to bread, syrup

α-amylase activity, β-amylase activity

Takadiastase Aspergillus oryzae

Digestive aid; supplement to bread, syrup

Contains many other enzymes, protease, R-Nase

Amylase Bacillus subtilis

Desizing textiles, syrup, alcohol fermentation industry, glucose production

Crude preparation contains protease

Acid-resistant amylase

Aspergillus niger Digestive aid Optimum pH 4-5

Cont' ( starch-saccharifying amylase)Name Source Application

Amyloglucosidase

Rhizopus niveus, Aspergillus niger, Endomycopsis fibuliger

Glucose production

Cont' ( animal and vegetable protease)Name Source Application

Trypsin Animal pancreas Medical uses, meat tenderizers, beer haze removalPepsin Animal stomach Digestive aid, meat tenderizerα-Chymotrypsin Animal stomach medical uses

Rennet Calf stomach cheese manufacturePancreas protease Animal pancreas Digestive aid, cleaning, leatherbathing, dehairing, feed improvement

Papain Papaya digestive aid, medical uses, beer haze removal, meat tenderizerBromelain, ficin PIneapple, fig digestive aid, medical uses, beer haze removal, meat tenderizer

Cont' ( microbial protease)Name Source Application Notes

Protease

Aspergillus oryzae Flavoring of sake, haze removal in sake -

Aspergillus niger feed, digestive aidacid resistant protease, optimum pH

Bacillus subtilisdetergents, removal of gelatin from film (recovery of silver), fish solubles, meat tenderizer

optimum pH 7

Streptomyces griseus

detergents, removal of gelatin from film (recovery of silver), fish solubles, meat tenderizer

optimum pH 8

Varidase Streptococcus sp. medical use lederle

Streptokinase Streptococcus sp. medical use profibrinolysin

APPLICATION OF HYDROLYTIC ENZYMES

Hydrolytic Enzymes ???

An enzymes that catalyzes hydrolysis.

Protein Carbohydrate Fat molecules Polymers

Simplest Units

Hydrolysis Process

Hydrolytic Enzymes ???

Hydrolytic enzymes

Hydrolytic EnzymesEnzyme Substrate Hydrolysis productEsterases:Lipase Glycerides (fats) Glycerol + fatty acids

Phosphatases:LecithinasePectin esterase.LecithinPectin methyl ester

.Choline + H3PO4 + fatmethanol + polygalacturonic acidCarbohydrases:Fructosidaseα-glucosidase (maltase)β-glucosidase (cellobiase)β-galactosidase (lactase)AmylasesCellulaseCytasePolygalacturonase

.SucroseMaltoseCellobioseLactoseStarchCellulose.Polygalacturonic acid

.fructose + glucoseglucoseglucosegalactose + glucosemaltose or glucose + maltooligosaccharidescellobiosesimple sugarsgalacturonic acidNitrogen-carrying compounds:ProteinasesPolypeptidases proteinsproteins

polypeptidesamino acids

Desaminases:UreaseAsparaginaseDeaminaseureaasparagineamino acids

CO2 + NH3Aspartic acid + NH3NH3 + organic acids

Hydrolysis of Starch & Cellulose

Amylasescan hydrolyze the glucosidic bonds in starch and related glucose-containing compounds

Saccharifying enzyme?

Hydrolysis of Starch & Cellulose

α-amylase

β-amylase

Starch-liquefying enzyme

Industry UseGlucose & syrup

total or partially hydrolysis of corn starch to give a large quantity of sweeteners.

Brewing conversion of crushed grain starch to maltose

Breadmaking leavening: conversion of sufficient starch to fermentable saccharides needed for CO2 generation

Fruit juice hydrolysis of starch causing turbidity due to insolubility

Papermaking alpha-amylase action to liquefy starch coating to a desired viscosity for application to fibers

TextilesSizing: alpha-amylase activity to liquefy starchDesizing: alpha,beta-amylse action to remove size from woven material so that all threads will dye uniformly and fabric will have desired texture.

Candy production of candy of desired softness

Common applications of amylse preparations

Proteolytic enzymeSynthesized in an inactive form suitable either for STORAGE or for TRANSPORT from the site of synthesis to the desired site of activityHOW to activate the proteolytic

enzyme?From a precursorBy presence of a metal ion

OHRCOOHROHCOORR 2121

Esterase ApplicationsTo synthesize of various ester bonds to yield an acid and an alcohol.

Hydrolytic enzyme applications which are expanding (in development)Enzyme ProcessPenicillin acylase Production of semysynthetic penicillin core from nature penicillin GLactase Removal of lactose from whey, milkRibonuclease Production of 5'-nucleotides from RNADextranase Removal of tooth plaqueIsoamylase Production of maltose from starchKeratinase Modification of wool, hair, leatherTannase Removal of tannic acid from foods

ENZYME IMMOBILIZATION TECHNOLOGY Methods of

enzyme immobilization

Immobilized enzyme kinetics

Immobilized Enzymes?

Introduction

Enzyme Immobilization????

To restrict enzyme mobility in a fixed space.

Enzyme Immobilization????

Immbolized enzymes are ATTACHED

to an insoluble suppport medium or enclosed by the support medium which is also known as a CARRIER

In some cases, the enzymes molecules are cross-linked to each other so that their movement is restricted but their catalytic

activities are retained.

Needed for Immobilizaton

ECONOMICAL

CONVENIENCE

STABILITY

continuous use of bio-catalyst is possible

separation of bio-catalyst & product is much easier than conventional batch process

Immobilized enzymes typically have greater thermal

& operational stability than the soluble form of the

enzyme

Methods for Enzyme Immobilization

Methods for immobilization enzyme

Carrier-binding Method

The oldest immobilization method. Carriers (water insoluble) such as polysaccharide derivatives, synthetic polymers, and porous glass, etc.

Cross-linking Method

Based on the formation of chemical bonds, but water-insoluble carriers are not used in this method. Reagents such as glutaldehyde, bisdiazobenzidine, and hexamethylene diisocyanate, etc.

Entrapping Method

Based on confining enzymes in the lattice of a polymer matrix or enclosing enzymes in semipermeable membranes. A chemical polymerization reaction e.g. cllagen, gelatin, cellulose etc.

Preparation & Characteristics of immobilized

enzyme

Preparation & Characteristics of immobilized enzyme

Benefits of immobilizing and ENZYME

Multiple or repetitive use of a single batch of enzymes.

The ability to stop the reaction rapidly by removing the enzyme from the reaction solution (or vice versa).

Enzymes are usually stabilized by bounding. Product is not contaminated with the enzyme

(especially useful in the food and pharmaceutical industries).

Analytical purposes - long 1/2-life, predictable decay rates, elimination of reagent preparation, etc.

Immobilized Enzyme Kinetics