BY: MOSESBRUCE.M.DESAI M.Sc BT ROLL NO: 39 SEM III

Enzyme

engineering can be defined as the modification of enzyme structure with recombinant DNA technology or chemical treatment to get a desirable function for better use in medicine, industry and agriculture.

The

objectives of enzyme engineering is as follows (a) to create a superior enzyme to catalyze the production of high value specific chemicals. (b) to produce enzyme in large quantities. (c) to produce biological compounds(include synthetic peptide, storage protein, and synthetic drugs) superior to natural one.

Kinetic

properties of enzyme-turnover and Michaelis constant, Km. Thermo stability and the optimum temperature for the enzyme. Stability and activity of enzyme in nonaqueous solvents. Substrate and reaction specificity. Cofactor requirements Optimum PH. Molecular weight and subunit structure.

TWO

MAIN METHODS

1.RATIONAL METHODS 2.RANDOM METHODS

RATIONAL

METHODS INCLUDES:

1.SITE-DIRECTED MUTAGENESIS 2.COMPUTATIONAL MODELING

SITE-DIRECTED

MUTAGENESIS, ALSO CALLED SITE-SPECIFIC MUTAGENESIS OR OLIGONUCLEOTIDE -DIRECTED MUTAGENESIS, IS A MOLECULAR BIOLOGY TECHNIQUE OFTEN USED IN BIOMOLECULAR ENGINEERING IN WHICH A MUTATION IS CREATED AT A DEFINED SITE IN A DNA MOLECULE.

TWO

COMMON METHODS FOR SITE DIRECTED MUTAGENESIS

1.OVERLAP EXTENSION METHOD. 2. WHOLE PLASMID SINGLE ROUND PCR METHOD.

This method involves two primer pairs. where one primer of each primer pair contains the mutant codon with a mismatched sequence. These four primers are used in the FIRST PCR. where two PCRs take place, and two dsDNA products are obtained. Upon denaturation and annealing of them, two heteroduplexes are formed. This heteroduplex involves the desired mutagenic codon. Second PCR takes place using the nonmutated primer set to amplify the mutagenic.

This

method requires two oligonucleotide primers with the desired mutation(s). This are complementary to the opposite strands of a double-stranded DNA plasmid template. Both strands of the template are replicated without displacing the primers and a mutated plasmid is obtained with breaks that do not overlap.

DpnI

methylase is then used for selective digestion to obtain a circular, nicked vector with the mutant gene.

Computational

methods employs the three-dimensional coordinates of protein structures. Computer modeling is particularly valuable for estimating short-range interactions.

Use

of methods for the rational mutagenesis of proteins is often limited by the difficulty of predicting what specific amino acid replacements will lead to the preferred altered selectivities, even when high resolution structural data are available.

Random

methods includes two major methods:

1.DIRECTED MUTAGENESIS 2.RANDOM MUTAGENESIS

DIRECTED

MUTAGENESIS INCLUDES :

1.DNA SHUFFLING 2.FAMILY SHUFFLING 3.NONHOMOLOGOUS RECOMBINATION

Developed

by Stemmer. In this method group of genes ,relatively similar sequence is obtained from different organism. These genes are then randomly cleaved into small fragments by digestion with the restriction enzyme DnaseI. The fragments are then purified and reassembled in a PCR in the presence of a thermostable, error-prone DNA polymerase.

Family

shuffling is done recombining a set of naturally occurring homologous genes.

Random

mutagenesis is inducing mutation randomly in DNA. It can be done by uv radiation, base analogs , alkylation.

AN

IMMOBILIZED ENZYME IS ONE WHICH HAS BEEN ATTACHED TO OR ENCLOSED BY AN INSOLUBLE SUPPORT MEDIUM, WITHOUT LOSS OF CATALYTIC ACTIVITY.

ATTACHMENT

TO INSOLUBLE SUPPORT MEDIUM ENTRAPPING BY AN INSOLUBLE SUPPORT MEDIUM CROSS-LINKING OF ENZYME MOLECULE

COMMONLY

USED ENTRAPMENT MEDIA ARE POLYACRYLAMIDE, CALCIUM ALGINATE, AND GELATIN. ENZYMES ARE WELL MIXED WITH MONOMERS/POLYMERS AND CROSSLINKING AGENTS IN A SOLUTION. THE SOLUTION IS THEN EXPOSED TO POLYMERIZATION PROMOTERS TO START THE PROCESS OF GEL FORMATION.

STABILITY

OF ENZYME CHANGES. PH OPTIMUM CAN BE CHANGED. APPERENT Km CHANGES.

Human

butyrylcholinesterase expressed in mammalian cells. Shows increased activity towards cocaine hydrolysis. This enzyme is used for treating severe cases of cocaine toxicity.

Toluene

4-monooxygenase, tolueneoxylene monooxygenase and toluene-4monooxygenase. Produced by DNA shuffling method. Improved activities towards benzene, toluene, nitrobenzene, nitrophenols, catechols, o-methoxyphenol, and o-Cresol.

FOOD

INDUSTRY DETERGENT INDUSTRY MEDICAL APPLICATION BIOPOLYMER PRODUCTION

Enzyme

produced by enzyme engineering have properties like thermostability, specificity and catalytic efficiency.

Used

to remove stains. Enzyme produced by enzyme engineering are thermotolerant and cryotolerant. Eg : subtilisin

THE

USE OF ENZYME ENGINEERING IN CANCER TREATMENT. PRODUCTION OF MULTIFUNCTIONAL AND SMART DRUG. Eg: Recombinant human erythropoietin.

BIOMATERIALS

USED BECAUSE OF THEIR SPECIFIC PHYSICAL, CHEMICAL AND BIOLOGICAL PROPERTIES. ENZYME ENG USED TO CREATE AND IMPROVE PROTEIN DOMAINS. EG.ELASTIN-LIKE POLYPEPTIDES, SILK-LIKE POLYMERS, ETC.

ENZYMES: Biochemistry, Biotechnology and clinical chemistry by Trevor Palmer and Philip Bonner Altering protein specificity: techniques and applications by Nina M. Antikainen and Stephen F. Martin* Protein Engineering Methods and Applications by Burcu Turanli-Yildiz1,2, Ceren Alkim1,2 and Z. Petek Cakar ENZYME IMMOBILIZATION BY GEL ENTRAPMENT by Nam Sun Wang Engineering Enzymes for Biocatalysis Paul A. Dalby Site Directed Mutagenesis and Protein Engineering by Dr. Marcia E. Roye Engineering of Therapeutic Proteins by Mariusz Kamionka.

THANK YOU