Part

Chapter 3

Biotransformations

Outline
3.1 Introduction

3.2 Biocatalyst selection

3.3 Biocatalyst immobilisation and performance
3.4 Biotransformation application

3.1 Introduction

Biocatalysis: transformations involving isolated enzymes Biotransformations: procedures involving whole cells

Biocatalytic manufacture: scale up of enzymecatalysed and whole cell transformations

3.1 Introduction

isolated enzymes resting whole cells dead microorganism immobilised enzymes or cells

The biological catalyst

3.1 Introduction

Biotransformation: the process whereby a substance is converted into a product in a limited number of enzymatic steps by the use of biological catalysts.

Biotransformation process requirement:

optimal biocatalysts reaction media bioreactors

The Biotransformation process

3.1 Introduction

Traditional hydrolytic reactions, e.g. starch and protein hydrolysis

Isomerisation reactions, e.g. glucose conversion to fructose

Industrial use of biotransfor -mations

Synthesis of chiral compounds Reversal of hydrolytic reactions Complex synthetic reactions, such as aromatic hydroxylations and enzymatic group protection chemistry

Degradation of toxic and environmentally

harmful compounds

3.1 Introduction

The advantages of biotransformations:

regioselectivity and stereospecificity

energy effective catalysts working at moderate temperatures, perssures and pH values safe and environmentally friendly

3.1 Introduction

A key issue: the availability of suitable biocatalysts.

Screening and selection techniques are required to:
isolate

biocatalysts select and design catalysts

Enhancing of the predictability and performance of biocatalysts: immobilised biocatalysts.

3.1 Introduction

The biotransformations have two purpose:

one is to remove them from effluents and convert them to less toxic products; the other is to convert them into products with economic value.

3.2 Biocatalyst selection

Advantages of enzyme (or whole cell) biocatalysis:
Substrate specificity Selectively use a substrate in a mixture of feed Substrate flexibility (non-specificity) Can often be used to catalyse a reaction on a similar, but non-native, substrate Relatively mild reaction conditions, environmentally friendly (green) Minimal side reactions (comparing to high temperatures or living cell processes) Rigioselectivity (stereoselectivity)

3.2 Biocatalyst selection

It is necessary to select the appropriate biocatalyst with suitable activity, selectivity and stability.
screening for novel biocatalysts

Strategies

use of existing biocatalysts genetic modification of existing biocatalysts

3.2.1 Screening for novel biocatalysts

Selection of new micro-organisms with novel activities is still worthwhile taking into account the overwhelming biochemical diversity present in nature. One gram of soil may contain up to 4000 different species, however, current estimates indicate that less than 1% of these organisms have been isolated.

Metagenome approach involved in techniques to directly extract, clone and recombinantly express genomic DNA from entire uncultivated microbial communities provides genetic access to the uncultured majority of microbial diversity and its enzymatic constituents, and serve as a rich source for isolation of novel biocatalysts.

Steps involved in a metagenomics experiment.

Construction and screening of metagenomic libraries.

To screen large numbers of organisms,

cheap, simple, rapid and selective detection methods, perferably capable of some automation, are required.

3.2.2 Use of existing biocatalysts

A well-known way to accomplish a desired biotransformation is the use of existing biocatalysts on natural and unnatural substrates. The exploitation of existing biocatalysts under different reaction conditions could lead to the finding of a biocatalyst for the desired biotransformation.

3.2.3 Genetic modification of existing biocatalysts

In vivo: metabolic pathway engineering

Transfer of genes Gene duplication Gene fusion Recombination between genes Deletion or insertion of gene segments One or more single site mutations Combination of these activities

An analytic part of metabolic engineering

A synthetic part of metabolic engineering

In vitro: protein engineering

Alter properties (substrate specificity and pH
activity profile)

Improve thermal and oxidative stabilities

3.3 Biocatalyst immobilisation and performance

Why? Enzyme Re-use allows for a continuous process (in packed bed reactor) allows for a batch recirculation process, so that enzyme catalysts can be used at the end of the batch process Increased enzyme concentration, especially in a packed bed process

3.3.1 Biocatalyst immobilisation

Advantages on the use of immobilised biocatalysts
General aspects Retention of the biocatalyst in the bioreactor Specific aspects Possible biocatalyst re-use Product contamination avoided High dilution rates allowed without biocatalyst washout
Increased volumetric productivity Rapid conversion of unstable substrates Minimised side-reactions

High biocatalyst concentration

Control of biocatalyst micro- Manipulation of biocatalyst activity and specificity environment Stabilisation of biocatalyst activity Protection of shear-sensitive biocatalysts Facilitated separation of the Precise control of bioreaction time biocatalyst from the product Minimisation of further product transformation

Immobilized enzymes

3.3.2 Methods of immobilization

Entrapment:
enzymes and gel precursor or monomer are mixed, then gel is allowed to form, or to polymerize from monomer, thus entrapping the enzyme (cells)

Adsorption:
enzymes are added to porous polymer matrix and adsorbed to the internal surfaces through different interactions (charge, hydrostatic, etc.)

Covalent Binding:
enzymes are added to porous matrix and covalently linked to the matrix

Membrane retention:
enzymes are either entrapped in one compartment and retained in the reactor be selection of the pore size of the membrane, or enzymes are covalently bound to the membrane.

Methods of immobilization

3.4 Biotransformation application

The stages of a biotechnological process.

3.4.1 Sugar industry

Great industrial enzymes: consumed in the sugar industry.

Used for the production of glucose from starch.

-amylase
starch glucoamylase

dextrins

glucose

Invert suger from sucrose as well as for the isomerization of glucase to fructose.

3.4.2 Dairy industry

Hydrolysis of lactose Cheese production Sterillization of dairy products

3.4.3 Amino acids production

L-amino acids: produced by using chemical and biosynthetic methods: Chemical synthesis: a racemic mixture of the L- and Disomers; Biosynthetic methods: specifically produced.
L-amino acids: hydrolysis of proteins with proteases and peptidases.

3.4.4 Wine industry

Lactobacillus Pediococcus or

malic acid

Leuconostoc

lactic acid

oxidate sulfites

innocuous sulfates

3.4.5 Pharmaceutical industry

In the pharmaceutical industry, many chemical transformation are carried out by biocatalysts, such as

purified enzymes or whole microbial cells.

Questions

1. What is biotransformation? 2. Describe the biotransformation process? 3. What is the advantages of biotransformations and biocatalysis? 4. List three strategies for biocatalyst selection? 5. Why and how is biocatalyst immobilized? 6. List several examples of industrial enzyme biocatalysis process?

Textbooks and references

Colin Ratledge, Bjrn Kristiansen. Basic Biotechnology (Second Edition) 2003. . Liese A, Seelbach K, Wandrey CIndustrial Biotransformations2008. . 2006. Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B. Industrial biocatalysis today and tomorrow. Nature, 2001, 409, 258-266 Lorenz P, Eck J. Screening for novel industrial biocatalysts. Eng. Life Sci., 2004, 4(6), 501-504. Zeyaullah Md, Kamli MR, Islam B, Atif M, Benkhayal FA, Nehal M, Rizvi MA, Ali A. Metagenomics-An advanced approach for non-cultivable micro-organisms. Biotechnology and Molecular Biology Reviews, 2009, 4(3): 49-54.