Biotransformations

1、BiotransformationsPart Chapter 3Outline3.1 Introduction3.2 Biocatalyst selection3.3 Biocatalyst immobilisation and performance3.4 Biotransformation application3.1 IntroductionBiocatalysis: transformations involving isolated enzymes Biotransformations: procedures involving whole cells Biocatalytic ma

2、nufacture: scale up of enzyme-catalysed and whole cell transformations 3.1 IntroductionThe biological catalyst immobilised enzymes or cells resting whole cells dead microorganism isolated enzymes3.1 IntroductionBiotransformation: the process whereby a substance is converted into a product in a limit

3、ed number of enzymatic steps by the use of biological catalysts.Biotransformation process requirement: optimal biocatalystsreaction mediabioreactorsThe Biotransformation process3.1 IntroductionIndustrial use of biotransfor-mationsTraditional hydrolytic reactions, e.g. starch and protein hydrolysis I

4、somerisation reactions, e.g. glucose conversion to fructose 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

5、regioselectivity and stereospecificityenergy effective catalysts working at moderate temperatures, perssures and pH valuessafe and environmentally friendly The advantages of biotransformations:3.1 IntroductionA key issue: the availability of suitable biocatalysts.Screening and selection techniques a

6、re required to:Enhancing of the predictability and performance of biocatalysts: immobilised biocatalysts.isolate biocatalystsselect and design catalysts3.1 IntroductionThe biotransformations have two purpose:one is to remove them from effluents and convert them to less toxic products;the other is to

7、 convert them into products with economic value. 3.2 Biocatalyst selectionAdvantages 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-n

8、ative, substrate Relatively mild reaction conditions, environmentally friendly (green) Minimal side reactions (comparing to high temperatures or living cell processes) Rigioselectivity (stereoselectivity)3.2 Biocatalyst selectionIt is necessary to select the appropriate biocatalyst with suitable act

9、ivity, selectivity and stability.Strategies screening for novel biocatalystsuse of existing biocatalystsgenetic modification of existing biocatalysts3.2.1 Screening for novel biocatalystsSelection of new micro-organisms with novel activities is still worthwhile taking into account the overwhelming b

10、iochemical 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

11、 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 metageno

12、mic 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 biocatalystsA well-known way to accomplish a desired biotransformation is the use of existing biocatalysts on natural an

13、d 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 biocatalystsIn vivo: metabolic pathway engineeringTransfer of genesGene duplication

14、Gene fusionRecombination between genesDeletion or insertion of gene segments One or more single site mutationsCombination of these activitiesAn analytic part of metabolic engineeringA synthetic part of metabolic engineeringIn vitro: protein engineeringAlter properties (substrate specificity and pH a

15、ctivity profile)Improve thermal and oxidative stabilitiesWhy? 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 be

16、d process3.3 Biocatalyst immobilisation and performance3.3.1 Biocatalyst immobilisationGeneral aspectsSpecific aspectsRetention of the biocatalyst in the bioreactorPossible biocatalyst re-useProduct contamination avoidedHigh dilution rates allowed without biocatalyst wash-outHigh biocatalyst concent

17、rationIncreased volumetric productivityRapid conversion of unstable substratesMinimised side-reactionsControl of biocatalyst micro-environmentManipulation of biocatalyst activity and specificityStabilisation of biocatalyst activityProtection of shear-sensitive biocatalystsFacilitated separation of t

18、he biocatalyst from the productPrecise control of bioreaction timeMinimisation of further product transformationAdvantages on the use of immobilised biocatalystsImmobilized enzymes3.3.2 Methods of immobilization Entrapment: enzymes and gel precursor or monomer are mixed, then gel is allowed to form,

19、 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

20、 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 immobilization3.4 Biotransformation applicationThe stages of a biotechnological process.st

21、arch-amylasedextrinsglucoamylaseglucose3.4.1 Sugar industryGreat industrial enzymes: consumed in the sugar industry. Used for the production of glucose from starch. Invert suger from sucrose as well as for the isomerization of glucase to fructose. 3.4.2 Dairy industryHydrolysis of lactoseCheese prod

22、uctionSterillization of dairy products3.4.3 Amino acids productionL-amino acids: produced by using chemical and biosynthetic methods:Chemical synthesis: a racemic mixture of the L- and D-isomers;Biosynthetic methods: specifically produced. L-amino acids: hydrolysis of proteins with proteases and pep

23、tidases. 3.4.4 Wine industryLactobacillusPediococcus orLeuconostoc malic acid lactic acid oxidate sulfites innocuous sulfates 3.4.5 Pharmaceutical industryIn the pharmaceutical industry, many chemical transformation are carried out by biocatalysts, such as purified enzymes or whole microbial cells. Questions1.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 bioc