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Biotransformations. Part Ⅱ Chapter 3. 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
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Biotransformations Part ⅡChapter 3
Outline 3.1 Introduction 3.2Biocatalyst selection 3.3Biocatalyst 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 enzyme-catalysed and whole cell transformations
3.1 Introduction The biological catalyst • isolated enzymes • resting whole cells • dead microorganism • immobilised enzymes or cells
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
3.1 Introduction • Traditional hydrolytic reactions, e.g. starch and protein hydrolysis Industrial use of biotransfor-mations • Isomerisation 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 • regioselectivity and stereospecificity • energy effective catalysts working at moderate temperatures, perssures and pH values • safe and environmentally friendly • The advantages of biotransformations:
3.1 Introduction • A key issue: the availability of suitable biocatalysts. • Screening and selection techniques are required to: • Enhancing of the predictability and performance of biocatalysts: immobilised biocatalysts. • isolate biocatalysts • select and design catalysts
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. • Strategies screening for novel biocatalysts 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.
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
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
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.
α-amylase glucoamylase glucose starch dextrins 3.4.1 Sugar industry • Great 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 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 D-isomers; • Biosynthetic methods: specifically produced. • L-amino acids: hydrolysis of proteins with proteases and peptidases.
3.4.4 Wine industry Lactobacillus Pediococcus or Leuconostoc malic acid 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, Bjørn Kristiansen. Basic Biotechnology (Second Edition)（影印版），科学出版社，2003. • 欧阳平凯、林章凛主译. Liese A, Seelbach K, Wandrey C，Industrial Biotransformations工业生物转化过程（原著第二版），化学工业出版社，2008. • （德）博马留斯、里贝尔著，孙志浩、许建和译. 生物催化——基础与应用，化学工业出版社，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.
