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662 431 Molecular Biotechnology Application of molecular biotechnology in biocatalysis อ. ดร. วีระ ปิยธีรวงศ์

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งานนำเสนอเรื่อง: "662 431 Molecular Biotechnology Application of molecular biotechnology in biocatalysis อ. ดร. วีระ ปิยธีรวงศ์"— ใบสำเนางานนำเสนอ:

1 Molecular Biotechnology Application of molecular biotechnology in biocatalysis อ. ดร. วีระ ปิยธีรวงศ์

2 Advantages of enzymes as biocatalysts  การเร่งปฏิริยาเคมีความจำเพาะสูง  ทำงานที่อุณหภูมิไม่สูง  ใช้พลังงานไม่มาก  ทำงานได้ตั้งแต่ช่วง pH 2-12  ปฏิกิริยาเคมีส่วนใหญ่จะให้ผลิตภัณฑ์เป็นหลัก มี byproducts ออกมาน้อย  ไม่มีความเป็นพิษ (ถ้าใช้อย่างถูกวิธี)  สามารถนำกลับมาใช้ใหม่ได้  ถูกย่อยสลายโดยธรรมชาติ

3 Disadvantages of enzymes as biocatalysts  การทำงานไม่มีเสถียรภาพที่อุณหภูมิสูง, pH ต่ำ หรือสูงจนเกินไป และทำงานในสารละลาย อินทรีย์  เอนไซม์บางชนิดจะถูกยับยั้งการทำงานโดย ไอออนของโลหะหนัก  ถูกย่อยสลายเอนไซม์ proteases  เอนไซม์บางชนิดยังมีราคาแพง

4 Approaches to engineering enzyme activity  Rational protein design (computer-aided molecular modeling and site-directed mutagenesis)  Directed evolution (random mutagenesis / recombination and screening / selection method)  Semi-rational protein design

5 Applications of enzyme engineering  Improving enzyme activity  Changing enzyme substrate specificity and selectivity  Enhancing enzyme stability  Altering enzyme mechanism

6 Rational protein design (I)  Usually requires both the availability of the structure of the enzyme and knowledge about the relationships between sequence, structure and mechanism  Using molecular modeling, it has been possible to predict how to increase the selectivity, activity and the stability of enzymes

7 Rational protein design (II)  Amino acid substitutions are often selected by sequence comparison with homologous sequences.  Comparison of the three-dimensional structures of mutant and wild-type enzymes are necessary to ensure that a single mutation is really site-directed.

8 Rational protein design (III) Protein structure Planning of mutants & Site-directed mutagenesis Vector containing mutated genes Transformation in E. coli Protein expression & purification Mutant enzymes

9 Rational Design Using Site- Directed Mutagenesis (I)  Saturation mutagenesis is basically a site- directed mutagenesis protocol adapted to the use of degenerate oligonucleotides (NNN or NNK mutagenic cassettes, with N = A, T, G, C and K = G, T for instance) to introduce a full diversity (the 20 amino acids) at a given position.

10 Genetic code

11 Rational Design Using Site- Directed Mutagenesis (II) (A)degenerated codons introduced by PCR; (B) overlap PCR assembly (C) set of degenerated gene fragments (D) cloning into an expression vector.

12 Examples of enhance thermostability  The removal of asparagine residues in α- amylase  The introduction of more rigid structural elements such as proline into α-amylase and D-xylose isomerase  Addition of disulfide bridges to stabilize hen lysozyme

13 Directed evolution (I)  It mimics the process of Darwinian evolution in the test tube, combining mutagenesis and recombination with selection or screening for improved variants with the desired characteristics.  The main advantage is that the enzyme’s properties and functions can easily be engineered even without any knowledge of the structure.

14 Directed evolution (II)  Random mutagenesis of the gene encoding the catalyst or recombination of gene fragments  The variants are analysed on the basis of the properties of interest by either screening or selection.  The gene(s) encoding the improved variants are identified and then used to parent the next round of directed evolution

15 Directed evolution (III)  The ultimate goal of directed evolution is to accumulate improvements through repetitive rounds of mutagenesis and identification.

16 Directed evolution (IV)

17 Random Mutagenesis Using Error- Prone PCR  A starting gene is amplified over a million fold in an imperfect copying process that generates uncontrolled errors.  The technique is a variation of standard PCR using unbalanced deoxyribo- nucleotides concentrations, high Mg2+ concentration, Mn2+, low annealing temperatures, or a high number of cycles which are all error-triggering factors

18 Error-Prone PCR  (A) gene amplification under error triggering conditions;  (B) set of mutated gene fragments;  (C) cloning into an expression vector

19 PCR & ep-PCR

20 Recombination of gene fragment using Gene Shuffling  The recombination of homologous genes harvested from nature.  the parental genes have been preselected by natural evolution as functional; hence their progeny has a good chance of containing improved genes due to additive or synergistic combinations.  Fragmentation step of the parental genes followed by the random reassembly of parental gene segments

21 Gene Shuffling (A)DNAse I fragmentation of parental genes; (B) assembly of recombined genes using outer primers; (C) cloning into an expression vector.

22 Directed evolution of some enzymes


ดาวน์โหลด ppt 662 431 Molecular Biotechnology Application of molecular biotechnology in biocatalysis อ. ดร. วีระ ปิยธีรวงศ์

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