662 431 Molecular Biotechnology Application of molecular biotechnology in biocatalysis

1. 662 431 Molecular BiotechnologyApplication 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 ต่ำหรือสูงจนเกินไป และทำงานในสารละลายอินทรีย์ • เอนไซม์บางชนิดจะถูกยับยั้งการทำงานโดยไอออนของโลหะหนัก • 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) • 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 • DNAse I fragmentation of parental genes; • assembly of recombined • genes using outer primers; • (C) cloning into an expression vector.

22. Directed evolution of some enzymes