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

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

Disadvantages of enzymes as biocatalysts การทำงานไม่มีเสถียรภาพที่อุณหภูมิสูง, pH ต่ำหรือสูงจนเกินไป และทำงานในสารละลายอินทรีย์ เอนไซม์บางชนิดจะถูกยับยั้งการทำงานโดยไอออนของโลหะหนัก ถูกย่อยสลายเอนไซม์ proteases เอนไซม์บางชนิดยังมีราคาแพง

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

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

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

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.

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

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.

Genetic code

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.

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

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.

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

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

Directed evolution (IV)

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

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

PCR & ep-PCR

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

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

Directed evolution of some enzymes