Endo-1,4-beta-xylanase is the main enzyme in xylan-backbone hydrolysis and has received attractable research interest due to its significant application in various industrial processes such as food, feed, waste treatment, fuel and chemical production, paper and pulp industries; but these applications require thermostable xylanase enzymes. Error-prone polymerase chain reaction (PCR) and site-directed mutagenesis were used to engineer new thermostable mutants of Bacillus halodurans. The results showed that the rate of mutagenesis in the error-prone PCR was at least 1%, resulting in more than 1000 mutated colonies. About one third of mutants lost the enzymes’ activities and no sign of thermal stability improvement was observed in other mutants; less than 2% of mutants were active at temperature higher than 75°C and they lost their activities quickly at temperature higher than 80°C. In site-directed mutagenesis at position 274, threonine (T) amino acid changed to proline (P) but the extent of thermostability improvement in newly engineered mutants were not sufficient. Various screening, clustering, decision tree and generalized rule induction models used to search for patterns of thermostability and the frequency of glutamine was the most important feature in many bioinformatics models. The importance of these methods has been discussed here.
Key words: Bioinformatics, modeling, protein, thermostability.
Abbreviations: PCR, Polymerase chain reaction; GPCR, G protein-coupled receptor;LB, Luria-Bertani ; IPTG, isopropyl-β-D-thiogalactopyranoside; FPLC, fast protein liquid chromatography; DNS, 3,5-dinitrosalicylic acid; T, threonine; P, proline; IMAC,immobilized metal affinity chromatography; GRI, generalized rule induction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; RBB, Ramazol brilliant blue
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