利用兩步驟序列比對法提升β-Glucosidase BgxA與D-Aminoacylase DA1熱穩定性之研究

Abstract

高熱穩定性蛋白質具有讓生化製程的效率提升與抗微生物汙染的優勢，因而被廣泛應用於食品、化工、製藥等產業。針對胺基酸序列的保守程度進行酵素改造以提升該酵素熱穩定性，保守設計(Consensus Method)為提升酵素熱穩定性方法中最具效率的方法之一，然而該方法需突變大量鹼基位置且無法知曉所有能有效提升蛋白質熱穩定性的突變位置。因此本研究目的為以改進保守設計的缺點為概念，並以雙功能纖維素水解酶BgxA (β- glucosidase/xylosiase)為模型來開發一新方法，藉由第一步驟的同屬蛋白質序列比對與第二步驟的NCBI的Conserved domain序列比對，分別篩選適合變異的位置與替代胺基酸。篩選出的9個單點突變株中，Q290E與Q421L於45˚C成功提升BgxA半衰期1.5到1.7倍，根據這兩個突變株的熱穩定性結果，可歸納出新的篩選條件並將其加入原本篩選規則後，單點突變株S14R和S441V成功將另一酵素D- aminoacylase (DA1) 於60˚C下提升半衰期分別至2.1倍與2.6倍，所以本研究是可以有效提升蛋白質熱穩定性的新方法。

Nowadays, thermostable enzymes are widely utilized in foods, chemical and therapeutic drugs industry. Consensus method is one of the effective methods for improving enzymes’ thermostability by designing amino acids sequence. Nevertheless, it need mutate all residues at once and can’t tell us truly valid mutations for improving thermostability. Therefore, the new method was developed in this study using β-glucosidase/xylosidase (BgxA) as a monomeric model. Two amino acid sequence alignments of BgxA, based on its same family enzymes’ alignment and conserved domain alignment from NCBI, were used to select the suitable residues for mutations and substitutions, respectively. Upon selected 9 single mutants, Q290E and Q421L showed a 1.5 to 1.7-fold increase in half-life. It displayed that relative residues in the second alignment were all charged or hydrophobic. After adding the new screening requirement into original screening conditions, modified D-aminoacylases (DA1), S14R and S441V, showed separately the 2.1-fold and 2.6-fold increase in half-life. Thus, this new method is successful for effectively improving thermostability of protein in protein engineering.