Construction Of Highly Efficient Secretory Expression Of Heat - Resistant β - Glucosidase Engineered Bacteria

The thermophile Thermotoga maritima P-glucosidase (Tm-BglA) has good thermal stability and hydrolysis characteristics. It has important application value in industrial productions such as cellulose degradation, food flavor improvement, preparation of soy isoflavone glycosides and so on. In this study, we committed to construct efficient secretion expression recombinant strain of Tm-BglA to developed its application of food processing and bio-transformation.By means of genetic engineering, the chromosomal DNA of B. subtilis was used as a template, the size of amplified fragment amylase (amyE) promoter and signal peptide sequence is 600 bp, then the fragment was inserted into the vector pET-20b(+) and the shuttle vector pHY300PLK, thus successfully constructing the three different secretion expression vectors pET-20b-Pamy, pET-20b-T7-Pamy and pHY-Pamy.This study successfully constructed pET-20b-Pamy-bglA,pET-20b-T7-Pamy-bglA, pHY-Pamy-bglA, pET-28a-thrombm-bglA and pET-20b-pelB-bglA, and transformed into Escherichia coli JM109 (DE3) respectively, the SDS-PAGE electrophoresis analysis showed that Tm-BglA band was clearly visible. The activity analysis indicated there was more than 50% of the Tm-BglA were secreted into culture supernatant after 36 h induction, excepted recombinant plasmid pET-20b-Pamy-bglA, just 0.18 U/ml was in culture medium, accounting for 12.37% of the total activity. Owing to a sequence in pET-28a(+) vector encoded 23 amino acids were added at the N-terminal of the Tm-BglA, received an efficient secretory expression recombinant strain:E. coli JM109(DE3)/ pET-28a-thrombin-bglA. After 60 h induction, about 13.11 U/ml Tm-BglA was secreted in the culture supernatant, accounting for 67.63% of the total activity. All of these illustrated that the Tm-BglA had a successful secretory expression in E. coli and the correct folding of the native protein structure, which provides a method of constructing E. coli expression system efficiently.The results of SDS-PAGE for Bacillus subtilis WB600/pHY-Pamy-bglA showed that the Tm-BglA was expressed and secreted into the extracellular successfully. After incubated 60 h, the Tm-BglA in culture supernatant reached 6.61 U/ml, about 85.06% of the total activity. This study successfully constructed a secretion expression vector in B. subtilis. Due to the characteristics of the B. subtilis, such as simple culture conditions, rapid growth, non-pathogenic, secreted variety of enzymes antibiotics, excellent fermentation foundation, this study can lay the foundation for the expression of the industrial enzymes and their applications in the field of foods.