类芽孢杆菌属β-葡萄糖苷酶在大肠杆菌中可溶性重组表达的优化

Optimization of soluble expression of recombinant Paenibacillus spβ- glucosidase in Escherichia coli

目的:β-葡萄糖苷酶在食品工业领域具有广泛的应用价值,但重组表达时容易形成无活性的包涵体。通过传统诱导条件优化无法完全解决包涵体积累问题,需探索新的策略。方法:从类芽孢杆菌属(Paenibacillus sp)基因组中克隆获得了β-葡萄糖苷酶基因bgl,构建到大肠杆菌表达载体p ET28a获得重组质粒p ET-bgl,转化大肠杆菌宿主细胞BL21(DE3)获得重组菌BL-ETbgl,并进行诱导条件优化。进一步通过复制起始位点替换,构建了新型大肠杆菌表达载体p ACYT-bgl,转化大肠杆菌宿主细胞BL21(DE3)后得到改进型重组菌BL-ATbgl。结果:BL-ETbgl经诱导表达后,所表达重组蛋白具有β-葡萄糖苷酶活性。经诱导条件优化后,仍有40%蛋白以包涵体存在。而BL-ATbgl经诱导表达后,可溶性的重组β-葡萄糖苷酶约占80%。自诱导培养基中β-葡萄糖苷酶产量可达2.31×106U/L。结论:通过降低质粒拷贝数、优化培养条件等手段,可以大幅度提高类芽孢杆菌β-葡萄糖苷酶在大肠杆菌中的可溶性表达水平。

Objective： β-glucosidase has great application value in food industry. However, a major problem with !5-glucosidase recombinant expression is inclusion body formation.Common methods to reduce inclusion body formation are not always effective,thus new strategy is needed.Methods.β-glucosidase encoding gene bgl was amplified by PCR from Paenibacillus sp genome, and then was constructed into pET28a. The resulting vector pET-bgl was transformed into Escherichia coil BL21 （ DE3 ） to obtain recombinant strain BL- ETbgl, and the induction conditions were optimized.By replacing the replication origin of pET-bgl,pACYT-bgl was developed and transformed into BL21 （DE3） to obtain strain BL-ATbgI.Results ：The recombinant expression product of BL- ETbgl has β- glucosidase active. However, 40% of product was expressed as inclusion body. ConverseLy, most recombinant product of BL-ATbgl was soluble,and the maximum yield of recombinant β-glucosidase could reach to 2.31 × 10^6 U/L by conditions optimized.Conclusion： By combined with reducing plasmid copy number and optimizing induction conditions, the soluble expression level of Paenibacillus sp β-glucosidase was improved significantly.