外切型琼胶酶AgaO的高效表达及酶学性质表征

High Expression and Enzymatic Characterization of an Exo-type Agarase AgaO

根据大肠杆菌的密码子偏好性,优化外切型琼胶酶AgaO的基因并人工全合成,克隆入表达载体质粒pET-30a(+),转化大肠杆菌BL21(DE3),优化表达条件即诱导温度、诱导时间和诱导剂IPTG终浓度,纯化重组蛋白rEAgaO,并分析其酶学性质变化。结果表明,重组酶rEAgaO在16℃、IPTG终浓度0.1 mmol/L条件下诱导20 h时表达量最高;95%以上的重组蛋白质为水溶性表达,产量约为1432.7 mg/L,较优化前原始基因的产量提高了10.9倍;重组酶rEAgaO的最适反应温度为45℃,在低于40℃的温度下预处理2 h后,仍然具有90%以上的残余活性,最适pH为7.0,在pH6.0～10.0不同缓冲体系4℃处理2 h仍保留69.5%以上的活性;该酶降解琼脂糖后的最终产物经TLC分析鉴定为新琼二糖。基因密码子优化虽不改变蛋白质氨基酸序列,但可明显提高水溶性重组蛋白质的产率及酶活,对促进相关酶类的生产和应用具有借鉴意义。

Based on the codon preference of Escherichia coli,the gene of AgaO from Flammeovirga sp.MY04 was optimized and artificially synthesized.The new gene was cloned into the expression vector pET-30 a(+)and transformed into E.coli BL21(DE3).Under the optimized expression condition,the recombinant protein rEAgaO was purified and its enzymatic properties were analyzed.The results showed that the recombinant agarase rEAgaO had the highest expression levels when induced by 0.1 mmol/L IPTG at 16℃for 20 h.SDS-PAGE analysis indicated that more than 95%of the recombinant proteins were expressed in water-soluble form with a yield of approximately 1432.7 mg/L,which was increased by 10.9 folds compared with that of the original gene before optimization.The recombinant enzyme rEAgaO showed its optimal agarose-degrading activity at 45℃and pH7.0 respectively.After an incubation below 40℃for 2 h,rEAgaO retained more than 90%activity.It was stable in environments ranging from pH6.0 to 10.0 and retained more than 69.5%of its residual activity after incubation at 4℃for 2 h.Moreover,the final degradation product of agarose by rEAgaO was identified as neoagarobiose by TLC analysis.Although codon optimization did not change the amino acid sequence of protein,it could significantly improve the yield and enzyme activity of water-soluble recombinant protein,which would benefit the production and application of related enzymes.