全细胞催化剂pelB-Xln-DT构建及其在水解三七皂苷R1中的应用

Construction of whole cell catalyst pelB⁃Xln⁃DT and its application in biotransformation of Panax notoginsenoside R1

设计带有EcoRV和XhoI酶切位点的上下游引物PCR扩增嗜热菌Dictyoglomus thermophilum DSM 3960的GH39家族的β-木糖苷酶Xln-DT编码基因,利用基因拼接技术将其重组至p ET-20b质粒中pelB信号肽基因的下游,重组质粒转化大肠杆菌表达宿主Escherichia coli BL21,构建分泌融合表达型基因工程菌pelB-Xln-DT。经异苯基-β-D-硫代半乳糖苷诱导,pelB-Xln-DT酶活为2.1 U/m L。粗酶液pelB-Xln-DT的最适温度为85℃,较原酶提高了10℃,最适p H为5.5,比原酶降低了0.5;pelB-Xln-DT在85℃下的热稳定性比原酶有了一定提高,保温1 h后酶活基本不变,保温2 h其剩余酶活为82.0%,而Xln-DT在85℃下保温1 h后其剩余酶活为70.4%;在p H 4.0~7.0的范围内,pelB-Xln-DT具有良好的p H稳定性。引入pelB信号肽后不但能够提高蛋白的可溶性表达,还能提高酶蛋白的热稳定性。以pelB-Xln-DT作为全细胞催化剂转化三七皂苷R1生成人参皂苷Rg1,研究了转化温度、转化时间、三七皂苷R1添加量、重复次数等因素对转化效率的影响。结果表明,全细胞催化剂用量1.0 U/m L,在温度37℃下,3 g/L的三七皂苷R1反应3 h后的摩尔转化率达到91.6%,重复利用10次后,转化率仍能达到46.7%,显示出pelB-Xln-DT良好的重复催化性能。

As a kind of green biocatalyst,biological enzyme has the advantages of mild reaction conditions,strong specificity,short-term and high efficiency,which is widely used in the industrial catalytic conversion and biosynthesis.In order to achieve the soluble expression of recombinant enzyme protein,the pelB signal peptide was introduced into the recombinant expression plasmid to guide the expression of enzyme protein into the periplasmic space.Primes with EcoR V and Xho I restriction enzyme sites were designed to amplify the mature peptide ofβ-xylosidase Xln-DT coding gene from D.thermophilum DSM 3960 by polymerase chain reaction(PCR).The amplified gene was spliced to the downstream of the pelB signal peptide gene and inserted into pET-20b plasmid to obtain the recombinant plasmid,and then the recombinant plasmid was transformed into the expression host E.coli BL21.The engineering strain pelB-Xln-DT was induced by isopropyl-β-D-thigalactoside,as the enzyme activity of pelB-Xln-DT was 2.1 U/mL.Then the expressed enzyme protein was analyzed by SDS-PAGE.The SDS-PAGE analysis showed that the soluble expression ofβ-xylosidase Xln-DT was greatly improved.The enzymatic properties of the originalβ-xylosidase Xln-DT and pelB-Xln-DT were compared,and the results showed as follows:the optimum temperature of crude pelB-Xln-DT was 85℃,which was 10℃higher than that of the original enzyme Xln-DT;the optimum pH value was 5.5,which was 0.5 lower than that of the original enzyme.The temperature stability of crude pelB-Xln-DT at 85℃was obviously increased.The residual enzyme activity of Xln-DT was 70.4%after holding the temperature at 85℃for 1 h,while that of pelB-Xln-DT was almost unchanged at 85℃in 1 h and the residual enzyme activity of pelB-Xln-DT was 82.0%after 2 h.In the range of pH 4.0-7.0,crude pelB-Xln-DT showed excellent pH stability.All the above results indicated that the pelB signal peptide could not only improve the soluble expression of protein,but also enhance the temperature stability of enzyme protein.In addition,using pelB-Xln-DT as a whole-cell catalyst and the Panax notoginsenoside R1 as the catalytic substrate,the biotransformation conditions of conversion temperature,conversion time,Panax notoginsenoside R1 concentration and reuse numbers were optimized by the single factor experiment.The results showed that the molar conversion of Panax notoginsenoside R1 reached 91.6%in 3 h with the addition of 3 g/L of Panax notoginsenoside R1 at 37℃,and 46.7%of conversion rate after 10 times of reuse,which suggested a good catalytic capability of pelB-Xln-DT for repeated use.