源自嗜热菌的α-L-鼠李糖苷酶交联聚集体的制备及应用

Preparation and application of α-L-rhamnosidase cross-linked aggregates from Thermotoga petrophila DSM 13995

研究了一种制备嗜热菌Thermotoga petrophila DSM 13995来源的α-L-鼠李糖苷酶交联聚集体的方法。试验结果表明:在酶蛋白与Pluronic F127质量比为1∶8、沉淀剂硫酸铵质量浓度为75 mg/mL、戊二醛浓度为40 mmol/L时固定α-L-鼠李糖苷酶,酶活回收率可达49.45%。以硫酸铵-戊二醛(CLEA)的常规顺序制备固定化酶(PAG-R)时,酶活回收率仅为30.87%,而在Pluronic F127-硫酸铵-戊二醛(PL-AS-GL)顺序下制备PAG-R,酶活回收率达到60.90%,固定化剩余酶活力为34 U/mL。此外,研究了固定化酶PAG-R的酶学性质,结果表明PAG-R的最适温度为90℃,最适pH为5.0,与游离酶TpeRha的酶学性质相近,但在不同温度和pH条件下PAG-R的比酶活更高。PAG-R在90℃保温3 h后剩余酶活力为77.11%,而游离酶TpeRha则完全丧失活力,说明固定化酶的温度稳定性得到了较大的提升。以芦丁为底物,比较了PAG-R与TpeRha催化芦丁生成异槲皮素的产率差异。在温度80℃、pH 6.5、加酶量4 U/mL、反应时间90 min时,PAG-R能完全转化3 g/L底物,摩尔转化率为100%,产物的生成量是游离酶的2.31倍。连续反应10次后,芦丁相对转化率仍能保持在60%以上,由此可知固定化后的鼠李糖苷酶其催化效率得到了提高,且重复使用性能良好,更有利于工业化应用。

Immobilization of enzymes is an effective method for improving the stability and catalytic efficiency of enzymes.The ratio of their materials has a decisive effect on the recovery of enzyme activity.α-L-rhamnosidase from Thermotoga petrophila DSM 13995(TpeRha)has high catalytic activity,but its thermal stability is poor.Therefore,it is desired to overcome the drawbacks of TpeRha by immobilization.The cross-linked enzyme aggregate(CLEA)is a kind of technology for protein precipitation and cross-linking to form insoluble and stable immobilized enzyme.Based on the immobilized enzyme(PAG-R)prepared by the immobilization of the conventional ammonium sulfate-glutaraldehyde of CLEA,this study improved the sequence of Pluronic F127-ammonium sulfate-glutaraldehyde(PL-AS-GL).Pluronic F127 is the polyoxyethylene polyoxypropylene ether block copolymer,which can improve the stability and activity of the enzyme through the interaction between the hydrophobic segment of the polymer and the hydrophobic region on the enzyme surface.It is demonstrated that the optimal condition of the immobilization ofα-L-rhamnosidase was as follows.When the mass ratios of enzyme to Pluronic F127 were 1∶8,the mass concentration of ammonium sulfate was 75 mg/mL and the concentration of glutaraldehyde was 40 mmol/L.At this time,the enzyme activity,protein fixation rate and enzyme activity recovery of the immobilized enzyme were 34 U/mL,52.53%and 60.90%,respectively.After optimizing the ratio of materials of immobilized enzyme,the enzyme properties were improved significantly,i.e.,the residual activity was 77.11%at 90℃for 3 h,while the TpeRha of free enzyme lost its activity completely in 30 min.The residual activity was 72.41%at pH 8 and 70℃for 3 h,while under the same condition,the activity of free enzyme was only 23.35%.Simultaneously,the recovery of enzyme activity(34 U/mL)was 23.2%higher than that of the conventional method.Using rutin(3 g/L)as the substrate,the optimal converting conditions were obtained with the highest enzyme expression,which were the enzyme concentration of 4 U/mL,the induction temperature of 80℃,the pH of 6.5,and the induction time of 90 min,and the yield of products was 2.31 times higher than that of free enzymes.After 10 times of repeated reaction,the conversion of rutin remained above 60%,which showed a well improvement of the catalytic efficiency and repeated utilization of the immobilizedα-L-rhamnosidase.