一种代谢芦荟苷的微生物筛选及其酶学性质初步研究

Microbiological screening for aloin metabolism and preliminary study of its enzymatic properties

摘要: 通过基因工程手段筛选到代谢芦荟苷的菌种——大肠杆菌(E.coli BL21(DE3),PET28(a))(ECUST0416,CGMCC NO.6114),发酵产生的酶液能够水解芦荟苷为芦荟大黄素。30℃、250r/min条件下发酵11h,产酶量可以达到148U/L发酵液。初步研究了酶液在不同温度、pH、底物浓度、PBS浓度、NaCl浓度、反应时间下的活性,从而确定酶活测定的最佳条件为:30℃、pH7.5、5mL粗酶液与5mL 40mmol/LPBS(pH7.5)混合组成反应体系、底物浓度3mg/mL、Nacl0.6%、反应时间为20min。酶反应产物经高效液相检测,纯度为98.5%,酶催化反应转化率为73%。质谱和核磁共振图谱结果表明,酶解产物为芦荟大黄素。

Abstract: A E. coli (BL21 (DE3) , PET28 (a) ) (ECUST0416, CGMCC NO. 6114) was obtained by Genetic Engineering Technology as the target bacteria which produced protease converting aloin into aloe-emodin. 148U of protease in 1L fermented culture medium could be achieved under the fermentation conditions as 30℃, 250r/min for 11h. The enzymatic activity was studied and the best activity determination method was:5mL crude enzyme, 5mL 40mmol/L Phosphate Buffer (pH7. 5) , Snbstrate concentration 3mg/mL, NaCl 0. 6%, reaction at 30℃ and pH7. 5 for 20min. The enzymatic biotransformed product was determined by HPLC with a purity of 98. 5% and identified as aloe-emodin by MS and NMR.