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

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。

A E. coil（ BL21 （ DE3）, PET28 （a） ） （ ECUST041 6, CG MCC 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 for11 h.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 300℃ 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.