Cloning,Expression And Characterization Of ?-Glucosidase From Aspergillus Niger And Its Enzymatic Conversion Of Geniposide

Genipin is regarded as a natural biological crosslinking agent and has a bright application prospect.In nature,Genipin is rare and stays in the form of its precursor-Geniposide.Currently the ways of producing Genipin is direct extraction from the plant,chemical catalysis by acid,biotransformation of microbial cells,bioconversion by ?-glucosidase.among these four methods.Bioconversion by ?-glucosidase has a lot of advantage,such as higher conversion efficiency,less by-product.however ?-glucosidase has a high cost which imposes restrictions on its application.In our laboratory we have screened a Aspergillus niger strain named B2 which has a strong ability of hydrolyzing glycosides.In the experiment the gene bgll encoding the mature protein of ?-glucosidase obtained from Aspergillus niger B2 was cloned,and then expressed in Pichia pastoris to investigate the characterizations of the recombinant ?-glucosidase,the results are as follows:(1)Clone of bgll gene:The RNA was extracted from the fermented mycelia of Aspergillus niger B2,we cloned two bgll genes,bgll A and bgllB,using the RT-PCR technology.The gene bgllB had an intron in it.(2)Expression of the bgll A and bgllB in Pichia pastoris:The gene bgll A and bgllB was inserted into the expression vector of pPICZ?A and transformed into Pichia pastoris GS115 using electrotransformation.The positive transformants of NO1 strain which integrated bgll A into the genome and NO2 strain which integrates bgllB were screened.Two strains were induced with methanol in 30?,the concentration of methanol was 1%.However,the enzyme activity of both fermented supernatant was low.The cells were broken to measure enzyme activity of intracellular enzyme,in which no activity was found.Finally obvious activity was found in yeast cells and cell debris by mechanical disruption.So we got a conclusion that both recombinant enzymes are bound to the cell wall.(3)The characterizations analysis recombinant BGL1A and BGL1B by concentration:BGL1A had a optimum pH of 4.2,the optimum temperature was 50?,the residual activity was 46.93%after 50? 60min incubation compared with the activity of the enzyme with Omin incubation,the enzyme was stable with pH 3-6.BGL1B had an optimum pH of 4.4,the optimum temperature was 60?,the residual activity was 63.03%after 50? 60min incubation compared with the activity of the enzyme with 0min incubation,the enzyme was stable with pH 3-6.(4)Hydrolysis of Geniposide by fermented supernatant and cell suspensions:hydrolyzate was analysis by TLC:the fermented supernatant showed a weak activity towards geniposide.However geniposide was completely hydrolyzed by cell suspensions of NO 2 strain which integrates bgllB.