The Catalytic Properties Of Cell Surface Display CALB On Aspergillus Niger And Its Application

Aspergillus niger,certified as a generally recognized as safe(GRAS)microorganism,has extraordinarily capacity of protein secretion and is an important strain for production of industrial enzymes.Cell surface display is a powerful tool for endowing novel functions on the host cell by displaying functional proteins on the cell surface.Among them,displaying enzymes on the cell surface showed excellent performance in biocatalysis and preparation of biochemical products.Chiral alcohol is the center of some complex natural products and an important chiral building block for producing drug intermediates.Catalytic resolution of secondary alcohols by enzymes to produce chemicals is a promising green chemistry procedure.Currently,resolution of chiral alcohol based on cell-surface display enzyme technology has some problems,such as long reaction time,low efficiency,and difficulty in industrial application and so on.Therefore,it is necessary to explore and evaluate new cell surface display enzyme technology for the resolution of chiral alcohol.The kinetic resolution of secondary alcohols was catalyzed by cell surface display CALB on A.niger for the first time.This article studies and results are as follows:First of all,to investigate surface hydrophobicity and ester hydrolysis activity of different carbon chain lengths on cell surface display CALB on A.niger.The results showed that it has a strong surface hydrophobicity,and it is speculated that it may have a stronger catalytic activity in the non-aqueous system;the optimal hydrolysis substrate is p-nitrophenol butyl ester for cell surface display CALB on A.niger.Secondly,a series of acetic esters were obtained by chemical synthesis.Moreover,methods for the detection of substrates and products for the kinetic resolution of secondary alcohols in non-aqueous systems have been established.These provide an experimental basis for the kinetic resolution of secondary alcohols catalyzed by cell surface display CALB on A.niger.Finally,cell surface display CALB on A.niger was used to kinetic resolution of secondary alcohols.Taking 2-Octanol as the model substrate,the effects of acyl donor,substrate molar ratio,the amount of cell surface display CALB on A.niger,water activity,reaction solvent and reaction temperature on the kinetic resolution ability of cell surface display CALB on A.niger were investigated.The optimized process conditions for the kinetic resolution of the secondary alcohol catalyzed by cell surface display CALB on A.niger are isopropenyl acetate as acyl donor,molar ratio of alcohol to acyl donor is 1:1,the amount of cell surface display CALB on A.niger are 50 g/L,initial water activity of cell surface display CALB on A.niger is 0.11,methylbenzene as solvent and reaction temperature is 45?.Under these conditions,to study the time course of kinetic resolution of 2-octanol catalyzed by cell surface display CALB on A.niger.the substrate 2-octanol catalyzes the reaction for 12 h using cell surface display CALB on A.niger,The substrate 2-octanol conversion rate of 47.4%,ee_p more than 99%,E value more than 600.Moreover,in the first seven batches of the continuous catalytic reaction of the process of 8 batches,the molar conversion rate can be maintained at more than 70%,with a relatively good reusability.cell surface display CALB on A.niger continues to use its optimized process conditions to expand its application to catalyze the kinetic resolution of seven other secondary alcohols.The substrate conversion rate of 2-pentanol,1-(4-chlorophenyl)-ethanol and 1-(4-bromophenyl)-ethanol reached over 47%,and the respective ee_p reached 99.6%,and the E value was more than 600;The substrate conversion rates of 3-heptanol,1-phenylethyl alcohol,and 1-(4-methylphenyl)-ethanol were 18%,39.3%and 43.7%,respectively,and the respective ee_p reached 99.9%,and E value was more than 600;The substrate conversion of 4-methoxy-?-methylbenzyl alcohol reached 48.8%,while the ee_p and E value were 97.8%and 311.3,respectively.Compared with the already reported catalysed resolution of 1-phenylethanol by cell surface display lipase,the space time yield of 1-phenylethanol catalyzed by cell surface display CALB on A.niger is more than twice as high as that reported in the literature.It has a good resolution effect of 1-phenylethanol.Overall,the kinetic rosolution of secondary alcohols catalyzed by cell surface display CALB on A.niger showed good stereoselectivity.For 2-pentanol,1-(4-chlorophenyl)-ethanol,1-(4bromophenyl)-ethanol,1-phenylethanol,1-(4-methylphenyl)-ethanol,4-Methoxy-?-methylbenzyl alcohol has a good resolution effect.This work provides a new biocatalyst alternative for economic preparation of chiral secondary alcohols.