| Aspergillus niger,a versatile platform microbe in biotechnology,is certified as a generally recognized as safe(GRAS)filamentous fungus by the Food and Drug Administration(USA).Because of its extraordinarily high production level of secreted proteins,A.niger has been used as a host for large-scale production of homologous and heterologous enzymes required in food industries.Using microbial cell-surface displayed enzymes to produce chemicals is a promising green chemistry procedure,but few studies evaluated filamentous fungi cell-surface display systems.Though the major components of fungal cell walls are glycoproteins,glucan and chitin polymers,there are still considerable differences in the cell wall compositions and contents between filamentous fungi and yeasts during submerged fed-batch fermentation.The feasibility of using the homologous and heterologous cell wall proteins to functionally display enzyme proteins on the cell surface of A.niger needs to be confirmed.To illustrate the relationships among the A.niger cell surface displayed lipases,substrates and reaction systems,the details about the practical catalytic properties of the A.niger cell surface displayed lipases were characterized in esterification and transesterification reaction system.The contents and results are listed as follows.Using the homologous and heterologous anchor module proteins,new cell surface display systems were developed de novo in A.niger.The selective marker amdS from Aspergillus nidulans,the endogenous glucoamylase promoter sequence with the signal sequence and prosequence from A.niger SH-1,the alpha-glucosidase terminator region from Aspergillus oryzae were ligated sequentially with a fragment from the plasmid pUC19(the section of AMP and Ori)to construct the basal plasmid pUAGA.The coding sequence for Candida antarctica lipase B(CALB)and the coding sequence for the anchored protein CwpA from A.niger SH-1 were digested and ligated with the longer fragment of pUAGA to construct the A.niger cell surface display vector pCALB-CwpA,The coding sequence for CALB and the coding sequence for the anchored protein SED1 from Saccharomyces cerevisiae were digested and ligated with the longer fragment of pUAGA to construct the A.niger cell surface display vector pCALB-SED1.These two CALB display expression cassettes with the amdS selective marker were transformed into A.niger SH-1 respectively,to develop the CALB-displaying recombinants A.niger/CALB-CwpA and A.niger/CALB-SED1.Integration of the gene cassettes for the cellsurface display of CALB into the A.niger genome was confirmed by genomic DNA PCR.Localization of CALB on the A.niger mycelium surface was confirmed by enzymatic assay and immunofluorescence analysis using laser scanning confocal microscopy.After induction by maltose for 48 h,the hydrolytic activity of the CALB-displaying recombinants A.niger/CALB-CwpA and A.niger/CALB-SED1 reached 400 U/g of dry cell and 440.30 U/g of dry cell,respectively.The results presented here indicate that both the homologous cell wall protein CwpA and the heterologous cell wall protein SED1 from S.cerevisiae can serve as highly efficient anchor modules to display industrial enzymes in the A.niger expression system.Synthesis of fatty acid esters and characterization of the synthetic capacity of the A.niger mycelium?surface displayed CALB were conducted.In solvent-free esterification system,A.niger/CALB-CwpA whole-cell biocatalyst showed great synthetic activity and afforded high substrate mole conversions,which amounted to 87 % for ethyl hexanoate after 2 h,89 % for ethyl laurate after 2 h,and 84 % for ethyl stearate after 3 h.Adding appropriate amount of water at the start of the reaction can benefit the esterification and shortened the time to reach maximum conversion by alleviating the essential-water-depriving effect of ethanol.When compared with 3 % water(mole percentage of fatty acid),30 % water initially added led to 3.53-fold higher initial reaction rate for the synthesis of ethyl hexanoate,6.89-fold higher initial reaction rate for the synthesis of ethyl laurate,and 9.58-fold higher initial reaction rate for the synthesis of ethyl stearate.A significant lag time and a rather lower initial reaction rate were observed in the esterification of stearic acid and ethanol catalyzed by A.niger/CALB-CwpA whole-cell biocatalyst when only 3% water was added to the reaction medium at the beginning of the reaction,and water formed during the course of esterification was also found to dramatically enhance the reaction rate.Due to the lower polarity of isopropanol and the less destruction of the water layer,when the A.niger/CALB-SED1 whole-cell biocatalyst promoted the esterification of myristic acid and isopropanol,the initially added water amount ranging from 0 to 50%,does not significantly affect this esterification.The initial reaction rate did not drop sharply to present a significant lag time in the reaction process even when the esterification reaction was started in a dry state.Higher initial reaction rate was attained at 10% initially added water amount.Acetone washing helped to remove water formed during the esterification and benefitted the operational stability.After being reused for 5 batches,the A.niger/CALB-SED1 whole-cell biocatalyst showed good stability with no significant decrease in esterification activity during the production of isopropyl laurate,isopropyl myristate,and isopropyl palmitate,the relative molar conversions of fatty acid for these three isopropyl esters in batch 5 still reached 99.79,99.10,96.83%,respectively,of the values achieved in batch 1.The results presented here indicate that the CALB displaying A.niger whole-cell biocatalysts showed great organic substrate tolerance,high synthetic activity and excellent operational stability.Production of L-alpha glycerylphosphorylcholine(GPC)and characterization of the transesterification capacity of the A.niger mycelium?surface displayed CALB were conducted in non-aqueous system.A.niger/CALB-CwpA whole-cell biocatalyst can serve as an efficient alternative for the preparation of GPC by alcoholysis of phosphatidylcholine(PC).At the conditions of Isooctane 2mL,PC 12mg/mL,ethanol/PC 7:1(mol/mol),deionized water added 40%(based on the weight of PC and ethanol),A.niger/CALB-CwpA whole-cell biocatalyst 20mg/mL,60°C 200 r/min,94.48% yield of GPC was obtained after 12 h.High excess of ethanol in the reaction mixture enhances the preferential selectivity of A.niger mycelium?surface displayed CALB to sn-1 acyl groups over sn-2 acyl groups of PC,and accumulation of intermediate,lysophosphatidylcholine became significant.Adsorption of GPC onto the A.niger mycelium surface was observed,washing the recovered CALB displaying A.niger whole-cell biocatalyst with the tertiary butanol-methanol mixture(4/3,v/v)facilitated great operational stability during the repeated-batch reaction.After 9 batche reactions,the PC conversion and GPC yield at 12 h were retained at 94.45% and 89.79%,respectively.The results presented here indicate that the CALB displaying A.niger whole-cell biocatalyst possessed high transesterification activity and good operational stability. |
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