Thermal Stability Modification Of Candida Antarctica Lipase B And Its Expression In Aspergillus Niger

Lipase（EC 3.1.1.3）can catalyze the hydrolysis of oil to produce fatty acid,glycerol and monoglyceride or diester.Among them,lipase B（CALB）from Candida antarctica is widely employed in enantiomer resolution,synthesis of modified esters,and biodiesel synthesis due to its high stereoselectivity,organic solvent tolerance and substrate tolerance.In the industrial application of CALB（such as catalyzing the synthesis of glycerol carbonate from glycerol and dimethyl carbonate）,higher reaction temperature contributes to mass transfer and product synthesis efficiency.As a result,developing CALB with excellent thermal stability has long been a prominent research topic at home and abroad.To improve the thermal stability of CALB,this study improved the thermal stability of CALB derived from C.antarctica LF058 through proline scanning and consensus sequence modification.Meanwhile,its expression level in A.niger AG11-PK was improved through optimizing of expression elements and fermentation conditions.The main research findings are as follows:（1）Proline scanning and consensus sequence modification to improve the thermal stability of CALBFirst,a proline scan of CALB was conducted using the Rosetta Cartesian＿ddg script to identify mutations that could reduce the folding free energy.Then,eight mutants were selected based on this scan and expressed in E.coli.The enzymatic properties of these mutants were characterized,resulting in the identification of two variants,S31P and K98P,which exhibited enhanced stability.Compared with wild-type CALB,the residual enzyme activities of S31P and K98P were increased by 4.4%and8.5%after treatment at 50℃for 5 min,respectively.To further improve the conservation of CALB sequence,a comparison was made between CALB and 48 lipase sequences obtained from different sources,leading to the identification of a consensus sequence.Finally,14 mutants were then expressed in E.coli to enhance the conservation of CALB sequence,and their enzymatic properties were determined Among them,the residual activities of mutants I87V,G114A,A148G,T159A,and A284N increased by 10%,10.1%,2.6%,10.5%,and 13.4%compared to the wild-type,respectively.Then,these above mutants were combined to obtain a highly stable mutant A284N/G114A（CALBm-E）.Compared with wild-type CALB,the half-life of CALBm-E increased by 74.7%at 50℃,reaching14.5 min.However,the specific activity(16.1 U·mg^-1)and k_cat/K_m(5932.3 m M·min^-1)of CALBm-E decreased by 36.3%and 23%,respectively.Molecular dynamics simulation indicated that the improved stability of CALBm-E was attributed to increased hydrophobic interactions and hydrogen bonding,which enhanced the overall rigidity of the mutant enzyme.（2）Optimization of expression elements to improve CALB expression in A.nigerThe CALB gene expression cassette with P_bgl（β-glucosidase promoter from A.niger）as the promoter was constructed.Using the aforementioned vector as a template,a gene fragment P_bgl-calb-hyg B containing the CALB gene expression cassette and hygromycin resistance gene（hyg B）was obtained through amplification.Converting P_bgl-calb-hyg B into A.niger AG11,a recombinant strain with randomly integrated CALB gene in its genome was obtained.After 96 h of fermentation at 30℃,the extracellular enzyme activity was 2.7 U·m L^-1.SDS-PAGE and Western Blot analysis showed that the molecular mass of CALB expressed by A.niger is 38 k Da.To improve the expression level of CALB,P_bgl was replaced by inducible(P_{gla A},P_{amy A},and P_{amm A})and constitutive(P_{mbf A},P_{pki A},and P_{gpd A})promoters respectively.Among them,the highest expression level of CALB was obtained using P_{gla A}as the promoter(3.42 U·m L^-1),an increase of 22.4%compared to before optimization.Based on the optimal promoter conditions,the N-terminus anterior portion（50-500）and all amino acid sequences of A.niger AG11 glycosylase were fused to the N-terminus of CALB.Among them,the expression level of CALB fused the first 500 amino acids of glycosylase reached 11.2 U·m L^-1,which was 226.2%higher than before optimization.Based on the CRISPR-Cas9 system,the optimized CALB expression cassette was integrated into amy A,amm A,and bgl loci in the genome of A.niger AG11-PK（a non homologous terminal repair gene kus deficient strain）.Among them,the expression level of CALB in the amy A locus reached 17.7 U·m L^-1,which was 58%higher than before optimization.（3）A284N/G114A expression and enzymatic property analysis in A.nigerBased on the CRISPR-Cas9 system,the A284N/G114A gene fused with the best expression elements was integrated into the amy A locus of AG11-PK;recombinant strain had a CALBm-A activity of 18.7 U·m L^-1 after fermentation for 96 h.To improve the expression level of CALBm-A,fermentation conditions were optimized in recombinant A.niger.Under the optimal conditions（inoculation amount of 10%,fermentation temperature of 32℃,p H 5.5）,CALBm-A enzyme activity reached 22.4 U·m L^-1,which was 19.8%higher than before optimization.On this basis,the optimal culture medium was determined as follows:corn starch 20 g·L^-1,malt extract 30 g·L^-1,corn slurry:beef extract=1:2(total addition amount 30 g·L^-1),Tween 80 0.15%.Under this medium condition,the CALBm-A enzyme activity reached 34.8 U·m L^-1,which was 55.4%higher than before optimization.Further analysis was conducted on the differences in enzymatic properties between wild-type CALB and A284N/G114A（CALBm-A）expressed in A.niger.the results showed that the half-life(t_1/2)and melting temperature（T_m）of CALBm-A reached 54.7 min and 59.6℃at 50℃,respectively,which were 91.9%and 1.8℃higher than those of wild-type CALB;however,the K_mvalue of CALBm-A（15.3 m M）increased by 28.8%,while k_cat/K_m(7810 m M·min^-1)decreased by21.4%.