High-efficiency Expression And Molecular Modification Of Bacillus Subtilis L-asparaginase

L-asparaginase?EC.3.5.1.1;ASN?catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia,and is used for the treatment of acute leukemia in children and the production of acrylamide-free food.Currently,the commercialized ASN is mainly produced by non-food grade Escherichia coli or Aspergillus Niger with long fermentation period.Therefore,constructing a food grade host with short fermentation period and high ASN yield has attracted much attention of many researchers.In addition,the low specific activity,protease degradation and poor substrate specificity of these commercialized ASN restricts their widely applications.In this study,the Bacillus subtilis WB600 was used as the host for the high efficiency expression of ASN from B.subtilis 168 and for the modification of enzyme application performance.The main results are as follows:?1?Improvement of extracellular production of L-asparaginase by optimizing the expression elementsThe encoded wap A signal peptide gene was fused to B.subtilis 168 ASN(amino acid ^20-375)gene and expressed in B.subtilis WB600 previously.Herein,the expression of ASN in B.subtilis WB600 was improved from the level of transcription and translation through screening and modification of the promoter and the 5'untranslated region?UTR?.Among the choosed 5promoters?PgroE,Pyxi E,PsecA,PylbP and P43?,P43 promoter achieved the highest extracellular ASN activity?39.25 U/m L?.Eror-prone PCR was performed on P43,and the mutant B2[A?-13?G,A?-28?G and C?-34?T]was obtained.As a result,compared with the wild type,the ASN activity and transcription of B2 was increased by 30.9%and 1.5-fold,respectively.On the basis of the B2 promoter,the best UTR HpaII was screened from the 5'UTR of 8 genes?sacB,wapA,gsiB,lepA,aprE,HpaII,spaC and amyE?,which made the ASN activity up to 65.78 U/m L.Then,a 5'UTR?AAAGGGGGGATTTACAT?was obtained by rational design using the UTR design and RBS Calculator,and the ASN activity reached 75.64U/mL,which was 47%higher than that of B2.?2?Improvement of ASN secretory expression by N-terminal truncationSDS-PAGE and mass spectrometry assays showed that there were two ASN bands with molecular weight of 43 kDa and 40 kDa in the fermentation supernatant when using the wapA signal peptide to secrete the ASN.As indicated by N terminal sequencing and signal peptide prediction,partial ASN was generated by removing 25 residues from the N-terminus,and the cutting site was a typical type two signal peptidase recognition site.When using natural signal peptide?ans Z?to secrete ASN,only one ASN band with a molecular weight of 40 kDa was observed in the fermentation supernatant,and its N terminal sequence is consistent with the sequence of the band of 40 kDa obtained by wap A signal peptide.The above results confirmed that the removed 25 amino acids were a part of the C terminal of ansZ signal peptide,which corrected the previous mistake understanding of the sequence?the removed 25 amino acids belong to the N-terminal of ASN?.To detect the role of the N-terminus in the secretion of ASN,the mutant B₂H-D₂₅ was constructed by delete the N-terminal 25-residues.As a result,the extracellular ASN activity of B₂H-D₂₅ was 1.12-fold higher than wild-type,and reached 112.15U/mL.To increase the ASN production,fed-batch fermentation was performed.Correspondingly,the ASN activities of B₂H-D₂₅ in 3 L and 50 L fermentor reached 407.6 U/mL and 872 U/mL,respectively.This is the highest yield of ASN in a food-grade host ever reported.The optimal temperature,the half-life at 65°C and specific activity of ASN were 65°C,61 min and 132 U/mg,respectively.The Michaelis constant K_m and k_cat were 5.3 mM and 54.4 s^-1,respectively.The experimental analysis shows a 74%elimination in final acrylamide content by ASN pretreated.?3?Enhancement of the catalytic efficiency of ASN by optimizing the high flexible loopAs suggested by structural simulation and sequence alignment,a continuous non-conserved region?20-ADQSKTSTTE-29?between the two active residues Thr16 and Tyr30was found at the highly flexible N-terminal loop of the B.subtilis ASN.To improve the catalytic activity,the residues 20-29 of B.subtilis ASN were replaced with the structurally equivalent residues of 6 different ASNs.The results showed that reducing flexibility of loop2 could increase the catalytic efficiency and thermal stability of ASN.Among which,L6 was constructed by replacing the continuous non conservative region of loop2 with the corresponding region Pectobacterium carotovorum ASN.Correspondingly,compared with the wild type enzyme,the activity and thermal stability of the mutant L6 were increased by 94%and 282%,respectively.To further increase the specific activity,the saturation mutagenesis were performed on the two highly flexible residues?Ala26 and Gly29?of L6.As a result,the specific activity of the mutant L6-A26N,L6-G29F and double mutant L6-A26N/G29F,with decreased flexibility,were 1.31-fold,1.86-fold and 2.44-fold higher than that of the wild type enzyme.Furthermore,compared with wild type enzyme,the half-life at 65°C for L6-A26N,L6-G29F and L6-A26N/G29F increased by 4.02-fold,5.25-fold and 6.76-fold,respectively.In contrast to wild-type enzyme,the distances between the active sites Thr16 and Tyr30 in L6 and L6-A26N/G29F were shorted and the interactions between the loop2 and substrate pocket?loop7 and loop10?were increased by 2 and 3,respectively.As indicated by the above results,the enhanced specific activity was obtained mainly by intensifing the interaction of catalytic active site and facilitating the closing of substrate pocket through modification of loop2.Based on this suppose,the interactions between loop2 and loop10 were increased by mutant the C terminal loop10.As a result,the mutant B2H-D25-A291E was obtained and its ASN activity was 46%higher than that of the wild type enzyme.?4?Improvement of the anti-degradation ability of ASN by optimizing the high flexible loopThere were two ASN bands with molecular weight of 40 kDa and 37 kDa of L6-A26N/G29F observed after fermentation for 96 h,indicating that the L6-A26N/G29F was degraded by protease.According to the N-terminal sequencing,the N-terminal first 29-residues were deleted in the small band of L6-A26N/G29F.The two bands could not be effectively separated by Ni²⁺column affinity purification,and the enzyme activity decreased with the increase of 37 kDa band.In addition,only one small band?37 kDa?and tiny ASN activity was detected from supernatant of L6-A26N/G29F when using the B.subtilis 168 with high protease activity as the host.As indicated by the structural analysis,the above 37 k Da ASN could be no catalytic activity in theory due to the active site Thr16,containing in the part of the N terminal,was removed.Based on this,the degradation of L6-A26N/G29F in the late stage of fermentation was not conducive to its production and application.To inhibit the degradation of the N terminal,saturation mutagenesis was conduced at the Phe29,which located at the left side of degradation site of the L6-A26N/G29F.As a result,the mutant L6-A26N/F29E was obtained and the 37 kDa band was remarkably decreased.To further inhibit the degradation of the N terminal,saturation mutagenesis was conduced at the adjacent region?27-TTEYK-31?of the degradation site.Finally,tiny 37 kDa band was observed in L6-A26N/F29E-T27F.Compared with L6-A26N/G29F,the half-life of L6-A26N/F29E-T27F degraded by B.subtilis 168 protease was increased by 8.49 times to 186 min.Structural analysis showed that the interactions of L6-A26N/F29E-T27F at the protease degradation site were increased and the flexibility was decreased significantly,which may increase the steric hindrance of protease degradation of ASN.?5?Elimination of the GLN activity of ASN by multiple loops modificationBased on structural simulation and sequence alignment,it was found that 3 loops,including loop2,loop5 and loop3 in B.subtilis ASN,were directly related to ASN catalyzing glutamine.By optimizing these 3 loops,the GLN activity of ASN was reduced by adjusting the closure of the substrate pocket and changing the interactions between the active sites and the substrate.Compared with B₂H-D₂₅,the L6 was constructed by the molecular modification of loop2 and its relative GLN activity was decreased by 46%,which only remained 0.84%of relative GLN activity.Then,on the basis of L6,the mutant L6-M122V was screened from the saturation mutagenesis libraries of loop5.Correspondingly,Its GLN specific activity was only0.33 U/mg and about 0.26%of the relative GLN activity was remained.Structural analysis showed that these two groups of mutants were not conducive to the closure of substrate pocket for glutamine,thereby inhibiting the GLN activity of ASN.Besides,on the basis of L6-M122V,the mutant L6-M122V-G61A/T63A was obtained by saturation mutagenesis the residues Gly61and Thr63 in loop3.Compared with B₂H-D₂₅,the GLN activity of the compound mutant L6-M122V-G61A/T63A was only 6%and the GLN specific activity was 0.127 U/mg.Notably,these mutant were not sacrificed theri ASN specific activity.