Molecular Modification,Heterologous Expression Study Of XynZF-2 From Aspergillus Niger XZ-3S

BackgroundXylanase[EC.3.2.1.8]is a group of enzymes that can degrade xylan into xylo-oligosaccharides and xylose.Xylanase is widely used in brewing,feed,papermaking,energy and other fields.High thermal stability of xylan is required in industrial applications,however,the thermal stability of natural mesophilic xylanase is poor,so molecular modification and thermostability improvement have become an urgent need.Objectives1 To obtain mutant enzyme with improved thermal stability.2 Obtain the optimal fermentation conditions of xylanase gene engineering bacteria at laboratory level.3 To explore the expression system difference of mutant enzymes in Bacillus subtilis and Pichia pastoris.Methods1 Bioinformatics analysis of xylanase geneXylanase gene xyn-225 was aligned and analyzed by DNAMAN 6.0 and NCBI BLAST.The spatial structure,molecular physicochemical properties and intermolecular interactions of the enzyme molecule were comprehensively analyzed by the software of The Phyre 2,DS ViewerPro 6.0,Prosite and ProScale,and the mutation sites were determined.2 Construction of mutant genes expression vectorsTarget genes were obtained by overlap extension PCR and whole gene mutation.The expression vectors of mutant genes in Pichia pastoris and Bacillus subtilis were constructed.3 Induced expression of mutant xylanaseThe recombinant mutant enzyme was successfully expressed in Pichia pastoris and Bacillus subtilis by induced fermentation.4 Analysis of enzymatic properties of mutant enzymesSDS-PAGE analysis was carried out on the induced expression products of recombinant engineering bacteria.The optimum temperature,thermal stability,optimum pH,pH stability of mutant enzyme were determined by DNS color reaction.5 Optimization of fermentation conditions of recombinant engineering bacteriaThe fermentation conditions of recombinant engineering bacteria,such as fermentation medium,induction temperature,seed age,induction time,initial pH and methanol concentration,were optimized by single factor experiment.The optimal fermentation conditions of engineering bacteria were explored by combining orthogonal experiment and response surface analysis.Results1 A disulfide bond was introduced into XynZF-2 C-terminal of xylanase.Recombinant strain GS115/pPIC9K-xynZFTA and BL21?DE3?/pET-28a-xynZFTA were successfully constructed.were successfully constructed.2 The 48 N-terminal amino acids were replaced of XynZF-2 with 34 N-terminal amino acids of Xylanase EvXyn11 successfully,and aromatic amino acids P9Y and H14F,glycosylation modification sites E42N and F17S was introduced into XynZF-2;A disulfide bonds Cys38-Cys191 was introduced in C-terminal;hydrophobic amino acids K164M,G166A,N160I were introduced in?-helix area,and V111A,G109A were introduced in core regions.The last two bases of xyn ZF-2 were substituted?AA?with?GC?and a fragment of polyclonal region sequence of the yeast expression vector pPIC9K was added into xyn ZF-2.The heterozygous enzyme Pichia pastoris expression system GS115/pPIC9K-xynZL+was successfully constructed.The optimal fermentation conditions of engineering bacteria GS115/pPIC9K-xyn ZL+at laboratory level were obtained:induction temperature 29.4?,induction time 180 h,initial pH 5.5 and methanol concentration 1.16%.3 The molecular weight of XynZFTA,XynZL+,XynZF-2 were 24.10 kDa,24.60 kDa and 24.10 kDa,respectively.SDS-PAGE electrophoresis was used to analyze the induced crude enzyme solution,and the target bands were successfully detected at the corresponding positions.4 The protoenzyme Bacillus subtilis expression system WB600/pP43NMK-xynZF-2were successfully constructed.Xylanases were successfully expressed by induction.5 The optimum temperature of XynZFTA in BL21?DE3?expression system was 10? higher than that of original enzyme?40??.The residual relative enzyme activity of the mutant enzyme was 55.36%after 5 min at 50?,which was 22.74% higher than that of the original enzyme?32.62%?.6 The optimum temperature of XynZFTA in pichia pastoris expression system was15? higher than that of the original enzyme XynZF-2,and the optimum temperature of XynZL+was 5?higher than that of XynZF-2,t_1/2^60? of XynZFTA was 9 min,t_1/2^60? of XynZL+was 10 min.The original enzyme was inactivated at 60?.The optimum temperature and pH of GS115/pPIC9K-xyn ZFTA and GS115/pPIC9K-xynZL+were 60%,50%,pH5.0 and pH6.0,respectively.7 The K_m of recombinant strains BL21?DE3?/pET-28a-xynZFTA,GS115/pPIC9K-xynZFTA and GS115/pPIC9K-xynZL+were 0.063,4.15 and 2.52,respectively.The V_m were 120?moL/mL/min?350?moL/mL/min?253?moL/mL/min,respectively.Conclusions1 The thermostability of xylanase can be improved by introducing disulfide bond in C-terminal,and multipoint mutation and modification?N-terminal substitution,glycosylation modification sites and hydrophobic amino acids in the?-helix and core regions respectively?.2 As the expression host,Pichia pastoris is more conducive to reflect the enzymatic modification characteristics than Escherichia coli and Bacillus subtilis.