Molecular Engineering Of Pullulanase For Enhancement Of Catalytic Efficiency And Its Extracellular Expression Regulation

Pullulanase?EC 3.2.1.41?is a starch-debranching enzyme that catalyzes the hydrolysis of a-1,6-glucosidic linkages of amylopectin,pullulan,and other oligosaccharides.Owing to the characteristics of the pullulanase,the amount of glucoamylase that used in saccharification,as well as the reaction time,will be reduced through adding pullulanase to increase the hydrolysis efficiency of starch.Therefore,pullulanase has important commercial value in the fields of food,pharmacy,energy and polymer materials.Moreover,as a tool for studying the structure of carbohydrates,pullulanase has expanded its popularity.However,most of the pullulanase showed low catalytic efficiency and stability,the increased production cost makes it difficult to apply the enzyme in industry.Pullulanase from Bacillus naganoensis has characteristics of acid and thermal stability,and the highest enzyme activity is at pH 4.5 and 60°C.In this thesis,the protein engineering of B.naganoensis pullulanase was carried out based on sequence-structrue information.The enzyme activity,specific activity,catalytic efficiency and application effect of B.naganoensis pullulanase have been improved by means of mutation strategies,including truncation of disorder amino acid sequence,evolutionary coupling saturation mutagenesis and triple codon saturation mutagenesis.In addition,the extracellular secretion efficiency of the enzyme in E.coli was increased by fusing negatively charged polypeptide to the N-terminus of the protein and overexpressing the D,D-carboxypeptidase.The main results are listed as follows:?1?Disorder-based analysis and truncation of B.naganoensis pullulanase amino acid sequence.The N-terminal or C-terminal disorder regions of the pullulanase was truncated,and eight mutants were constructed.The specific activities of the DN5 and DN106 were 410U·mg^-1 and 490 U·mg^-1,respectively,which were 1.1 times and 1.3 times that of the wild-type,respectively.Further studies on Michaelis-Menten parameters and enzymatic properties showed that the catalytic efficiency of the mutants DN5 and DN106 was improved,the k_cat/K_M values increased 2.2 times and 1.4 times that of the wild-type,respectively.The optimum reaction temperature and pH of the DN5 was 60°C,pH 4.5,which was consistent with the wild-type.The optimum reaction temperature and pH of the DN106 was 55°C,pH 5.0.The half-lives of the DN5 and DN106 were both longer than that of the wild-type by one hour.?2?Evolutionary coupling analysis of amino acid sequence from B.naganoensis pullulanase and saturation mutagenesis of covariance residue pairs.According to the theory that the amino acid sequence of a protein exhibits evolutionary coupling to maintain the structure-function relationship of the protein in the process of natural evolution,the full-length amino acid sequence of B.naganoensis pullulanase was analyzed by the evolutionary coupling analysis tool EVcouplings to detect evolutionary coupling sites of the enzyme.The residue pairs with strongest evolutionary coupling were partitioned in the spatial structure of the enzyme,and the residue pairs were selected according to the evolutionary coupling strength and the spatial position in the protein to construct mutation library.The mutant A232G/I226A with improved catalytic efficiency was screened.The specific activity increased to 550 U·mg^-1,which was 1.5 times that of the wild-type,and the k_cat/K_M value was2.3 times that of the wild-type.The optimum reaction temperature and pH of A232G/I226A was 60°C,pH 5.0.All of the mutants displayed a shorten half-life than that of the wild-type.?3?Evolutionary coupling analysis of catalytic domain,substrate binding domain CBM48 of B.naganoensis pullulanase and saturation mutagenesis of covariance residue pairs.The evolutionary coupling residue pairs in the catalytic domain,the polypeptide segment spanning the catalytic domain and CBM48,the polypeptide segment spanning the catalytic domain and C-terminus,were analyzed by EVcouplings,respectively.Moreover,the evolutionary coupling residue pairs between the catalytic domain and CBM48 were analyzed by EVcomplex.The residue pairs that showed strongest evolutionary coupling were selected according to the evolutionary coupling strength and the spatial position in the protein to conduct saturation mutagenesis and construct mutation library.The double mutant V328L/I565L exhibited highest specific activity of 870 U·mg^-1,which was 2.3 times that of the wild-type.And the k_cat/K_M value of the mutant K631V/Q597S was 5.6 times that of the wild-type.Five double mutants with the highest specific activity were combined.The quadruple mutant K631V/Q597S/V328L/I565L showed a higher specific activity of 1,000U·mg^-1,which was 2.7 times that of the wild-type.Moreover,the k_cat/K_M value of it was also the highest among the mutants,which was 6.3 times that of the wild-type.The optimum reaction pH of mutants were consistent with the wild-type,while the optimum reaction temperature of the mutants D541I/D473E,D541I/D473Q,K631V/Q597K/D541I/D473E,K631V/Q597S/D541I/D473E were reduced to 55°C.Mutations at sites D541/D473 caused a negative influence on half-life of the pullulanase,the half-life of other mutants were substantially similar to the wild-type.?4?Combinational saturation mutagenesis of active sites lining the catalytic pocket of B.naganoensis pullulanase.Aligned the homologous pullulanases that had known 3D crystal structures with the B.naganoensis pullulanase,nine potential functional sites lining the catalytic pocket were selected for mutation.Based on the multiple sequence alignment of homologous pullulanase,we chose highly conserved tryptophan,tyrosine and asparagine as building block for triple-code saturation mutagenesis.According to the experimental results,we inferred that the unknown functional sites D787 and M621 were related to enzymatic catalysis,then saturation mutagenesis was performed at the two sites and corresponding mutant libraries were constructed.The mutant D787C with improved catalytic efficiency was screened,the specific activity of it was increased to 550 U·mg^-1 which was 1.5 times that of WT,and the k_cat/K_M value of the enzyme was 1.8 times that of the wild-type.The optimum reaction temperature and pH,as well as the half-life,of the mutant D787C were consistent with the wild-type.?5?Secretion regulation of B.naganoensis pullulanase in Escherichia coli and application of the mutant.By fusing negatively charged polypeptide at the N-terminus of B.naganoensis pullulanase,e.g.the aspartic acid tag,the first 20 amino acids of the N-terminus of the cellulase catalytic domain,and super-folded green fluorescent protein,we improved the extracellular secretion of the pullulanase in E.coli.In addition,the cell wall structure of E.coli was modified by overexpressing D,D-carboxypeptidase,thereby increasing the secretion efficiency of the pullulanase in E.coli.The mutants DN5,DN106,K631V/Q597S/V328L/I565L,K631V/Q597K/D541I/D473E,and D787C,were combined and generated a mutant DN106-K631V/Q597K/D541I/D473E/D787C showing the highest specific activity of all the mutants.The mutant had the specific activity of 1,100 U·mg^-1,which was 2.9 times that of wild-type.The optimum reaction pH and temperature of the mutant was pH 5.0,55°C,and the half-life of the mutant was consistent with the wild-type.Then,We mixed the mutant DN106-K631V/Q597K/D541I/D473E/D787C,the wild-type pullulanase,and the commercial pullulanase with glucoamylase,respectively.When the reaction time reached 72 h,the DE value of the reaction liquid containing the mutant protein was close to that of the reaction liquid containing the commercial pullulanase,and the DE value was increased from 99%to 99.4%compared with adding the wild-type pullulanase.Moreover,in the middle and late stages of the saccharification reaction,the IM value was much lower with the mutant,indicating that lower level of reverse oligomerization from glucose to isomaltose was obtained by the mutant enzyme.