Preparation method and application of pullulanase mutant PulB-d99-D436H

Abstract

The invention discloses a preparation method and application of a pullulanase mutant PulB-d99-D436H. The preparation method comprises the steps that 100-926 amino acid peptide fragments of bacillus naganoensis pullulanase are cut, aspartic acid (Asp) at the 436 locus of the amino acid peptide fragments is mutated into histidine (His), and then the novel pullulanase mutant is obtained through transformation. The preparation method and application of the pullulanase mutant PulB-d99-D436H have the advantages that the mutant enzyme has the excellent characteristics compared with an enzyme before mutation, wherein the enzyme expression quantity is 3.8 times of that of a parent enzyme, the optimum temperature is increased by 2.5 DEG C, and the catalytic activity is 1.36 times of that of the parent enzyme. The mutant gene is integrated into chromosomes of bacillus subtilis to construct and recombine the bacillus subtilis, and therefore pullulanase can be stably fermented and produced. The hydrolysis efficiency of starch and pullulan can be improved through the mutant enzyme.

Description

technical field [0001] The invention belongs to the field of genetic engineering and molecular enzyme engineering, in particular to a preparation method and application of a pullulanase mutant PulB-d99-D436H. Background technique [0002] Starch is a high molecular natural compound and is the most widely used industrial raw material. Natural starch raw material is a mixture of amylose and amylopectin, of which amylopectin is the main one, accounting for about 80%. Amylopectin is a highly branched macromolecular polysaccharide composed of many short chains. It is usually connected by α-D-glucose residues through α-1,4-glycosidic bonds to form a straight chain, and on the straight chain it can be connected by α -1,6-glycosidic bonds form side chains, and another branched short chain will appear on the side chain to form dendrites. Each short chain contains about 20-30 α-D-glucose units. The reduction of the short chain The ends are also connected by α-1,6-glycosidic bonds, a...

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Problems solved by technology

[0007] The patents of Jiangnan University (Application No. 201210256804.9 and Application No. 201310471021.7) disclose a pullulanase mutant and its preparation method. The pullulanase related to their two patents is derived from Bacillus demycetes, which is the opposite of the parent The active amino acid of the enzyme is replaced, and the mutant enzymes with different performances and improvements have been obtained, but the strain sources of these pullulanase genes are different from those described in this patent, and the DNA sequence and amino acid sequence are very different. The molecular weight is different, the enzymatic properties and activity are all very different, and there is no mutant with absolute advantage, and there is no report on its industrial production and application. The enzymatic properties are different, and the most suitable fields of application are also different, so It is also completely different from the mutants described in this patent

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