CN-121227670-B - Mutant pullulanase and uses thereof

Abstract

The invention reveals the action mechanism of key amino acids in the pullulanase PulA active framework through a structure analysis-site-directed mutation-function verification path, obtains mutant enzyme with high catalytic efficiency, and provides a new direction for the development of industrial starch processing enzyme resources. The invention successfully obtains purified mutant protein, N98D improves the enzyme activity of a wild type to 296%, T175S improves the activity of the wild type to 157%, N379D improves the activity of the wild type to 170%, and three mutants, N98D, T S and N379D, have important contribution to the improvement of the enzyme activity and have wide application prospect.

Inventors

• ZENG JUNHAO
• WANG LUSHAN
• ZENG QINGMING
• WU XIUYUN
• JI FUQUAN
• XIA GUANGMING
• GU XUDONG
• MA YING

Assignees

• 山东弥美生物科技股份有限公司

Dates

Publication Date

20260512

Application Date

20250825

Claims (10)

1. 1. A mutated pullulanase has a mutation at any one of the amino acid positions 98, 175 and 379 selected from SEQ ID No.1, wherein the 98 th amino acid is mutated to D, the 175 th amino acid is mutated to S and the 379 th amino acid is mutated to D, compared with SEQ ID No. 1.
2. 2. A gene encoding the mutated pullulanase of claim 1.
3. 3. A recombinant vector comprising the gene of claim 2.
4. 4. The recombinant vector according to claim 3, wherein the recombinant vector is a recombinant expression vector.
5. 5. A recombinant strain comprising the recombinant vector of claim 3 or 4.
6. 6. Use of a mutated pullulanase according to claim 1, or a gene according to claim 2, or a recombinant vector according to claim 3 or 4, or a recombinant strain according to claim 5, for degrading a material comprising a polysaccharide with an alpha-1, 6 glycosidic bond.
7. 7. The use according to claim 6, wherein the material of the polysaccharide containing alpha-1, 6 glycosidic bonds is starch.
8. 8. A method of hydrolyzing a material comprising an alpha-1, 6 glycosidic linkage polysaccharide, the method comprising the step of treating the material comprising an alpha-1, 6 glycosidic linkage polysaccharide with the mutant pullulanase of claim 1.
9. 9. The method of claim 8, wherein the temperature of the treatment is 50 ℃ to 80 ℃.
10. 10. The method according to claim 8 or 9, wherein the material of the polysaccharide containing alpha-1, 6 glycosidic bonds is starch.

Description

Mutant pullulanase and uses thereof Technical Field The invention belongs to the field of genetic engineering, and particularly relates to a mutant pullulanase. Background Starch is a high molecular carbohydrate and is formed by connecting alpha-D-glucopyranose through alpha-1, 4 and alpha-1, 6 glycosidic bonds, so that a composite structure of amylose and amylopectin is formed. Amylose generally represents 20% -30% of the total starch, in a linear helix structure, whereas amylopectin represents 70% -80%, forming a highly branched tree structure by frequent alpha-1, 6 glycosidic bond branching. This unique molecular structure imparts unique physicochemical properties to starch, such as a semi-crystalline structure forming starch particles with crystalline regions alternating with amorphous regions, rendering the starch cold-insoluble, gelatinized, etc. However, natural starch cannot be directly applied to industrial processing due to its physical and chemical properties, has a high gelatinization temperature, causes energy waste and destruction of heat-sensitive components due to high-temperature treatment, has poor shearing resistance, is not suitable for food with high processing strength, and has a lack of functions such as surface activity and antibacterial property, so that the natural starch cannot meet the requirements of high-end fields, and therefore, has generally had to be modified to obtain better physical and functional properties, and thus, the enzyme modification for starch deep processing technology has been rapidly developed in recent years. The pullulanase is a starch debranching enzyme, can efficiently hydrolyze alpha-1, 6-glycosidic bonds in starch molecules, and can degrade complex polysaccharide into linear oligosaccharides (such as maltotriose and glucose), so that the saccharification efficiency is remarkably improved, the pullulanase can be used for producing high-concentration maltose syrup, can also be used for treating amylopectin to generate linear glucan chains and producing resistant starch, has important application value in the starch deep processing industry, but the natural pullulanase cannot be directly applied to industrial production due to the low enzyme activity and other problems, and therefore, the pullulanase needs to be designed and modified. Disclosure of Invention In order to overcome the defects in the prior art, the invention provides a mutant pullulanase (or referred to as a pullulanase mutant) and application thereof. In one aspect, the invention provides a mutant pullulanase having a mutation in the amino acid sequence of the mutant pullulanase compared to SEQ ID No.1 at any one or any several (e.g., 2 or 3) amino acid positions selected from SEQ ID No.1, position 98, 175, 379. The amino acid site is the amino acid site from the N end of SEQ ID No. 1. In one embodiment, the amino acid at position 98 is mutated to D, the amino acid at position 175 is mutated to S, and the amino acid at position 379 is mutated to D. In the present invention, amino acid residues may be represented by single letters or three letters, such as alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R). In another aspect, the invention also provides a coding gene of the mutated pullulanase. On the other hand, the invention also provides a recombinant vector containing the coding gene of the mutated pullulanase, preferably the recombinant vector is a recombinant expression vector, preferably pET series vectors, such as pET-15b, pET-22b and pET-28a. In another aspect, the present invention also provides a recombinant strain comprising the recombinant vector described above, preferably, the recombinant strain is escherichia coli, such as escherichia coli BL21. On the other hand, the invention also provides application of the mutant pullulanase, the coding gene, the recombinant vector or the recombinant strain in degrading a material containing alpha-1, 6 glycosidic bond polysaccharide. In another aspect, the present invention also provides a method of hydrolyzing a material containing an alpha-1, 6 glycosidic linkage polysaccharide, the method comprising the step of treating the material containing an alpha-1, 6 glycosidic linkage polysaccharide with the mutated pullulanase described above. In one embodiment, the temperature of the treatment is50 ℃ to 80 ℃, preferably 60 ℃ to 70 ℃. In one embodiment, the material comprising a polysaccharide having alpha-1, 6 glycosidic linkages is starch. The invention reveals the action mechanism of key amino acids in the pullulanase PulA active framework through a structure analysis-site-directed mutation-function veri