CN113862241B

CN113862241B - Chitosanase Csncv, mutant CsnB thereof and application of mutant CsnB

Info

Publication number: CN113862241B
Application number: CN202111459576.0A
Authority: CN
Inventors: 王建荣; 祝木金; 王平; 陈微; 钟斌; 余思; 曹革
Original assignee: Shenzhen Raink Plant Nutrition Technology Co ltd
Current assignee: Shenzhen Raink Plant Nutrition Technology Co ltd
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-03-18
Anticipated expiration: 2041-12-02
Also published as: CN113862241A

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a chitosanase Csnv, a mutant CsnB thereof and application thereof, wherein the mutant CsnB is obtained by mutating the chitosanase Csnv after codon optimization, the amino acid sequence and the nucleotide sequence of the optimized chitosanase Csnv are shown in SEQ ID NO.16-17, the amino acid sequence and the nucleotide sequence of the mutant CsnB are shown in SEQ ID NO.1-14, the thermal stability of the chitosanase is improved by constructing efficient expression engineering bacteria of CsnB, and chitosan is hydrolyzed to obtain a chitosan oligosaccharide product containing chitobiose, chitotriose, chitotetraose, chitopentaose and chitohexaose; the chitosanase Csncv and the mutant CsnB thereof obtained by the invention can be applied to the preparation of chitosan oligosaccharide and the industries of medicine, agriculture, food and the like, and lay a foundation for the industrialized application of the chitosanase.

Description

Chitosanase Csncv, mutant CsnB thereof and application of mutant CsnB

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to chitosanase Csncv, a mutant CsnB thereof and application thereof.

Background

The chitosan oligosaccharide as a biological stimulator plays an important role in green agricultural planting, and research and practical application show that the chitosan oligosaccharide can increase crop yield, improve the quality of agricultural products, improve the disease resistance of crops, activate the innate immune system of plants, improve the stress resistance of crops and the like. At present, the preparation of chitosan oligosaccharide is mainly divided into a chemical method and an enzymatic method. The chemical method has the defects of environmental pollution caused by waste water, incomplete product structure and the like, and limits the application of the chemical method in the field of chitosan oligosaccharide preparation. Compared with a chemical method, the process for preparing the chitosan oligosaccharide by the enzyme method has the advantages of mild reaction conditions, complete product structure, easy process control, no pollution to the environment and the like, so the enzyme method is generally known as the mainstream production and processing technology of the chitosan oligosaccharide, but the physiological activity of the chitosan oligosaccharide product prepared by the existing enzymolysis technology is lower, the enzymolysis reaction time needs to be strictly controlled, the enzymolysis product with higher proportion of the high-polymerization-degree oligosaccharide can be obtained, and the requirements on the production technology and equipment control are higher.

In order to better utilize chitosanase, develop and modify it, at present, chitosanase is divided into 5 family, 7 family, 8 family, 46 family, 75 family, 80 family and so on according to gene sequence similarity, the research on 46 family chitosanase is the most, including the research on its three-dimensional structure and catalytic mechanism, such as Wang Yani to the complex of two 46 family chitosanaseThe structure of the compound is analyzed, a chitosan mutant is obtained, and the proportional content of the chitosan oligosaccharide product obtained by hydrolyzing chitosan with higher polymerization degree is improved (the structural analysis and the catalytic mechanism research of Wang Yani, GH46 family chitosanase); chinese patent 113493781A discloses chitosanase CsnH, the chitosanase yield is 665.3U/mL, the optimum reaction temperature is 70 ℃, the heat-resistant stability is better, and the hydrolysate is chitobiose; chinese patent 105602921 discloses a chitosanase EAG1, the product hydrolyzed at 45 ℃ for 4 hours is chitosan oligosaccharide 2-8 sugar, and the hydrolysis rate is correspondingly improved; chinese patent 111235131 discloses a chitosanase, its relative enzyme activity is 898.6U/mg at pH 10 and 50 deg.C, the hydrolysate is GlcN- (GlcN)₄(ii) a It can be seen that chitosanase is highly specific and has different application ranges, on the other hand, the most reported chitosanase from the 46-family published is bacillus chitosanase and streptomyces chitosanase, while chitosanase from other sources is relatively less studied. Therefore, targeted site-directed mutagenesis is carried out based on the structural information to obtain the chitosanase with high catalytic activity and wide adaptability, and the engineering bacteria for high yield of the chitosanase is constructed by utilizing genetic engineering, thereby having important significance for exploring and enriching the chitosanase and industrial production.

Disclosure of Invention

In order to solve the existing technical problems, the purple bacillus CV1192 chitosanase is firstly expressed in a recombinant mode, and analysis and measurement show that the chitosanase has industrial application value, but the heat stability of the chitosanase is poor, and the chitosanase is easy to denature and inactivate when the treatment temperature is higher than 55 ℃, so that the industrial application of the chitosanase is limited.

The invention aims to provide codon-optimized purple bacillus chitosanase Csncv and a mutant CsnB thereof;

the mutant CsnB is obtained by mutation of optimized chitosanase Csncv and comprises V105Q, G134N, G240I, G240I/G134N, G240I/V105Q, G134N/V105Q or G240I/G134N/V105Q, the amino acid sequences of the mutants are respectively shown as SEQ ID NO. 1-7, and the gene sequences of the mutants are respectively shown as SEQ ID NO. 8-14;

SEQ ID NO.1

V105Q amino acid sequence

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLQNKPEQDSLEWTKFYGYCENIGDKRGYTIGIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIGSFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK-

SEQ ID NO.2

G134N amino acid sequence

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLVNKPEQDSLEWTKFYGYCENIGDKRGYTINIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIGSFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK

SEQ ID NO.3

G240I amino acid sequence

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLVNKPEQDSLEWTKFYGYCENIGDKRGYTIGIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIISFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK

SEQ ID NO.4

G240I/G134N amino acid sequence

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLVNKPEQDSLEWTKFYGYCENIGDKRGYTINIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIISFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK-

SEQ ID NO.5

G240I/V105Q amino acid sequence

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLQNKPEQDSLEWTKFYGYCENIGDKRGYTIGIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIISFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK-

SEQ ID NO.6

G134N/V105Q amino acid sequence

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLQNKPEQDSLEWTKFYGYCENIGDKRGYTINIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIGSFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK-

SEQ ID NO.7

G240I/G134N/V105Q amino acid sequence

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLQNKPEQDSLEWTKFYGYCENIGDKRGYTINIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIISFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK

SEQ ID NO.8

V105Q gene sequence

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGCAGAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCGGTATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCGGTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

SEQ ID NO.9

G134N gene sequence

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGGTCAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCAACATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCGGTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

SEQ ID NO.10

G240I gene sequence

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGGTCAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCGGTATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCATTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

SEQ ID NO.11

G240I/G134N gene sequence

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGGTCAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCAACATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCATTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

SEQ ID NO.12

G240I/V105Q gene sequence

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGCAGAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCGGTATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCATTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

SEQ ID NO.13

G134N/V105Q gene sequence

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGCAGAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCAACATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCGGTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

SEQ ID NO.14

G240I/G134N/V105Q gene sequence

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGCAGAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCAACATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCATTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

The encoding chitosanase Csncv protogenecsncvThe nucleotide sequence of (A) is shown as SEQ ID NO. 15;

SEQ ID NO.15

CAGGGCTCGACGGCCGGCAGCCCCGAAAGACAAGCCGTTCGCCTGCCCGACTGCGCCGCCGCGTGGAGCCCCGCCTCGCCCTATCAAGCCGGCAGCGTCGCCAGCCGCGGCGGCGTCAACTACACCGCGGCATTCTGGACCCAGGGCAATCCTCCGGAACAGGGTCAGGCCTGGCAGGCCGGCAAGCGCTGTCGTCCGGCCGCGCAGGCCAAGGCCGCCGACCACGACGCCAACTTTCCGCCCGCCACGCTGAAATTCCTGAAAGCCAACACCGGCCTGGACGGCGAGCAGTGGGACAACATCATGAAGCTGGTCAACAAGCCGGAGCAGGATTCGCTGGAGTGGACCAAGTTCTACGGCTACTGCGAGAACATCGGCGACAAGCGCGGATACACGATAGGCATCTTCGGCGCCACCACCGGCGGCCCGAACGACGAGGGTCCGGACGGTCCGACGCTGTTCAAGGAGTTCGACGCTTCCAGCGGCGCGTCCAATCCGTCCATCACCGGCGGACTGGCCCGCGCCGGCGTGCACGGCAGCATGCAGGGCAAGATCCTGAAGATCAGCGACAGCGCCAAGGTGTTCTGCGACAAGATCGGCAACCTGCAGAACAATCCCGCCTGGCGCGACGCGATGTGGAACACCTTCTACAAGGTATACATCCAATACAGCGTGCAGCAGGCCCGCCAGCGCGGCTTCTCCAGCGCGCTGACCATAGGCTCCTTCGTCGACACCGCGCTGAACCAGGGCGCAACCGGCGATTCCGGCACGCTGCAGGGCCTGCTGTCCCGCTCCGGCAACAGCGGCGACGAGAAAACCTTCATGACCGCCTTCTACGCGCAGCGCAGCAAGATCGTCGACACCAACGACTACAATCAGCCGCCCAACGGCAAAAACCGCGTCAAGCAATGGAGCACGCTGTTGAACATGGGCGAGACCGACTTGAAGAATGCCGACGCCGCGGTGCAGAAAGTCACCAACTGGGAAATGAAGTAA

the amino acid sequence of the optimized chitosanase Csncv is shown in SEQ ID NO. 16;

SEQ ID NO.16

QGSTAGSPERQAVRLPDCAAAWSPASPYQAGSVASRGGVNYTAAFWTQGNPPEQGQAWQAGKRCRPAAQAKAADHDANFPPATLKFLKANTGLDGEQWDNIMKLVNKPEQDSLEWTKFYGYCENIGDKRGYTIGIFGATTGGPNDEGPDGPTLFKEFDASSGASNPSITGGLARAGVHGSMQGKILKISDSAKVFCDKIGNLQNNPAWRDAMWNTFYKVYIQYSVQQARQRGFSSALTIGSFVDTALNQGATGDSGTLQGLLSRSGNSGDEKTFMTAFYAQRSKIVDTNDYNQPPNGKNRVKQWSTLLNMGETDLKNADAAVQKVTNWEMK

the optimized encoding chitosanase Csncv genecsncvThe nucleotide sequence of (A) is shown as SEQ ID NO. 17;

SEQ ID NO.17

CAAGGTTCTACTGCTGGTTCTCCAGAGAGACAGGCTGTTAGATTGCCAGATTGTGCTGCTGCTTGGTCACCAGCTTCTCCATATCAAGCTGGTTCTGTTGCTTCCAGAGGTGGTGTTAACTACACTGCTGCTTTCTGGACTCAAGGTAACCCACCAGAACAAGGTCAAGCTTGGCAAGCTGGTAAGAGATGTAGACCAGCTGCTCAAGCTAAGGCTGCTGATCACGATGCTAACTTTCCACCAGCTACCTTGAAGTTCTTGAAGGCCAACACTGGTTTGGACGGTGAACAGTGGGACAACATCATGAAGTTGGTCAACAAGCCAGAGCAGGACTCTTTGGAGTGGACTAAGTTCTACGGTTACTGCGAGAACATCGGTGACAAGAGAGGTTACACCATCGGTATCTTCGGTGCTACTACTGGTGGTCCAAACGATGAAGGTCCAGATGGTCCAACCTTGTTCAAAGAGTTCGACGCTTCTTCCGGTGCTTCTAACCCATCTATTACTGGTGGTTTGGCTAGAGCTGGTGTTCACGGTTCTATGCAGGGTAAGATTCTGAAGATTTCCGACTCCGCCAAGGTGTTCTGTGACAAGATTGGTAATTTGCAGAACAACCCAGCTTGGAGAGATGCTATGTGGAACACCTTCTACAAGGTTTACATCCAGTACTCCGTTCAGCAGGCTAGACAAAGAGGTTTCTCTTCCGCTTTGACCATCGGTTCCTTCGTTGACACTGCTTTGAACCAGGGTGCTACTGGTGATTCTGGTACTCTGCAAGGTTTGTTGTCCAGATCCGGTAACTCTGGTGACGAAAAGACTTTCATGACTGCCTTCTACGCCCAGAGATCCAAGATTGTCGACACCAACGACTACAACCAGCCACCAAACGGTAAGAACAGAGTTAAGCAGTGGTCCACCTTGCTGAACATGGGTGAAACTGACTTGAAGAACGCTGACGCTGCTGTTCAGAAGGTTACCAACTGGGAGATGAAGTAA

the invention also aims to provide a method for efficiently expressing the purple bacillus chitosanase Csncv and the mutant CsnB thereof by using pichia pastoris;

the gene of the inventioncsncvThe recombinant expression vector is pPICZαA-csncv；

The gene of the inventioncsnbThe recombinant expression vector is pPICZαA-csnb；

The gene of the inventioncsncvOr a genecsnbThe recombinant strain of (1); preferably, the host bacteria of the recombinant strain are pichia pastoris engineering bacteria;

the invention further aims to provide the enzymological characteristics and the hydrolysate composition of the chitosanase Csncv and the mutant CsnB thereof.

The invention is realized by the following technical scheme:

(1) according to the codon preference of pichia pastoris, the purple bacillus CV1192 chitosan enzyme gene of partial sequence of the signal peptide is optimized and removed, thereby obtaining the codon optimized sequencecsncv；

(2) Construction of expression vector pPICZαA-csncvAnd transferring into pichia pastoris X33, and obtaining purple bacillus CV1192 chitosanase Csncv through fermentation expression;

(3) analyzing and determining the enzymatic characteristics of the chitosanase Csncv;

(4) obtaining a mutant CsnB with improved thermal stability by optimizing b-factor of the chitosanase Csncv, and determining the enzymatic characteristics of the mutant CsnB;

(5) efficiently preparing chitosanase Csncv and a mutant CsnB thereof through fermentation expression;

(6) and (3) hydrolyzing chitosan by using the chitosan enzyme Csncv and the mutant CsnB to prepare chitosan oligosaccharide.

The materials used in the technical scheme of the invention are described as follows:

strain and carrier: the Escherichia coli strain Top10 is conventionally preserved in laboratory, and contains Pichia pastoris X33 and pPICZαAll a were purchased from Invitrogen.

Enzyme and kit: q5 high fidelity Taq enzyme MIX was purchased from NEB; plasmid extraction, gel purification kit purchased from Tiangen Biotechnology (Beijing) Ltd; restriction enzymes were purchased from daisies technologies (beijing) ltd; zeocin was purchased from Invitrogen; the Ni-IDA protein purification kit was purchased from Shanghai Bioengineering Co., Ltd.

The Escherichia coli culture medium is LB: 1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, pH 7.0;

LBZ is LB culture medium added with 25 mug/mL Zeocin (bleomycin);

the yeast culture medium is YPD: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose). The yeast screening medium is YPDZ (YPD +300mg/L zeocin);

yeast induction medium BMGY: 1% (w/V) yeast extract, 2% (w/V) peptone, 1.34% (w/V) YNB, 0.00004% (w/V) Biotin, 1% glycerol (V/V);

the YNB is a Yeast Nitrogen source Base (Yeast Nitrogen Base);

the Biotin is Biotin;

the reagents used for the determination of the activity of chitosan include the following:

sodium acetate solution: 0.2 mol/L, pH = 5.0;

chitosan substrate: 0.5% (w/v) chitosan was dissolved in sodium acetate solution;

DNS reagent: 6.3 per thousand (w/v) of 3, 5-dinitrosalicylic acid, 18.2 percent (w/v) of sodium potassium tartrate tetrahydrate, 5 per thousand (w/v) of phenol and 5 per thousand (w/v) of anhydrous sodium sulfite.

The method for measuring the activity of the chitosan enzyme comprises the following steps:

firstly, preheating a chitosan substrate and an enzyme solution at 50 ℃; mixing the preheated enzyme solution with a chitosan substrate, reacting at 50 ℃, adding a DNS reagent to terminate the reaction, carrying out color development in a boiling water bath, cooling, centrifuging, taking the supernatant, and measuring the light absorption value at 540 nm; the definition of the enzyme activity unit is as follows: the amount of enzyme used to produce 1. mu. mol of reducing sugar per minute is defined as one activity unit.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention obtains the chitosan enzyme gene from purple bacillus by codon optimizationcsncvCompared with the original gene, the gene has the advantages of high gene expression,csncvthe GC content of the chitosan enzyme gene is optimized from 64.4 percent to 48.7 percent, the expression adaptation index is increased from 51 percent to 78 percent, and the chitosan enzyme genecsncvThe sequence stability and codon adaptability of the recombinant human chitosanase are obviously improved, high-efficiency expression can be performed in a pichia pastoris expression strain, and the production cost of the chitosanase Csncv is reduced.

(2) The chitosanase Csncv and the mutant CsnB provided by the invention are derived from purple bacilli and belong to bacterial chitosanase.

(3) According to the constructed recombinant engineering bacterium C1 containing the chitosanase Csncv, the fermentation enzyme activity can reach 12U/mL, the chitosan enzyme activity is gradually improved along with the increase of the induction time in the fermentation process, when the induction culture is carried out for 192 hours, the C1 fermentation enzyme activity reaches 1652U/mL, compared with C1, the highest fermentation enzyme activity of the recombinant engineering bacterium C3 containing the triple mutant CsnB appears in the induction process for 168 hours, the enzyme activity can reach 1765U/mL, and the obtained chitosanase mutant has higher enzyme activity and has good application prospect and industrial value.

(4) According to the invention, single mutation, double mutation and triple mutation are carried out on the chitosanase Csncv, so that the thermal stability of the obtained mutant is improved, particularly, the residual enzyme activity of the triple mutant CsnB after 30 minutes of heat preservation at 60 ℃ is respectively 19.5% and 11.2%, and compared with the chitosanase Csncv, the residual enzyme activity is improved by 140.7%, therefore, the thermal stability of the chitosanase Csncv is improved by optimizing the b-factor temperature factor, and the industrial application of the chitosanase Csncv is enlarged.

(5) The enzymolysis efficiency of the mutant CsnB is higher than that of the chitosan Csnv, and the mutant CsnB has directional hydrolyzability on chitosan hydrolysate through enzymolysis time, so that the chitosan Csnv and the mutant CsnB can be used for preparing chitosan oligosaccharides with different molecular weights, have great application potential in the field of enzymolysis preparation of the chitosan oligosaccharides, can be widely applied to industries such as medicine, agriculture and food, and lay a foundation for industrial application of the purple bacillus CV1192 chitosan.

Drawings

FIG. 1 is a temperature profile of chitosanase Csncv.

FIG. 2 temperature profile of mutant CsnB.

FIG. 3 shows the high-density fermentation curve diagram of chitosanase Csncv recombinant engineering bacteria (C1) and three-mutant CsnB recombinant engineering bacteria (C3) thereof.

FIG. 4 shows a thin layer chromatogram of chitosanase Csncv (A) and its three mutants CsnB (B) for enzymolysis of chitosan.

FIG. 5 is a high performance liquid chromatogram of a chitosan enzymatic hydrolysate of the chitosanase triple mutant CsnB.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the description is intended to be exemplary only, and is not intended to limit the scope of the present invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.

The invention is further described in the following examples, which are not intended to limit the scope of the invention.

Example 1 codon optimization of Violobacterium CV1192 chitosanase

The gene sequence of the purple bacillus CV1192 chitosanase (gene accession number: CP 024028.1) is obtained by analyzing NCBI database, the total length of the purple bacillus CV1192 chitosanase gene is 1083bp, and 360 amino acids are coded. Through the analysis of an online signal peptide software SignalP-5.0 Server, the first 29 amino acids of the violaceous bacillus CV1192 chitosanase are found to be the signal peptide sequence thereof. Since pichia is used as a recombinant expression host in this patent, the gene sequence of the violobacterium CV1192 chitosanase needs to be optimized according to the codon preference of pichia. In addition, since the signal peptide used in recombinant expression is an alpha signal peptide, the coding sequence of the signal peptide part of the violobacterium CV1192 chitosanase needs to be removed in gene optimization. Finally obtaining the chitosan enzyme gene by codon optimizationcsncvCompared with the original gene, the gene has the advantages of high gene expression,csncvthe GC content of the gene is optimized from 64.4% to 48.7%, the expression adaptive index is increased from 51% to 78%, 164 bases are optimized in total, and the sequence alignment before and after optimization is shown as SEQ ID NO.15 and SEQ ID NO. 17.

Example 2 construction and screening of recombinant engineering bacteria

Expression vector pPICZ alpha A-csncvThe construction of (A) is as follows:

(1) chitosan gene optimized in example 1csncvAs a template, by means of a primer (c 1-fw: 5' -AGTCGAATTCCAAGGTTCTACTGCTGGTTCT-3' and c 1-rev: 5' -ATCTCTAGACTTCATCTCCCAGTTGGT-3') performing PCR amplification, and purifying the amplification product by agarose gel;

(2) respectively carrying out enzyme digestion on the purified amplification product and the expression vector pPICZ alpha A by using restriction endonucleases EcoRI and XbaI;

(3) recovering the amplified product and the expression vector pPICZ alpha A which are subjected to enzyme digestion, and carrying out ligation reaction on the amplified product and the expression vector pPICZ alpha A;

(4) transforming the ligation reaction product into escherichia coli Top10, and finally obtaining an expression vector pPICZ through screening, verification and sequencing identification of recombinant transformantsαA-csncv。

The construction of the recombinant yeast engineering bacteria is as follows:

(1) the expression vector pPICZ alpha A-csncvLinearization is carried out by using restriction endonuclease SacI, and then the strain is transferred into pichia pastoris X33, and transformants are evenly coated on YPDZ plates (the concentration of bleomycin is 300 mg/L);

(2) inoculating the transformants into 50ml centrifuge tubes containing 5ml BMGY in the form of single colonies respectively, and culturing at 200rpm and 30 ℃;

(3) in the culture process, 0.5% (v/v) methanol is added every 24 hours for induction, and activity determination is carried out after 48 hours of induction;

(4) and (3) activity determination: firstly, preheating a chitosan substrate and an enzyme solution at 50 ℃; adding preheated 50 mu L of enzyme solution into a 1.5mL centrifuge tube, then adding 350 mu L of chitosan substrate, reacting at 50 ℃ for 10 minutes, adding 600 mu L of DNS reagent to stop the reaction, carrying out color development in a boiling water bath at 100 ℃ for 5 minutes, cooling, centrifuging, taking supernatant, and measuring the light absorption value at 540 nm. The definition of the enzyme activity unit is as follows: the amount of enzyme used to produce 1. mu. mol of reducing sugar per minute is defined as one activity unit;

(5) through screening 96 recombinant transformants, a recombinant engineering bacterium (named as C1) with dominant enzyme activity is finally obtained, and the fermentation enzyme activity of the recombinant engineering bacterium is 12U/ml.

Example 3 chitosanase Csncv temperature profiling

The recombinant engineered bacterium C1 obtained in example 2 was inoculated into a 500ml shake flask containing 100ml BMGY medium, induction-cultured for 120 hours, centrifuged to collect the supernatant enzyme solution, ultrafiltered through a 10kDa ultrafiltration tube, the supernatant enzyme solution was concentrated, the ultrafiltered enzyme solution was purified with a Ni-IDA protein purification kit, and the purified chitosanase Csncv was subjected to temperature property measurement.

The chitosanase Csncv temperature characteristic determination steps are as follows:

under the condition of pH5.5, measuring the enzyme activity of Csncv at different temperatures of 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃, and calculating the relative enzyme activity at other temperatures by taking the highest enzyme activity as 100%; determining residual enzyme activity after water bath heat treatment for 30 minutes at different temperatures of 45-65 ℃, calculating relative residual enzyme activity at other temperatures by taking the enzyme activity of a sample without heat treatment as 100%, wherein the temperature characteristic of the chitosanase Csncv is shown in figure 1;

as can be seen from FIG. 1, the optimum reaction temperature of Csncv is 50 ℃, and the relative enzyme activity is more than 60% in the range of 45 ℃ to 60 ℃; as shown in the figure 1, the chitosanase Csncv has good stability in the range of 45-55 ℃, the residual enzyme activity is more than 75% after heat treatment for 30 minutes, when the treatment temperature is more than 55 ℃, the residual enzyme activity is rapidly reduced, and after heat treatment for 30 minutes at 60-65 ℃, the residual enzyme activity is only 8.1% and 5.1% respectively.

Example 4 optimization of b-factor temperature factor to improve Chitosan enzyme Csncv thermostability

Obtaining a chitosanase Cscncv three-dimensional MODEL through online modeling software SWISS-MODEL (https:// swisssmall.expasy.org /), predicting and analyzing B-factor values of different amino acids of the chitosanase Cscncv through bioinformation software B-FITTER, finding that the value of glycine G at position 240 is 0.96 at most through analyzing a prediction result, and finding that glycine G at position 134 (0.95) and valine V at position 105 (0.94) are positioned next, and selecting the three amino acid positions (Gly 240, Gly134 and Val 105) as experimental targets to carry out mutation screening;

the construction and screening methods of the different mutants are as follows:

(1) expression vector pPICZ alpha A-csncvPerforming PCR amplification by using degenerate primers (primers 240-fw: TCCGCTTTGACCATCNNNTCCTTCGTTGACAC and 240-rev: GTGTCAACGAAGGANNNGATGGTCAAAGCGGA; primers for Gly134 are Gly 134-fw: AGAGGTTACACCATCNNNATCTTCGGTGCTA and Gly 134-rev: TAGCACCGAAGATNNNGATGGTGTAACCTCT; and primers for Val105 are Val 105-fw: AACATCATGAAGTTGNNNAACAAGCCAGAGCA and Val 105-rev: TGCTCTGGCTTGTTNNNCAACTTCATGATGTT) as a template;

(2) carrying out enzymolysis on the amplified product by using restriction enzyme DpnI so as to decompose the template pPICZαA-csncv；

(3) Transferring the product after enzymolysis into escherichia coli Top10 by a heat shock method;

(4) screening of large intestine transformant adopts bacteria liquid PCR method, firstly picking recombinant transformant into LB culture medium in form of single colony, culturing 4 hours at 200rpm and 37 ℃, taking 2 microliter bacteria liquid as template, carrying out PCR amplification by using primer (5 'AOX-fw: GACTGGTTCCAATTGACAAGC and 3' AOX-rev: GGCACCTGGCATTCTGACATCC), sequencing the product which is verified to be correct, and determining mutation site according to sequencing result.

The constructed mutant expression vector is transferred into pichia pastoris X33, and the culture process of the recombinant pichia pastoris is consistent with that of the embodiment 2. And (3) carrying out thermal stability determination on fermentation enzyme liquid of different mutants so as to judge whether the mutant is an effective mutant, wherein the thermal stability testing method comprises the following steps: and (2) carrying out residual enzyme activity determination on the diluted enzyme solution after 30-minute water bath heat preservation at 60 ℃, taking the non-mutated chitosanase Csncv as a control group, and screening each site to respectively obtain 1 optimal mutant, namely G240I, G134N and V105Q, wherein the residual enzyme activities of the three mutants after 30-minute water bath heat preservation at 60 ℃ are respectively 13.2%, 11.3% and 12.2%, and the residual enzyme activity of the control group is 8.1%, so that the thermal stability of the mutants G240I, G134N and V105Q obtained after the chitosanase Csncv mutation is improved.

Example 5 combinatorial mutations further improve chitosanase Csncv thermostability

Based on the 3 efficient mutants obtained in example 4, G240I, G134N and V105Q, combinatorial mutations were performed to further improve thermostability. Since the effect of improving the thermostability of mutant G240I was most remarkable in example 4, the mutant was constructed by using it as a template, the construction process of the combined mutant was substantially the same as that of the mutant in example 3, and the construction process of double mutant G240I/G134N was as follows:

taking a mutant G240I expression vector as a template, amplifying by primers (G134N-fw: AGGTTACACCATCAACATCTTCGGTGCTACTACTG and G134N-rev: CAGTAGTAGCACCGAAGATGTTGATGGTGTAACCT), carrying out enzymolysis on the amplified product by DpnI, transferring the product after enzymolysis into escherichia coli Top10, and determining whether the double mutant G240I/G134N is successfully constructed by bacterial liquid PCR and sequencing;

the construction method of the double mutant G240I/V105Q is consistent with that of G240I/G134N, except that the amplification primers are changed to V105Q-fw (AACATCATCATCATCATATGAAGTTGCAGAACAAGCCAGAGCAGGA) and V105Q-rev (TCCTGCTCTGGCTTGTTCTGCAACTTCATGATGTT).

The construction process of the triple mutant G240I/G134N/V105Q is the same as that of the double mutant, except that the amplification template is changed to G240I/G134N, and the amplification primers are V105Q-fw and V105Q-rev.

The combinatorial mutants were screened in accordance with the method of example 3 and the effect of the combinatorial mutation was judged by measuring the thermostability at 60 ℃ for 30 minutes. Through screening and experiments, the residual enzyme activities of the combined mutants G240I/G134N, G240I/V105Q and G240I/G134N/V105Q after 30-minute incubation at 60 ℃ are respectively 16.2%, 15.3% and 19.5%, while the residual enzyme activity of the control (chitosanase Cscncv) is 8.1%, so that the thermal stability of the three-mutant G240I/G134N/V105Q is best, and therefore, the three-mutant is selected for the next experiment;

inoculating a recombinant yeast engineering bacterium (named as C3) containing the three-mutant CsnB into a 500ml shake flask containing 100ml BMGY medium, carrying out induction culture for 120 hours, centrifuging to collect supernatant enzyme liquid, carrying out ultrafiltration through a 10kDa ultrafiltration tube, concentrating the supernatant enzyme liquid, purifying the ultrafiltered enzyme liquid by using a Ni-IDA protein purification kit, and carrying out temperature characteristic determination on the purified three-mutant CsnB, wherein the temperature characteristic determination method of the three-mutant CsnB is consistent with the temperature characteristic determination of the embodiment 3, and the experimental result is shown in FIG. 2;

as can be seen from FIG. 2, the optimum reaction temperature of the triple mutant CsnB is 55 ℃; in the aspect of thermal stability, the residual enzyme activities of the three-mutant CsnB after 30-minute heat preservation at 60 ℃ and 65 ℃ are respectively 19.5% and 11.2%, and the residual enzyme activities of the chitosanase Csncv at the two temperatures are respectively 8.1% and 5.1%, so that the thermal stability of the three-mutant CsnB is remarkably improved compared with that of the unmutated chitosanase Csncv.

Example 6 high Density fermentation

The recombinant engineering bacteria C1 and C3 are subjected to high-density fermentation in a 5L fermentation tank, and the specific process is approximately as follows:

inoculating the single colony recombinant yeast engineering bacteria into a 250 mL triangular flask containing 50mL YPG medium, and carrying out shaking overnight culture at 30 ℃ and 200 rpm; inoculating the overnight cultured recombinant yeast engineering bacteria into a 500mL triangular flask containing 100mL YPG medium according to the inoculation amount of 1% (v/v), performing shaking overnight culture at 30 ℃ and 200rpm until OD 600 is more than 10, inoculating the recombinant yeast engineering bacteria into a 5L fermentation tank containing 2L BSM medium according to the inoculation amount of 10% (v/v), wherein the culture conditions of the recombinant yeast engineering bacteria in the 5L fermentation tank are as follows: the temperature was 30 ℃, the pH was 5.0, the stirring speed was 500 rpm, and the air flow rate was 40L/min. In the initial stage of culture, glycerol is used as a carbon source for growth of the thalli, when the wet weight of the thalli reaches 180 g/L, the glycerol supply is stopped, after the glycerol is completely absorbed by the thalli, methanol is used for inducing, the addition amount of the methanol is adjusted according to dissolved oxygen, in the culture process, sampling is carried out every 24 hours to determine the enzyme activity, and the experimental result is shown in figure 3;

as can be seen from FIG. 3, the activity of the chitosan enzyme is gradually improved along with the increase of the induction time in the fermentation process, when the induction culture is carried out for 192 hours, the activity of the C1 fermentation enzyme reaches 1652U/mL, compared with the C1, the highest fermentation enzyme activity of the C3 appears in the induction for 168 hours, and the activity of the C3 enzyme can reach 1765U/mL.

Example 7 analysis of chitosanase Cscnv and mutant CsnB hydrolysates

The chitosan hydrolyzing reaction of the chitosan enzyme Csncv and the three mutant CsnB is as follows: weighing 4g of chitosan, dissolving in 100ml of sodium acetate buffer solution (pH 5.5), respectively adding 1000U of chitosan Csncv and the triple mutant CsnB, carrying out hydrolysis reaction at 50 ℃ and 120rpm, respectively selecting hydrolysis samples of 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes and 40 minutes, and carrying out thin layer chromatography and hydrolysis rate determination;

the thin layer chromatography comprises the following steps: respectively dropping 1 μ L of hydrolysis reaction product and 4 μ L of chitosan oligosaccharide standard mixture onto Silica gel plate (Silica gel 60, Merck), placing the well dropped Silica gel plate in an expansion cylinder for expansion, taking out the expanded Silica gel plate from the expansion cylinder, blowing the expanded Silica gel plate, spraying 0.2% ninhydrin-ethanol solution, blowing the expanded Silica gel plate, and placing the Silica gel plate at 100 ℃ for high-temperature color development to obtain the result shown in figure 4;

as can be seen from A in FIG. 4, when the hydrolysis time is within the range of 5 to 15 minutes, the chitosanase Csncv hydrolysate is mainly composed of chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose. The hydrolysis time is in the range of 20 to 40 minutes, the chitosanase Csncv hydrolysate is mainly composed of chitobiose, chitotriose, chitotetrasaccharide and chitopentasaccharide.

As can be seen from B in FIG. 4, the three-mutant CsnB has different hydrolysis product compositions and chitosanase Csncv in different time periods, the hydrolysis time is within the range of 5-10 minutes, and the products mainly comprise chitobiose, chitotriose, chitotetraose, chitopentaose and chitohexaose; the hydrolysis time is within 15-20 minutes, and mainly comprises chitobiose, chitotriose, chitotetrasaccharide and chitopentasaccharide; the hydrolysis time is within the range of 25-40 minutes, and the hydrolysis product mainly comprises chitobiose, chitotriose and chitotetraose;

the three mutant CsnB hydrolysate can be seen in a high performance liquid chromatography map shown in an attached figure 5;

from the results, the enzymolysis efficiency of the three-mutant CsnB is higher than that of the chitosanase Csncv.

It should be noted that specific features, structures, materials or characteristics described in this specification may be combined in any combination, all possible combinations of technical features in the above embodiments are not described in order to simplify the description, and those skilled in the art may combine and combine features of different embodiments and features of different embodiments described in this specification without contradiction.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Shenzhen Runkang ecological environment shares Limited

<120> chitosanase Csncv, mutant CsnB thereof and application

<130> 2021.12.02

<160> 17

<170> PatentIn version 3.3

<210> 1

<211> 331

<212> PRT

<213> V105Q amino acid sequence

<400> 1

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Gln Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Gly Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Gly

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 2

<211> 331

<212> PRT

<213> G134N amino acid sequence

<400> 2

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Val Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Asn Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Gly

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 3

<211> 331

<212> PRT

<213> G240I amino acid sequence

<400> 3

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Val Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Gly Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Ile

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 4

<211> 331

<212> PRT

<213> G240I/G134N amino acid sequence

<400> 4

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Val Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Asn Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Ile

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 5

<211> 331

<212> PRT

<213> G240I/V105Q amino acid sequence

<400> 5

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Gln Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Gly Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Ile

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 6

<211> 331

<212> PRT

<213> G134N/V105Q amino acid sequence

<400> 6

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Gln Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Asn Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Gly

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 7

<211> 331

<212> PRT

<213> G240I/G134N/V105Q amino acid sequence

<400> 7

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Gln Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Asn Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Ile

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 8

<211> 996

<212> DNA

<213> V105Q Gene sequence

<400> 8

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tgcagaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatcg gtatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcggt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

<210> 9

<211> 996

<212> DNA

<213> G134N Gene sequence

<400> 9

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tggtcaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatca acatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcggt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

<210> 10

<211> 996

<212> DNA

<213> G240I Gene sequence

<400> 10

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tggtcaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatcg gtatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcatt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

<210> 11

<211> 996

<212> DNA

<213> G240I/G134N Gene sequences

<400> 11

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tggtcaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatca acatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcatt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

<210> 12

<211> 996

<212> DNA

<213> G240I/V105Q Gene sequences

<400> 12

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tgcagaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatcg gtatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcatt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

<210> 13

<211> 996

<212> DNA

<213> G134N/V105Q Gene sequence

<400> 13

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tgcagaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatca acatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcggt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

<210> 14

<211> 996

<212> DNA

<213> G240I/G134N/V105Q Gene sequence

<400> 14

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tgcagaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatca acatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcatt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

<210> 15

<211> 996

<212> DNA

<213> nucleotide sequence of original gene csncv

<400> 15

cagggctcga cggccggcag ccccgaaaga caagccgttc gcctgcccga ctgcgccgcc 60

gcgtggagcc ccgcctcgcc ctatcaagcc ggcagcgtcg ccagccgcgg cggcgtcaac 120

tacaccgcgg cattctggac ccagggcaat cctccggaac agggtcaggc ctggcaggcc 180

ggcaagcgct gtcgtccggc cgcgcaggcc aaggccgccg accacgacgc caactttccg 240

cccgccacgc tgaaattcct gaaagccaac accggcctgg acggcgagca gtgggacaac 300

atcatgaagc tggtcaacaa gccggagcag gattcgctgg agtggaccaa gttctacggc 360

tactgcgaga acatcggcga caagcgcgga tacacgatag gcatcttcgg cgccaccacc 420

ggcggcccga acgacgaggg tccggacggt ccgacgctgt tcaaggagtt cgacgcttcc 480

agcggcgcgt ccaatccgtc catcaccggc ggactggccc gcgccggcgt gcacggcagc 540

atgcagggca agatcctgaa gatcagcgac agcgccaagg tgttctgcga caagatcggc 600

aacctgcaga acaatcccgc ctggcgcgac gcgatgtgga acaccttcta caaggtatac 660

atccaataca gcgtgcagca ggcccgccag cgcggcttct ccagcgcgct gaccataggc 720

tccttcgtcg acaccgcgct gaaccagggc gcaaccggcg attccggcac gctgcagggc 780

ctgctgtccc gctccggcaa cagcggcgac gagaaaacct tcatgaccgc cttctacgcg 840

cagcgcagca agatcgtcga caccaacgac tacaatcagc cgcccaacgg caaaaaccgc 900

gtcaagcaat ggagcacgct gttgaacatg ggcgagaccg acttgaagaa tgccgacgcc 960

gcggtgcaga aagtcaccaa ctgggaaatg aagtaa 996

<210> 16

<211> 331

<212> PRT

<213> amino acid sequence of optimized chitosanase Csncv

<400> 16

Gln Gly Ser Thr Ala Gly Ser Pro Glu Arg Gln Ala Val Arg Leu Pro

1 5 10 15

Asp Cys Ala Ala Ala Trp Ser Pro Ala Ser Pro Tyr Gln Ala Gly Ser

20 25 30

Val Ala Ser Arg Gly Gly Val Asn Tyr Thr Ala Ala Phe Trp Thr Gln

35 40 45

Gly Asn Pro Pro Glu Gln Gly Gln Ala Trp Gln Ala Gly Lys Arg Cys

50 55 60

Arg Pro Ala Ala Gln Ala Lys Ala Ala Asp His Asp Ala Asn Phe Pro

65 70 75 80

Pro Ala Thr Leu Lys Phe Leu Lys Ala Asn Thr Gly Leu Asp Gly Glu

85 90 95

Gln Trp Asp Asn Ile Met Lys Leu Val Asn Lys Pro Glu Gln Asp Ser

100 105 110

Leu Glu Trp Thr Lys Phe Tyr Gly Tyr Cys Glu Asn Ile Gly Asp Lys

115 120 125

Arg Gly Tyr Thr Ile Gly Ile Phe Gly Ala Thr Thr Gly Gly Pro Asn

130 135 140

Asp Glu Gly Pro Asp Gly Pro Thr Leu Phe Lys Glu Phe Asp Ala Ser

145 150 155 160

Ser Gly Ala Ser Asn Pro Ser Ile Thr Gly Gly Leu Ala Arg Ala Gly

165 170 175

Val His Gly Ser Met Gln Gly Lys Ile Leu Lys Ile Ser Asp Ser Ala

180 185 190

Lys Val Phe Cys Asp Lys Ile Gly Asn Leu Gln Asn Asn Pro Ala Trp

195 200 205

Arg Asp Ala Met Trp Asn Thr Phe Tyr Lys Val Tyr Ile Gln Tyr Ser

210 215 220

Val Gln Gln Ala Arg Gln Arg Gly Phe Ser Ser Ala Leu Thr Ile Gly

225 230 235 240

Ser Phe Val Asp Thr Ala Leu Asn Gln Gly Ala Thr Gly Asp Ser Gly

245 250 255

Thr Leu Gln Gly Leu Leu Ser Arg Ser Gly Asn Ser Gly Asp Glu Lys

260 265 270

Thr Phe Met Thr Ala Phe Tyr Ala Gln Arg Ser Lys Ile Val Asp Thr

275 280 285

Asn Asp Tyr Asn Gln Pro Pro Asn Gly Lys Asn Arg Val Lys Gln Trp

290 295 300

Ser Thr Leu Leu Asn Met Gly Glu Thr Asp Leu Lys Asn Ala Asp Ala

305 310 315 320

Ala Val Gln Lys Val Thr Asn Trp Glu Met Lys

325 330

<210> 17

<211> 996

<212> DNA

<213> optimized nucleotide sequence encoding CSncv gene of chitosanase

<400> 17

caaggttcta ctgctggttc tccagagaga caggctgtta gattgccaga ttgtgctgct 60

gcttggtcac cagcttctcc atatcaagct ggttctgttg cttccagagg tggtgttaac 120

tacactgctg ctttctggac tcaaggtaac ccaccagaac aaggtcaagc ttggcaagct 180

ggtaagagat gtagaccagc tgctcaagct aaggctgctg atcacgatgc taactttcca 240

ccagctacct tgaagttctt gaaggccaac actggtttgg acggtgaaca gtgggacaac 300

atcatgaagt tggtcaacaa gccagagcag gactctttgg agtggactaa gttctacggt 360

tactgcgaga acatcggtga caagagaggt tacaccatcg gtatcttcgg tgctactact 420

ggtggtccaa acgatgaagg tccagatggt ccaaccttgt tcaaagagtt cgacgcttct 480

tccggtgctt ctaacccatc tattactggt ggtttggcta gagctggtgt tcacggttct 540

atgcagggta agattctgaa gatttccgac tccgccaagg tgttctgtga caagattggt 600

aatttgcaga acaacccagc ttggagagat gctatgtgga acaccttcta caaggtttac 660

atccagtact ccgttcagca ggctagacaa agaggtttct cttccgcttt gaccatcggt 720

tccttcgttg acactgcttt gaaccagggt gctactggtg attctggtac tctgcaaggt 780

ttgttgtcca gatccggtaa ctctggtgac gaaaagactt tcatgactgc cttctacgcc 840

cagagatcca agattgtcga caccaacgac tacaaccagc caccaaacgg taagaacaga 900

gttaagcagt ggtccacctt gctgaacatg ggtgaaactg acttgaagaa cgctgacgct 960

gctgttcaga aggttaccaa ctgggagatg aagtaa 996

Claims

1. The chitosanase mutant CsnB is characterized by being V105Q, G134N, G240I, G240I/G134N, G240I/V105Q, G134N/V105Q or G240I/G134N/V105Q, and the amino acid sequences of the mutants are respectively shown in SEQ ID NO. 1-7.

2. The chitosanase mutant CsnB according to claim 1, wherein a gene coding the amino acid of the mutant CsnB is CsnB, and the nucleotide sequences of the gene CsnB are respectively shown in SEQ ID No. 8-14.

3. A recombinant expression vector containing the gene of claim 2.

4. A recombinant strain comprising the vector of claim 3.

5. The application of the chitosanase mutant CsnB of any one of claims 1-2 in the preparation of chitosan oligosaccharide.