Chitosanase Csncv as well as mutant CsnB and application thereof

A technology of chitosanase and mutants, which is applied in the field of genetic engineering, can solve problems such as high production process and equipment control requirements, different application ranges, and incomplete product structures, and achieve good application prospects and industrial value. Pathway of origin, effect of high enzyme activity

Active Publication Date: 2021-12-31
深圳润康生态环境股份有限公司
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AI Technical Summary

Problems solved by technology

Due to the disadvantages of wastewater polluting the environment and incomplete product structure, the chemical method has limited its application in the field of chitosan oligosaccharide preparation.
Compared with the chemical method, the process of enzymatic preparation of oligochitosaccharides has the advantages of mild reaction conditions, complete product structure, easy control of the process, and no pollution to the environment. Therefore, the enzymatic method is recognized as the mainstream production and processing technology of chitosan oligosaccharides. However, the physiological activity of chitosan oligosaccharide products prepared by the existing enzymatic hydrolysis process is low, and the enzymatic hydrolysis reaction time needs to be strictly controlled to obtain the enzymatic hydrolyzate with a high proportion of high-polymerization oligosaccharides. Therefore, the production process and equipment control more demanding
[0003] In order to better utilize chitosanase, develop and transform it, at present, chitosanase is divided into 5 families, 7 families, 8 families, 46 families, 75 families, 80 families, etc. according to the similarity of gene sequence. The 46 family chitosan enzymes are the most studied, including their three-dimensional structure and catalytic mechanism. For example, Wang Yani analyzed the complex structures of two 46 family chitosan enzymes and obtained chitosan mutants The proportion of higher polymerization degree in the chitosan oligosaccharide products obtained by hydrolyzing chitosan has increased (Wang Yani, GH46 family chitosanase structure analysis and catalytic mechanism research); Chinese patent 113493781A discloses a chitosanase CsnH, its chitosanase yield is 665.3U / mL, the optimum reaction temperature is 70°C, has good heat resistance and stability, and the hydrolyzed product does not contain chitobiose; Chinese patent 105602921 discloses a chitosanase EAG1 , the product of hydrolysis for 4 hours at 45°C is chitosan oligosaccharide 2~8 sugars, and the hydrolysis rate is correspondingly increased; The activity is 898.6U / mg, and the hydrolysis product is GlcN-(GlcN) 4 It can be seen that the specificity of chitosanase is higher, and the scope of application is not the same. On the other hand, in the disclosed 46 families, the most reported are bacillus chitosanase and streptomyces chitosanase, However, chitosanase from other sources is relatively less studied

Method used

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  • Chitosanase Csncv as well as mutant CsnB and application thereof
  • Chitosanase Csncv as well as mutant CsnB and application thereof
  • Chitosanase Csncv as well as mutant CsnB and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Codon optimization of chitosanase from Violaceum violaceum CV1192

[0102] The chitosanase gene sequence of Bacillus violaceum CV1192 was obtained by analyzing the NCBI database (gene accession number: CP024028.1). The full-length chitosanase gene of Bacillus violaceum CV1192 is 1083bp, encoding 360 amino acids. Through the analysis of the online signal peptide software SignalP-5.0 Server, it was found that the first 29 amino acids of the chitosanase of Violaceum violaceum CV1192 were its signal peptide sequence. Since Pichia pastoris is used as the recombinant expression host in this patent, it is necessary to optimize the chitosanase gene sequence of Violaceum violaceum CV1192 according to the codon preference of Pichia pastoris. In addition, since the signal peptide used in recombinant expression is an α signal peptide, it is necessary to remove the coding sequence of the chitosanase signal peptide part of Violaceum violaceum CV1192 during gene optimization...

Embodiment 2

[0103] Example 2 Construction and Screening of Recombinant Engineering Bacteria

[0104] Expression vector pPICZαA- csncv The build is as follows:

[0105] (1) Chitosanase gene optimized with Example 1 csncv As template, by primer (c1-fw: 5'-AGTC GAATTC CAAGGTTCTACTGCTGGTTCT-3' and C1-rev:5'–ATC TCTAGA CTTCATCTCCCAGTTGGT-3') for PCR amplification, and the amplified product was purified by agarose gel;

[0106] (2) Digest the purified amplified product and the expression vector pPICZαA with restriction endonucleases EcoRI and XbaI, respectively;

[0107] (3) Recover the digested amplification product and the expression vector pPICZαA, and perform a ligation reaction between the two;

[0108] (4) Transform the ligation reaction product into E. coli Top10, and finally obtain the expression vector pPICZ through screening, verification and sequencing identification of recombinant transformants α A- csncv .

[0109] The construction of recombinant yeast engineering bacteri...

Embodiment 3

[0115] Embodiment 3 chitosanase Csncv temperature characteristic is measured

[0116] Insert the recombinant engineered bacteria C1 obtained in Example 2 into a 500ml shake flask containing 100ml of BMGY medium, and after inducing culture for 120 hours, centrifuge to collect the supernatant enzyme solution, ultrafilter through a 10kDa ultrafiltration tube, concentrate the supernatant enzyme solution, The ultrafiltered enzyme solution was purified with Ni-IDA protein purification kit, and the temperature characteristics of the purified chitosanase Csncv were measured.

[0117] The temperature characteristic determination steps of chitosanase Csncv are as follows:

[0118] Under the condition of pH 5.5, the enzyme activity of Csncv at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C was measured, and the relative activity at other temperatures was calculated with the highest enzyme activity as 100%. Enzyme activity: measure the remaining enzyme activity after heat treatment in wate...

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Abstract

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

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a chitosanase Csncv and its mutant CsnB and applications thereof. Background technique [0002] As a biostimulant, oligochitosan plays an important role in green agricultural planting. Research and practical application show that oligochitosan can increase crop yield, improve the quality of agricultural products, improve crop disease resistance, activate the plant's own innate immune system, Improve crop resistance and other effects. At present, the preparation of oligochitosan is mainly divided into chemical method and enzymatic method. Due to the disadvantages of wastewater polluting the environment and incomplete product structure, the chemical method has limited its application in the field of chitosan oligosaccharide preparation. Compared with the chemical method, the process of enzymatic preparation of oligochitosaccharides has the advantages of mil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12P19/14C12P19/00C12R1/84
CPCC12N9/2402C12N15/815C12Y302/01132C12P19/14C12P19/00
Inventor 王建荣祝木金王平陈微钟斌余思曹革
Owner 深圳润康生态环境股份有限公司
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