Fusion chitinase capable of efficiently degrading alpha-chitin and related biological material and application thereof

A technology of chitinase and biological materials, applied in application, microorganism, hydrolase and other directions, can solve the problems of loose structure, difficult hydrolysis and utilization, etc.

Inactive Publication Date: 2021-07-23
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Colloidal chitin is chitin treated with artificial strong acid, which is different from the naturally occurring form of chitin. Dense, difficult to be hydrolyzed

Method used

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  • Fusion chitinase capable of efficiently degrading alpha-chitin and related biological material and application thereof
  • Fusion chitinase capable of efficiently degrading alpha-chitin and related biological material and application thereof
  • Fusion chitinase capable of efficiently degrading alpha-chitin and related biological material and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Preparation of Fusion Chitinases SaChiB-HEX and His-SaChiB-HEX-His and Measurement of their Catalytic Efficiency to α-Chitin

[0075] Delete the signal peptide (amino acid residues 1-26) of Streptomyces alfalfae chitinase from GenBank Accession Number WP_076682988 (12-APR-2018) to obtain a mature chitinase (amino acid sequence is GenBank Accession Number WP_076682988 (12-APR-2018) amino acid residues 27-296, named SaChiB); deletion of Streptomyces alfalfae β-N-acetyl The signal peptide of hexosaminidase (amino acid sequence is amino acid residues 1-25) was obtained from mature β-N-acetylhexosaminidase (amino acid sequence is GenBank Accession Number AYR18867.1 (12-NOV-2018) No. 26 -536 amino acid residues, it is named as SaHEX).

[0076] The above-mentioned mature chitinase SaChiB and the above-mentioned mature β-N-acetylhexosaminidase SaHEX were fused together to obtain a fusion protein, which was named SaChiB-HEX. The amino acid sequence of SaChiB-HEX is ...

Embodiment 2

[0110] Example 2. Chitinase activity analysis of fusion chitinase His-SaChiB-HEX-His

[0111] Use 3,5-dinitrosalicylic acid (DNS) method to measure chitinase activity: add 0.1mL enzyme solution (solvent is 50mM pH 7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer) and 0.2mL colloidal chitin to obtain an enzymatic reaction system, react at 30°C for 5min, add 0.4mLDNS after the reaction is completed, boil for 10min, measure OD540 after centrifugation. 1 Enzyme activity unit (U) is defined as the amount of enzyme needed to decompose colloidal chitin and release 1 μmol N-acetylamino-D-glucose (GlcNAc) per minute under the above conditions. Experiments were repeated three times.

[0112] The result shows that the chitinase specific activity (U / mol enzyme protein) of the His-SaChiB-HEX-His of the molecular sieve purification obtained in embodiment 1, the His-SaChiB-His of the molecular sieve purification and the His-SaHEX-His of the molecular sieve purification are...

Embodiment 3

[0113] Example 3, Determination of the properties of the fusion chitinase His-SaChiB-HEX-His

[0114] The properties of the molecular sieve-purified His-SaChiB-HEX-His obtained in Example 1 were determined as follows.

[0115] 1. Determination of the optimal pH and pH stability of the fusion chitinase His-SaChiB-HEX-His

[0116] 1.1 Determination of the optimal pH of the fusion chitinase His-SaChiB-HEX-His

[0117] The molecular sieve-purified His-SaChiB-HEX-His obtained in Example 1 was subjected to enzymatic reactions in buffer solutions of different pH to determine its optimum pH. The buffers used are: 50mmol / L disodium hydrogen phosphate-citric acid buffer (pH4.0-7.0), 50mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.0-8.0), 50mmol / L LTris-HCl buffer (pH 8.0-9.0), 50mmol / L glycine-sodium hydroxide buffer (pH 9.0-10.0). Using the 3,5-dinitrosalicylic acid (DNS) method in Example 2 to measure the chitinase activity of His-SaChiB-HEX-His in the...

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Abstract

The invention discloses fusion chitinase capable of efficiently degrading alpha-chitin and a related biological material and application thereof. The fusion chitinase is a protein obtained by fusing beta-N-acetyl hexosaminase and chitinase, wherein the chitinase activity of the fusion chitinase is higher than that of the beta-N-acetyl hexosaminase and that of the chitinase; the chitinase is D1) or D2), wherein the D1) is a protein with 53rd-322nd amino acid residues of which the amino acid sequence is shown in SEQ ID No.2; and the D2) is a fusion protein obtained by fusing a protein tag at the carboxyl terminal or/and the amino terminal of the protein shown in D1). The fusion chitinase has a degradation effect on various types of chitin, and has relatively high activity on colloid chitin, alpha-chitin and fungal cell wall chitin; and the fusion chitinase has the capability of completely hydrolyzing chitin and generating N-acetyl glucosamine. The fusion chitinase can be widely applied to biological medicine, biological agriculture, cosmetics, energy industry, etc.

Description

technical field [0001] The invention relates to a fusion chitinase for efficiently degrading α-chitin in the biological field, as well as related biological materials and applications. Background technique [0002] Chitin is a long-chain polymer connected by N-acetyl-D-glucosamine through β-1,4-glycosidic bonds. It is the second most abundant renewable substance on earth after cellulose. Natural carbon-based polymer compounds. At the same time, chitin is also the second largest nitrogen-containing compound after protein in the world. Chitin widely exists in the exoskeleton of marine organisms shrimp and crab, squid cartilage, fungi, insects and the internal structure of invertebrates. It is an important raw material for making chitosan and glucosamine. And health food has a wide range of uses. [0003] Chitin has different forms of existence. α-chitin is arranged in trans-parallel polymerized single chains and mainly exists in the exoskeleton of arthropods such as shrimps...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N9/24C12N15/70C12N15/56C12P19/14C12P19/26C12R1/19
CPCC12N9/2442C12N9/2402C12N15/70C12P19/14C12P19/26C12Y302/01014C12Y302/01052C07K2319/21C07K2319/00
Inventor 顾金刚赵国刚吕晨茵姚伟张艾迪马锐
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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