Chitinase Chit46 as well as expression and purification method and application thereof

A chitinase, expression and purification technology, applied in the field of bioengineering, can solve problems such as recombinant expression, separation and purification, and achieve the effect of increasing yield

Active Publication Date: 2019-04-26
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, no one has recombinantly expressed and purified Chit46 chitinase from Trichoderma harzianum, and no one has used it for hydrolysis of colloidal chitin; further obtaining new chitinases with higher yields and high enzymatic activity remains to be done. One of the research directions in this field

Method used

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  • Chitinase Chit46 as well as expression and purification method and application thereof
  • Chitinase Chit46 as well as expression and purification method and application thereof
  • Chitinase Chit46 as well as expression and purification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Heterologous Expression and Purification of Chitinase Chit46

[0059] (1) Trichoderma harzianum culture

[0060] Inoculate Trichoderma harzianum GIM 3.442 (purchased from Guangdong Microbial Culture Collection Center) into improved PDA solid medium (200 mL of potato flour filtrate, 4 g of colloidal chitin, 0.75 g of magnesium sulfate, 0.75 g of potassium dihydrogen phosphate, Agar powder 4g), cultured at 30°C for 2 days.

[0061] The preparation method of the colloidal chitin is as follows: Weigh 10.0 g of chitin (purchased from Sangon Bioengineering (Shanghai) Co., Ltd., article number A500659), add 100 mL of concentrated hydrochloric acid with a concentration of 36%, and keep stirring until Dissolve it completely, add 100mL ethanol and 300mL distilled water, at this time, a large number of flocs can be seen, then wash the obtained flocs with water until they are neutral, dry, weigh and store at 4°C for later use.

[0062] (2) Trichoderma harzianum total RN...

Embodiment 2

[0122] Example 2 Fermentation induction, purification and enzyme activity assay of chitinase Chit46

[0123] (1) Select the positive recombinant Pichia pastoris strain obtained in Example 1 to streak the MD plate, cultivate it for 2 days at 30° C., pick a single colony and inoculate it in 50 mL of BMGY culture solution (yeast extract 10 g, tryptone 20 g, YNB 13.4 g, glycerol 10mL, 1M potassium phosphate (pH 6.0) 100mL, distilled water to 1000mL) Erlenmeyer flask, 30°C, 250rpm shaking culture to OD600≈5.0. Then centrifuge to collect the bacterial cell precipitate, transfer the bacterial cell precipitate to 100mL BMMY culture medium (yeast extract 10g, tryptone 20g, YNB 13.4g, methanol 10mL, 1M potassium phosphate (pH6.0) 100mL, distilled water to volume to 1000 mL) in a Erlenmeyer flask at 28° C., 250 rpm shaking culture, adding 1.5% methanol solution every 24 hours to induce expression, inducing for 7 days, and finally obtaining a fermentation broth.

[0124] (2) The fermente...

Embodiment 3

[0145] The SDS-PAGE detection of embodiment 3 recombinant protein

[0146] SDS-PAGE gel electrophoresis was used to confirm the expression, purity and molecular mass of the recombinant chitinase obtained in Example 2. The concentration of the stacking gel used was 12% and the concentration of the separating gel was 5%, the sample volume was 20 μL, and a standard protein with a standard molecular weight was used as a marker. For the operation process of SDS-PAGE gel electrophoresis, please refer to "Protein Electrophoresis Experimental Technique". For the preparation of fermentation broth samples, the amount of recombinant chitinase produced by induced expression is relatively high. The fermentation broth can be directly diluted by 1 time and mixed with loading buffer. Perform electrophoresis.

[0147] The SDS-PAGE electrophoresis of the crude enzyme liquid (refers to the fermentation liquid that has not been purified by nickel column affinity chromatography) and the purified...

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Abstract

The invention provides chitinase Chit46 as well as an expression and purification method and application thereof. The chitinase Chit46 is derived from trichoderma harzianum and an amino acid sequenceis shown as SEQ ID NO. 1; the fermentation solution enzyme activity of the chitinase is 31.4 U / mL and the specific enzyme activity is 34.5 U / mg, which is higher than that of like reported chitinase. The invention further provides the expression and purification method of the chitinase Chit46; the yield of the chitinase is greatly improved, the protein yield of a crude enzyme solution can reach 1.26 g / L and the yield of purified protein reaches 0.77 g / L. The chitinase provided by the invention can be used for hydrolyzing colloid chitin and has good hydrolysis activity; hydrolysis products are stable and the vast majority of hydrolysis products are chitobiose; a subsequent complicated purification and separation technology of each polymerization degree is omitted and the chitinase has a goodapplication prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a chitinase Chit46 and its expression and purification method and application. Background technique [0002] Chitin is a natural macromolecular compound second only to cellulose on earth, and it is abundant in shrimp and crab shells (Arcidiacono and Kaplan, 1992). The content of chitin in shrimp shells is about 20%, while the current price of dried shrimp shells is only 450 yuan per ton, and the price of chitin extracted from shrimp and crab shells reaches 50,000 to 100,000 yuan per ton. However, the poor solubility of chitin limits its application, and the soluble oligosaccharides produced by its hydrolysis have great application value. Many recent studies have found that chitooligosaccharides (polyβ-1,4-N-acetylglucosamine), the hydrolyzed product of chitin, retain the natural acetyl groups of chitin, and are more effective than chitin oligosaccharides. Sugar (poly β-1...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/81C12N1/19C12P19/26C12P19/14
CPCC12N9/2442C12N15/815C12P19/14C12P19/26C12Y302/01014
Inventor 罗晓春邓俊劲李志伟史丹茅和花梁爽
Owner SOUTH CHINA UNIV OF TECH
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