Chitinase fused with carbohydrate binding module, preparation method for chitinase and application of chitinase

A carbohydrate and chitinase technology, applied in the field of bioengineering, to achieve the effect of rich distribution, growth support and easy digestion

Active Publication Date: 2020-11-17
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, no one has used chitinases fused with carbohydrate-binding modules for direct hydrolysis of shrimp shells

Method used

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  • Chitinase fused with carbohydrate binding module, preparation method for chitinase and application of chitinase
  • Chitinase fused with carbohydrate binding module, preparation method for chitinase and application of chitinase
  • Chitinase fused with carbohydrate binding module, preparation method for chitinase and application of chitinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Determination of various components of shrimp shell waste

[0057] Shrimp shell waste was purchased from Zhanjiang Guolian Aquatic Products Development Co., Ltd., crushed through a 50-mesh sieve to obtain shrimp shell waste powder, and stored at 4 °C for future use. The methods for determining protein, ash, chitin and moisture in shrimp shell waste are as follows:

[0058] (1) Protein: Because chitin contains nitrogen, in order to eliminate interference, the shrimp shell powder was first treated with 1M NaOH solution at 40°C for 3 hours, and the protein concentration of the supernatant was determined by the Kjeldahl method (GB 50095-2010). The protein content in the shrimp shell was measured to be 48.84%;

[0059] (2) Ash content: using the "First Method Determination of Total Ash Content in Food" in the National Food Safety Standard (GB5009.4-2016), the measured ash content in shrimp shells is 20.75%;

[0060] (3) Chitin: firstly treat the shrimp shell ...

Embodiment 2

[0063] Example 2: Fusion of CBM with chitinase Chit46

[0064] The primer F1(5'-CTGC GAATTC AGCCCCCTGGCCACAG-3') and R1

[0065] (5'-ACTT ACGCGT AACT GGGCCC GTTCAGACCGTTCCTGATGTT-3') (the underlined sequences represent the EcoRI restriction site, the MluI restriction site and the ApaI restriction site respectively), and the Chit46-C fragment was obtained by PCR (the amino acid and nucleotide sequences of Chit46 are respectively as in China SEQ ID NO.1 and SEQ ID NO.2 in the patent application CN201910064040.5); using the cDNA of Bacillus subtilis 168 strain as a template, the primer F3 (5'-AACT GGGCCC CAGGAATCATCCTCAGCT-3') and R3(5'-ACTT ACGCGT ATTTGGTTCTGTTCCCCCA-3') (the underlined sequences represent the ApaI restriction site and the MluI restriction site respectively), and the CBM3 with the connecting peptide was obtained by PCR method (derived from the sequence Genbank: CAA82317); with the Bacillus subtilis 168 strain cDNA as template, design primer F6

[0066...

Embodiment 3

[0107] Example 3 Chitinase hydrolysis of shrimp shell powder and hydrolyzate analysis

[0108] (1) In order to compare the efficiency of different fusion chitinases in hydrolyzing shrimp shell waste, 100 U fusion chitinases Chit46-CBM3, Chit46-CBM6 and Chit46-CBM26 were mixed with 1 g shrimp shell powder at 40 °C, and then Add 50mM pH 6.0 sodium phosphate buffer until the total volume of the mixture is 10mL. After hydrolysis for 12 hours, centrifuge at 5000rpm for 10 minutes to obtain chitin hydrolyzate and shrimp shell residue. Chitinase is used as a control.

[0109] Add the same volume of 95% (v / v) ethanol to the chitin hydrolyzate of shrimp shells and centrifuge at 12000rpm for 1min, the precipitate is protein, and the remaining solution contains chitooligosaccharide (hereinafter referred to as: chitooligosaccharide sugar solution). The precipitated protein was redissolved in the same volume of sterile water as the chitin hydrolyzate of shrimp shell, and the protein conte...

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Abstract

The invention provides chitinase fused with a carbohydrate binding module, a preparation method for the chitinase and application of the chitinase, and belongs to the technical field of biological engineering. The chitinase fused with the carbohydrate binding module comprises fusion chitinase formed by series connection of the carbohydrate binding module with a connecting peptide and a C end of chitinase Chit46. It is the first time for the invention to successfully apply the chitinase Chit46-CBM3, Chit46-CBM6 and Chit46-CBM26 fused with the carbohydrate binding module (CBM) to shrimp shell waste recovery. After the CBM is fused, the chitin in shrimp shells can be directly hydrolyzed by the fusion chitinase, and thus, the use of a strong alkali reagent and a complex pretreatment step in atraditional process are avoided.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a chitinase fused with a carbohydrate binding module and a preparation method and application thereof. Background technique [0002] Guangdong Province is a major aquatic province in China. According to statistics, the total production of aquatic products in Guangdong in 2016 reached 8.82 million tons, including a variety of shrimp and crabs. The output of shrimp alone was nearly 1 million tons. The edible part of shrimp is less than 50%, and a large amount of waste such as shrimp heads and shells will be produced in the process of shrimp processing. Shrimp heads account for more than one-third of the weight of the whole shrimp. Hundreds of thousands of tons. With the development of my country's seafood industry and artificially cultured shrimp and crab industries, the amount of these wastes will increase, causing serious environmental pollution (Ferraro et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N9/24C12N15/62C12N15/81C12N1/19A23K10/14A23K10/26C12R1/84
CPCC12N9/2442C12N9/24C12N15/815C12Y302/01014A23K10/26A23K10/14C07K2319/00
Inventor 罗晓春李宗球邓俊劲陆德林李志伟史丹
Owner SOUTH CHINA UNIV OF TECH
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