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Cellulose orthogenesis method of ampullaria crossean

A cellulase, directed evolution technology, applied in the field of bioengineering, can solve problems such as non-degradable and difficult to degrade

Inactive Publication Date: 2010-04-14
CHONGQING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Cellulose is the most abundant renewable resource on the earth, and it is a good renewable energy source, but it has disadvantages such as not easy to degrade and difficult to degrade

Method used

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  • Cellulose orthogenesis method of ampullaria crossean
  • Cellulose orthogenesis method of ampullaria crossean
  • Cellulose orthogenesis method of ampullaria crossean

Examples

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Embodiment Construction

[0023] Below take " apple snail " as an example to further illustrate the present invention.

[0024] The first step is to obtain the cellulase gene of apple snail:

[0025] (1) extracting the total mRNA of apple snail—messenger ribonucleic acid;

[0026] For the extraction of mRNA, about 600 mg of fresh gastric tissue samples were taken from apple snails, quickly frozen in liquid nitrogen, and fully ground with Promega System1000withMagneticStand extracts mRNA;

[0027] (2) Perform reverse transcription to amplify cDNA—complementary deoxyribonucleic acid, take 1.5 μL mRNA, 2 μL LigodT (highly polydeoxythymidine nucleotides) and 7.6 μL H 2 O mixed, heated to 65°C for 10 minutes, then ice-bathed for 2 minutes, added 4 μL 5M-MLV-buffer (avian myeloblastosis virus reverse transcription buffer), 2.5 μL dNTP (deoxynucleoside triphosphate) and AMV (avian Myeloblastoma virus) reverse transcriptase (Promega) 2mL, 41°C, 60 minutes, then 65°C to inactivate the activity of the enzyme...

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Abstract

The invention relates to a cellulase orthogenesis method of ampullaria crossean. The steps are as follows: step one, acquiring cellulase genes of ampullaria crossean: firstly extracting the total mRNA of ampullaria crossean; secondly conducting reverse transcription to amplify cDNA; then amplifying the cellulase genes of ampullaria crossean through PCR; and obtaining the cellulase gene fragments of ampullaria crossean; step two, cloning and sequencing: cloning PCR products and connecting the PCR products with pMD18-T carrier containing T-end; preparation and conversion of competent cell, and adopting a CaCL 2 method to prepare the competent cell; screening of positive clones, namely using X-ga1 and IPTG to primarily screen clones; extracting plasmid by an alkaline process; and sequencing; and step three: site-mutagenesis: using professional software and websites of molecular biology to conduct analytical prediction of functions and determine amino acid needing transforming; adopting a kit to conduct site-mutagenesis in the direction transformation of cellulase protein; and finally sequencing and determining. The method improves the activity of cellulase of ampullaria crossean, as well as the degradation action of cellulase of ampullaria crossean on cellulose.

Description

technical field [0001] The invention relates to bioengineering technology, in particular to a cellulase directed evolution method for apple snails. Background technique [0002] Cellulase is a general term for a group of enzymes that degrade cellulose to generate glucose. It is a class of biologically active proteins that catalyze the decomposition of cellulose into glucose. Cellulase was first studied in 1906 when Seilliere discovered cellulase that can decompose cellulose in the digestive juice of snails. It can convert abundant cellulose waste resources into renewable resources and solve the shortage of energy, feed and food supply. , and can be used in alcohol, liquor, soy sauce, feed and textile industries. [0003] In the past, the main focus was on the degradation and transformation of cellulose by microorganisms. In recent years, the application research of cellulase is very active, and a number of high-yield strains have been screened. But the cellulase activity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12P19/34C12R1/19
Inventor 王万能范新发
Owner CHONGQING UNIV OF TECH
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