Alkaline cellulase, and DNA (deoxyribonucleic acid) sequence and application thereof

A cellulase and cellulose degradation technology, applied in the field of peptides, can solve the problems of difficulty in glucose and the limitation of cellulose regeneration and utilization, and achieve the effects of good alkali-resistant cellulase activity and broad industrial application prospects.

Inactive Publication Date: 2015-01-14
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because cellulose has a water-insoluble, highly crystalline structure and is surrounded by a lignin layer, it is quite difficult to hydrolyze it into usable glucose, and the regeneration and utilization of cellulose has been limited.

Method used

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  • Alkaline cellulase, and DNA (deoxyribonucleic acid) sequence and application thereof
  • Alkaline cellulase, and DNA (deoxyribonucleic acid) sequence and application thereof
  • Alkaline cellulase, and DNA (deoxyribonucleic acid) sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The acquisition of metagenomic DNA in the soil sample of embodiment 1

[0044] Papermaking wastewater sediment samples were collected from the outfall of a paper mill in Xuchang City, Henan Province.

[0045] Take two 50 mL centrifuge tubes, weigh papermaking wastewater sediment samples, 6 g in each tube, add DNA extraction buffer 13.5 mL (containing 100 mM EDTA, 100 mM sodium phosphate, 1.5 M NaCl, 1% CTAB and 100 mM Tris-Hcl, pH 8.0), vortexed to mix, placed on a shaker at 220 rpm, activated at 37°C for 30 min, then added 1.5 mL of 20% SDS to each tube to make the final concentration 2% (w / v) . Then, bathe in a water bath at 65°C for 2 h, and gently invert up and down several times every 15 min to mix well. Centrifuge at 6,000 g at room temperature for 10 min. After collecting the supernatant, transfer it to a clean centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1, V / V), and gently invert up and down to mix. Then centrifuge at 11,000 g at ...

Embodiment 2

[0046] Example 2 Construction of the Metagenome Library of Papermaking Wastewater Sediments and Acquisition of DNA Sequences

[0047] 1. Construction of metagenomic library of papermaking wastewater sediment

[0048] use Eco The RI enzyme partially digested the metagenomic DNA of the papermaking wastewater sediment extracted in Example 1, and electrophoresis recovered a 2.5-7.5 kb digested fragment. Combine the recovered DNA fragments with the Eco RI digested and dephosphorylated pUC118 vector (Takara company) ligation, electroporation transformation E. coli DH5α competent cells were spread on LB solid plates containing 100 μg / mL ampicillin (Amp), 40 μg / mL X-gal and 0.5 mM IPTG, thereby constructing a library with a capacity of 10 7 Metagene library with multiple transformants and good diversity.

[0049] 2. Screening and sequence analysis of target genes

[0050] Randomly pick the clones on the plate and transfer them in parallel to CMC plates containing 100 μg / mL A...

Embodiment 3

[0052] Example 3 Obtaining of recombinant protein expressed by DNA sequence

[0053] 1. Inoculate the positive clones obtained in Example 2 into 5 mL LB medium (100 μg / mL Amp), culture on a shaker at 37 °C and 220 rpm for 12 h, and extract the plasmid.

[0054] 2. Design primers P1 and P2. Both ends of the primers are respectively introduced into the Saccharomyces cerevisiae expression vector pyes2 Eco R I and xho I Restriction site.

[0055] The nucleotide sequence of primer P1 (upstream primer) is shown in SEQ ID NO:3:

[0056] The nucleotide sequence of primer P2 (downstream primer) is shown in SEQ ID NO:4:

[0057] 3. Using the extracted plasmid as a template, PCR amplification was performed using primers P1 and P2.

[0058] The total volume of the PCR system is 50 μL, including Prime STAR TM Max Premix 25 μL (2.5 U), two primers each 2 μL (10 μM), sterilized distilled water 20 μL and template DNA 1 μL (1 ng).

[0059] PCR amplification conditions: 98°C pre-denat...

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Abstract

The invention discloses an alkaline cellulase gene and application thereof in the field of textiles or detergents. The nucleotide sequence is disclosed as SEQ ID NO:1, and the coding amino acid sequence is disclosed as SEQ ID NO:2. The gene is a new DNA sequence which is obtained from soil metagenome by a metagenome technique and has functions which are not researched. The researches on the structure and functions of the protein coded by the DNA sequence prove that the recombinant protein expressed by the DNA sequence has favorable alkali-resistant cellulose enzyme activity, and part of metal ions and surfactants can obviously enhance the activity of the recombinant protein decomposed cellulose substrate. The expression product of the DNA sequence can be used as a new enzyme source in textile or detergent industry, and has wide industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of polypeptides. More specifically, it relates to an alkaline cellulase and its DNA sequence and application. Background technique [0002] Cellulose is the main component of plant cell walls, and it is also the most abundant and cheapest renewable resource on the earth. The annual dry mass of plants produced by photosynthesis is as high as 22 billion tons, of which cellulose accounts for 50%, so it can use cellulose Microorganisms are characterized by species diversity and wide distribution. Cellulases are found in microorganisms, plants, insects and even mammals, among which cellulases derived from fungi, actinomycetes and bacteria have been studied to some extent. According to reports, so far, a variety of cellulase genes have been cloned from more than 40 kinds of bacteria and several fungi, among which the cellulase-producing fungi are roughly estimated to have reached 68 genera. However, because ce...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/63
Inventor 李刚肖月
Owner SUN YAT SEN UNIV
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