Schizosaccharomyces pombe engineering strain having cellulase activity and constructing method thereof

A technology of Schizosaccharomyces pombe and cellulase, applied to other methods of inserting foreign genetic materials, genetic engineering, botanical equipment and methods, etc., can solve the problems of few research reports, achieve high product expression level and low cost Low, the effect of improving screening efficiency

Inactive Publication Date: 2008-04-09
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of Schizosaccharomyces pombe as a host has not attracted attention until recently. There are many examples of foreign proteins being successfully expressed by using Schizosaccharomyces pombe, but there are very few domestic research reports

Method used

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  • Schizosaccharomyces pombe engineering strain having cellulase activity and constructing method thereof
  • Schizosaccharomyces pombe engineering strain having cellulase activity and constructing method thereof
  • Schizosaccharomyces pombe engineering strain having cellulase activity and constructing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the cloning of Trichoderma viride cellulase gene

[0054] 1. Extraction of Genomic DNA from Trichoderma viride (T.viride AS3.3711)

[0055] YPD medium (PH=5.3): 1% yeast extract, 2% tryptone, 2% glucose, 2% agar.

[0056] (1) Cultivation of mycelia: Cultivate Trichoderma viride with YPD solid medium for 2.5 days, then culture with YPD liquid medium for 3 days, at 28° C., 190 r / min.

[0057] (2) The thalline is collected by centrifugation, and the collected thalline is first washed three times with distilled water, and then vacuum-dried.

[0058] (3) Add 0.3ml of CTAB extract to a 1.5ml centrifuge tube, and then place it in a 65°C water bath to preheat.

[0059] (4) Take 0.03 g of the above-mentioned dried bacterial cells, grind them with liquid nitrogen and quickly grind them into powder.

[0060] (5) Quickly transfer the frozen powder into the extraction solution, mix it with a pipette gun as soon as possible, and place it in a water bath at 65°C for 3...

Embodiment 2

[0127] Example 2, Construction of cellulase gene recombinant Schizosaccharomyces pombe expression vector

[0128] The construction process of recombinant Schizosaccharomyces pombe expression vectors pESP-2-EGI, pESP-2-CBHII, and pESPUC-BGLI containing the target gene is shown in Figures 3 and 4, and the specific steps are as follows:

[0129] 1. Construction of recombinant Schizosaccharomyces pombe expression vectors pESP-2-EGI, pESP-2-CBHII, pESPUC-BGLI

[0130]A large number of recombinant cloning vectors pMD18-T-CBHII, pMD18-T-EGI, pMD18-T-BGLI and expression vectors pESP-2 (purchased from Stratagene, USA) and pESPUC (preserved in our laboratory) plasmid DNA were extracted in large quantities by alkaline lysis, and then The recombinant cloning vectors pMD18-T-CBHII, pMD18-T-EGI, pMD18-T-BGLI and the expression vectors pESP-2 and pESPUC of Schizosaccharomyces pombe were respectively double-enzymatically digested. Enzyme digestion system 20μl, pMD18-T-CBHII, pMD18-T-EGI and ...

Embodiment 3

[0136] Example 3, Transformation and identification of recombinant Schizosaccharomyces pombe expression vector

[0137] 1. A large amount of plasmid DNA of recombinant Schizosaccharomyces pombe expression vectors pESP-2-EGI, pESP-2-CBHII, pESPUC-BGLI was extracted by alkaline lysis method

[0138] 2. Lithium acetate (LiAc) conversion method

[0139] Host bacteria: SP-Q01

[0140] TE Buffer: 10 mM Tris·Cl, 1 mM EDTA (PH=8.0).

[0141] 0.2M LiAc: 0.21g LiAc was dissolved in 100ml distilled water.

[0142] 70% PEG4000: 35g PEG4000 dissolved in 50ml distilled water.

[0143] (1) Spread 30ml of the SP-Q01 bacterial solution taken out of the -80°C refrigerator on the YPD solid medium, and incubate at 30°C for 2-3 days until a single colony appears.

[0144] (2) Cultivate a single colony of yeast overnight in 10ml YPD liquid medium at 30°C, 240rpm, and transfer 10ml of the bacterial liquid into 100ml of YPD medium at 30°C, 240rpm the next day to make the OD 600 Reach 0.7 to 1.0....

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Abstract

The invention discloses an engineering bacteria of fission yeast schizosaccharomyces pombe which has cellulase activity and a construction method thereof. A constructed expression carrier of the fission yeast schizosaccharomyces pombe which contains cellulase genes is led into the yeast schizosaccharomyces pombe and the fission yeast schizosaccharomyces pombe provided by the invention is obtained. The construction method of engineering bacteria of fission yeast schizosaccharomyces pombe is that the fission yeast schizosaccharomyces pombe which contains cellulase genes is constructed and then led into the fission yeast schizosaccharomyces pombe to gain a recombinant yeast transformant, and then the recombinant yeast cultured to lead cellulase genes to express the carrier. The invention makes up the shortcomings of the traditional preparation method of cellulase, a prokaryotic expression system, and the expression system of saccharomyces cerevisiae and pichia. The invention is applied to the production of industrial cellulase and has advantages of high activity of cellulase, short growth cycle, and low cost of extraction.

Description

technical field [0001] The invention relates to a Schizosaccharomyces pombe engineering bacterium with cellulase activity and a construction method thereof. Background technique [0002] The enzymatic research on cellulase began in 1912, and it was not until 1954 that the American Reese proposed the concept of Cl-CX. Since then, research has proved that cellulase is a general term for a group of enzymes that degrade cellulose to generate glucose. Not a single enzyme, including a variety of hydrolytic enzyme members, constitute a complex cellulase family. It is generally believed that at least three different but complementary cellulase enzymes are required to completely degrade cellulose. [0003] (1) Endoglucanase (endo-1, 4-β-D-glucanase, EC 3.2.1.4, from fungi referred to as EG, from bacteria referred to as Len), also known as C1 enzyme, this type of enzyme acts on fiber The non-crystalline region inside the protein randomly hydrolyzes the β-1,4-glucosidic bond, truncat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/56C12N15/65C12N15/87C12N15/81
Inventor 王丕武付永平万丽娜
Owner JILIN AGRICULTURAL UNIV
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