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Schizosaccharomyces pombe engineering bacteria having hemicellulase activity and construction method thereof

A technology of Schizosaccharomyces pombe and hemicellulase, applied in the direction of microorganism-based methods, genetic engineering, botanical equipment and methods, etc., can solve the problems of few research reports and achieve high product expression level and low cost Effect

Inactive Publication Date: 2009-04-22
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 bacteria having hemicellulase activity and construction method thereof
  • Schizosaccharomyces pombe engineering bacteria having hemicellulase activity and construction method thereof
  • Schizosaccharomyces pombe engineering bacteria having hemicellulase activity and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the cloning of Aspergillus niger hemicellulase gene

[0044] 1. Extraction of Aspergillus niger genomic DNA

[0045] Cha's medium (PH=7.2): 30g sucrose, 3g sodium nitrate, 0.5g magnesium sulfate, 0.5 potassium chloride, 0.01g ferrous sulfate, 1g dipotassium hydrogen phosphate, 13g agar, add distilled water to 1L.

[0046] (1) Cultivation of mycelium: Cultivate Aspergillus niger with Zapei solid medium for 3-5 days, then culture with Zapei liquid medium for 3 days, at 28° C., 190 r / min.

[0047] (2) The thalline is collected by centrifugation, and the collected thalline is first washed three times with distilled water, and then vacuum-dried.

[0048] (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.

[0049] (4) Take 0.03 g of the above-mentioned dried bacterial cells, grind them with liquid nitrogen and quickly grind them into powder.

[0050] (5) Quickly transfer the frozen powder into the extr...

Embodiment 2

[0102] Example 2, Construction of hemicellulase gene recombinant Schizosaccharomyces pombe expression vector

[0103] The construction process of recombinant Schizosaccharomyces pombe expression vectors pESPUC-XYNB and pESPUC-XLND containing the target gene is as follows: Figure 2a , 2b As shown, the specific steps are as follows:

[0104] 1. Construction of recombinant Schizosaccharomyces pombe expression vectors pESPUC-XYNB and pESPUC-XLND

[0105] A large amount of recombinant cloning vectors pMD18-T-XYNB, pMD18-T-XLND and expression vector pESPUC (preserved in our laboratory) plasmid DNA were extracted in large quantities by alkaline lysis, and then the recombinant cloning vectors pMD18-T-XYNB, pMD18-T- XLND and S. pombe expression vector pESPUC. Digestion system 20μl, pMD18-T-XYNB and pESPUC were double-digested with restriction endonucleases XhoI and SalI, pMD18-T-XLND and pESPUC were double-digested with restriction endonucleases BamHI and NotI, and digested at 37°C...

Embodiment 3

[0111] Example 3, Transformation and identification of recombinant Schizosaccharomyces pombe expression vector

[0112] 1. Alkaline lysis method to extract a large amount of plasmid DNA of recombinant Schizosaccharomyces pombe expression vectors pESPUC-XYNB and pESPUC-XLND

[0113] 2. Electroporation conversion method

[0114] Host bacteria: SP-Q01

[0115] YCD medium: yeast extract powder 10g, glucose 20g, hydrolyzed casein 2g, sorbitol 218.6g dissolved in 1L distilled water

[0116] 1.2M Sorbitol: 218.604g sorbitol was dissolved in 1L of distilled water.

[0117] EMM Medium (L):

[0118] Sodium hydrogen phthalate 14.7mM Na 2 HPO 4 15.5mM

[0119] NH 4 Cl 93.5mM Glucose 111mM

[0120] Salts 2% (v / v) 50×stock Vitamins 0.1% (v / v) 1000×stock

[0121] Minerals 0.01% (v / v) 10000×stock

[0122] Salts(50×stock)(L)

[0123] MgCl 2 ·6H 2 O 0.26M CaCl 2 2H 2 O 4.99mM

[0124] KCl 0.67M Na 2 SO 4 14.1mM

[0125] Vitamins (1000×stock)

[0...

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Abstract

The invention relates to a Schizosaccharomyces pombe engineering bacteria with hemicellulase activity and a method for constructing the same. The Schizosaccharomyces pombe engineering bacteria is obtained by introducing a constructed recombinant Schizosaccharomyces pombe expression vector containing hemicellulase gene into Schizosaccharomyces pombe; and the construction method comprises the following steps: constructing a Schizosaccharomyces pombe expression vector containing the hemicellulase gene; introducing the Schizosaccharomyces pombe expression vector into the Schizosaccharomyces pombe to obtain a recombinant yeast transformant; and cultivating a recombinant yeast to secrete and express the enzyme gene of the hemicellulase. The invention makes up the defects of the prior method for preparing the hemicellulase, a prokaryotic expression system, and other yeast expression systems. When the Schizosaccharomyces pombe engineering bacteria is applied to the industrial production of the hemicellulase, the Schizosaccharomyces pombe engineering bacteria has the advantages of high enzyme activity, strong thermal stability, short growth cycle, and low extraction cost.

Description

technical field [0001] The invention relates to a Schizosaccharomyces pombe engineering bacterium with hemicellulase activity and a construction method thereof. Background technique [0002] Hemicellulase is a general term for a group of enzymes that degrade xylose, glucose, mannose and five-carbon sugars. Xylanase is one of the main enzymes of hemicellulase. Xylanase is a group of complex enzymes that can degrade xylan into oligosaccharides and xylose. Due to the complex molecular structure of xylan, it contains many different substituents. Therefore, the biodegradation of xylan requires the synergy between different components in a complex enzymatic system to convert it into basic monosaccharides. There are two enzymes acting on the backbone: β-1,4-endoxylanase and β-xylosidase. [0003] (1) β-1, 4-endo-xylanase (endo-1, 4-β-D-xylanxylanohydrolase, EC3.2.1.8.), in general, the role of endo-xylanase is to cut Opens the glycosidic chains within the xylan backbone and re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/56C12N15/81C12N9/42C12N9/24C12R1/85
Inventor 王丕武李晓婷付永平曲静
Owner JILIN AGRICULTURAL UNIV
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