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Yeast engineering strain expressing chaetomium thermophilum gla gene and constitution method

A technology of Chaetomium thermophilus and yeast engineering, which can be used in genetic engineering, microorganism-based methods, plant genetic improvement, etc., and can solve problems such as no reports of thermophilic saccharification enzymes.

Inactive Publication Date: 2007-11-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chaetomium thermophilum (Chaetomium thermophilum) is a thermophilic fungus that is widely distributed and has a high growth upper limit temperature. Various thermophilic enzymes have been isolated from this fungus, but no thermophilic glucoamylase has been found in this fungus. report

Method used

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  • Yeast engineering strain expressing chaetomium thermophilum gla gene and constitution method
  • Yeast engineering strain expressing chaetomium thermophilum gla gene and constitution method
  • Yeast engineering strain expressing chaetomium thermophilum gla gene and constitution method

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Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0083] Embodiment 1: Cloning method of Chaetomium thermophilium glucoamylase gla gene:

[0084] (1) Extraction of RNA: Total RNA was extracted according to the instructions of the Trizol kit.

[0085] (2) Synthesis of the first strand of cDNA: the first strand of cDNA was synthesized using Oligo(dT)20 as a primer according to the instructions of the Reverse Transcription Reaction kit from Promega. The reaction conditions are: 42°C for 1 hour; 95°C for 5 minutes.

[0086] (3) PCR reaction: polymerase chain reaction (PCR) reagents and conditions are:

[0087] First mix the following reagents together:

[0088] 10X reaction buffer 2.5μl

[0089] 25mM MgCl 2 2μl

[0090] 10mM dNTP 2μl

[0091] Upstream primer (10μM) 1μl

[0092] Downstream primer (10μM) 1μl

[0093] Template cDNA 2μl

[0094] Taq DNA polymerase 0.5μl

[0095] Sterilized double distilled water 14μl

[0096] Total volume 25μl

[0097] The PCR reaction conditions were: pre-denaturation at 95°C for 4 ...

Embodiment approach 2

[0103] Embodiment 2: For the nucleotide sequence and amino acid sequence of the Chaetomium thermophilium glucoamylase ebhl gene, see the "Summary of the Invention" section.

Embodiment approach 3

[0104] Embodiment 3: Construction of expression vector

[0105] (1) Design primers according to the full coding sequence of the mature peptide of cbhl gene, and introduce SnaB I and Not I respectively

[0106] Restriction sites:

[0107] Forward primer ep-gla5: 5'-GCG GCGGTCGATTCCTACATTG-3' (SnaB I)

[0108] Reverse primer ep-gla3: 5'-GTC TCACCAGTGGTCTTGACCAC-3' (Not I)

[0109] Polymerase chain reaction was carried out using the reverse-transcribed cDNA of Chaetomium thermophila total RNA as a template

[0110] (2) Take 2 μl of the PCR product and connect it to the pGEM-T easy vector, and the operation steps are carried out according to the product manual of Promega Company. Then the ligation product was transformed into Escherichia coli DH5α strain, and the surface was coated with 5-bromo-4-chloro-3-indole-β-D-galactoside and X-gal on the LB plate containing ampicillin (100 μg / mL) Grow overnight. Pick white colonies and culture them overnight in LB liquid medium. P...

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Abstract

This invention is a genetically engineered strain of yeast Pichia pastoris GS-GLA-22, RT-PCR and RACE Chaetomium from thermophilic bacteria chaetomium thermophilum was glucoamylase gla gene fragment was cloned into the Pichia pastoris secreted expression vector pPIC9K One of recombinant GS-GLA-22 by 6 days induction, glucoamylase protein expression levels of 0.86 mg / mL, its activity is 16.73 U / mL for starch processing and other industrial areas, has a very significant social and economic value.

Description

(1) Technical field [0001] The invention relates to a genetically engineered strain of yeast expressing a thermostable exoglucoamylase gene gla and a construction method thereof. (2) Background technology [0002] Glucoamylase, also known as glucoamylase [Glucoamylase, systematically named as starch α-1.4-glucan glucohydrolase, α-1.4-Glucanglucohydrolase (EC.3.2.1.3.)], is an enzyme with exolytic activity, It can hydrolyze starch, dextrin, glycogen, etc. from the non-reducing end of the α-1.4 glucosidic bond to obtain the final product β-D-glucose. It can also slowly hydrolyze the α-1.6 glucosidic bond and convert it into glucose. As one of the main enzymes in the process of converting starch into glucose, glucoamylase has important commercial value, and it has been widely used in brewing, food, medicine and other industrial and agricultural fields. Used in the production of liquor, rice wine, alcohol, vinegar, beer, calcium lactate, citric acid, monosodium glutamate, etc. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/56C12N15/81C12R1/84
Inventor 李多川陈静
Owner SHANDONG AGRICULTURAL UNIVERSITY
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