Glucoamylase MhglaA from myceliophthora heterothallica, gene for coding glucoamylase MhglaA, and application of glucoamylase MhglaA to glucose production

A technology of glucoamylase and myceliophthora, applied in the field of genetic engineering and biology

Active Publication Date: 2020-07-07
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Volkov PV et al. (Glucoamylases from Penicillium verruculosum and Myceliophthora thermophila: analysis of differences in activity against polymeric substrates based on 3D model structures of the interactenzymes. Biochimie, 2015, 110:45-51) studied the Myceliophthora thermophila glucoamylase 2M The enzyme was obtained by fractional purification dire

Method used

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  • Glucoamylase MhglaA from myceliophthora heterothallica, gene for coding glucoamylase MhglaA, and application of glucoamylase MhglaA to glucose production
  • Glucoamylase MhglaA from myceliophthora heterothallica, gene for coding glucoamylase MhglaA, and application of glucoamylase MhglaA to glucose production
  • Glucoamylase MhglaA from myceliophthora heterothallica, gene for coding glucoamylase MhglaA, and application of glucoamylase MhglaA to glucose production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Construction of FMDV 2A peptide-mediated system for recombinant expression of glucoamylase MhGlaA from Myceliophthora thermophila

[0035] 1. Construction of glycoamylase MhGlaA recombinant expression vector

[0036]The plasmid pAN52-bar (Gu SY, Li JG, Chen BC, Sun T, Liu Q, Xiao DG, TianCG. Metabolic engineering of the thermophilic filamentous fungus Myceliophthora thermophila to produce fumaric acid. Biotechnology for Biofuels. 2018, 11: 323.) was used as Backbone constructs expression vectors. Using Myceliophthora thermophila glucoamylase (Mycth_72393; XuGB, Li JG, Liu Q, Sun WL, Jiang M, Tian CG. Transcriptional analysis of Myceliophthora thermophila on soluble starch and role of regulator AmyR on polysaccharide degradation. Bioresource technology.2018, 265: 558-562.) sequence as a reference, the analysis and comparison of biological information in the protein sequence encoded by Myceliophthora heterothaliana revealed the glucoamylase MhGlaA (Myche_75600...

Embodiment 2

[0073] Example 2, Phenotype Analysis of Glucoamylase Production by Myceliophthora thermophila Recombinant Strain

[0074] 1. Western blot detection of MhGlaA secretion in the recombinant strain of Myceliophthora thermophila

[0075] The 8 recombinant strains OE-MhglaA-gfp (T2, T6, T9, T12, T14, T18, T20, T23) with strong eGFP fluorescence expression and the wild-type strain WT were induced to produce glucoamylase, and the induction culture conditions were : Cultivate in 2% (2g / 100mL) water-soluble starch medium (recipe: 50×Vogel's salt 2mL, water-soluble starch 2g, peptone extract 0.5g, constant volume to 100mL, autoclaved) at 45°C and 150rpm for 3d , the sample was centrifuged to take the supernatant, and the recombinant protein MhGlaA-9×His was detected by Western blot. The primary antibody used was His-Tag rabbit monoclonal antibody, and the secondary antibody was rabbit anti-IgG HRP antibody. The result is as figure 2 As shown in A, the recombinant protein MhGlaA-9×His ...

Embodiment 3

[0087] Example 3. Preparation of recombinant glucoamylase rMhGlaA and research on its enzymatic properties

[0088] 1. Preparation of glucoamylase rMhGlaA by fermentation

[0089] Place the recombinant strain T18 producing the highest level of glucoamylase on 2% (2g / 100mL) water-soluble starch medium (recipe: 50×Vogel's salt 2mL, water-soluble starch 2g, constant volume to 100mL, autoclaved) at 45°C For induction culture, the fermented liquid cultured for 5 days was taken at 4°C and centrifuged at 12,000×g for 30 min, the supernatant was collected and concentrated by 50kD ultrafiltration, and the protein concentrate was purified according to Qiagen’s Ni-NTA Matric operation manual.

[0090] Use buffer A (20mmol / L NaH 2 PO 4 , 500mmol / L NaCl and 20mmol / L imidazole, pH7.4) to equilibrate the column, the sample flows through the Ni-NTA purification column at a flow rate of 1mL / min, and then buffer B (20mmol / L sodiumphosphate, 500mmol / L NaCl and 500mmol / L Li imidazole (pH 7.4) ...

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Abstract

The invention discloses a thermophilic fungus glucoamylase MhGlaA having an amino acid sequence as shown in SEQID NO.1, a gene for coding the thermophilic fungus glucoamylase MhGlaA, an expression boxmediated with FMDV 2A peptide and containing the gene and a GFP gene, and a recombinant expression carrier, and further discloses a method for increasing yield of glucoamylase through myceliophthorathermophila, and an application of the glucoamylase to hydrolyzed starch. The glucoamylase provided by the invention is wide in pH value application range, good in heat stability and notable in hydrolyzing effects on starch; particularly, the yield of the glucoamylase produced through an obtained optimal myceliophthora thermophila recombinant strain is increased by about 12.5 times than that of the glucoamylase produced through a wild strain, the vitality of the glucoamylase produced through the optimal myceliophthora thermophila recombinant strain is improved by about 8.5 times than that of the glucoamylase produced through the wild strain, and the starch hydrolyzing effects of the fermentation liquid of the glucoamylase produced through the optimal myceliophthora thermophila recombinantstrain are notably better than those of the fermentation liquid of the glucoamylase produced through the wild strain, and therefore, the thermophilic fungus glucoamylase MhGlaA has higher applicationvalue.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biotechnology. Specifically, the present invention relates to a glucoamylase MhGlaA of Myceliophthora heterothaliana and its gene and production application. Background technique [0002] The full name of glucoamylase is Glucoamylase (Glucoamylase, EC 3.2.1.3). It is widely used in beer, monosodium glutamate, starch sugar, antibiotics and other industrial fields that require maximum conversion of starch into glucose. It is one of the most important enzymes in industrial production. . The main production strains of glucoamylase are molds, and Monascus, Aspergillus niger and Rhizopus are mostly used in my country. my country's glucoamylase is mainly refined and refined by submerged fermentation of excellent strains of Aspergillus niger. After decades of development, my country's starch processing enzyme industry has been able to independently produce bulk enzyme preparations such as glucoa...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/34C12N15/80C12P19/14C12P19/02
CPCC12N9/2428C12N15/80C12P19/02C12P19/14C12Y302/01003
Inventor 田朝光刘倩李芳雅刘丹丹张晨阳李金根孙涛孙文良
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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