Recombined pichia pastoris strain for commonly expressing glucamylase and alpha-amylase and construction method thereof as well as mixed enzyme preparation

A technology of glucoamylase and Pichia pastoris, applied in the biological field, can solve the problem of no simultaneous expression of α-amylase, and achieve the effects of reducing reverse reactions, improving hydrolysis efficiency, and reducing costs

Active Publication Date: 2013-05-22
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no one has expressed glucoamylase and α-amylase simultaneously in Pichia past

Method used

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  • Recombined pichia pastoris strain for commonly expressing glucamylase and alpha-amylase and construction method thereof as well as mixed enzyme preparation
  • Recombined pichia pastoris strain for commonly expressing glucamylase and alpha-amylase and construction method thereof as well as mixed enzyme preparation
  • Recombined pichia pastoris strain for commonly expressing glucamylase and alpha-amylase and construction method thereof as well as mixed enzyme preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Construction and transformation of recombinant expression vector pPIC9KGla

[0034] 1.1 Primer design

[0035] R.pGAf (SEQ ID NO.3): ATGCGTTATGCAACCCCGC

[0036] R.pGTr (SEQ ID NO.4): GGCGTTATTTATTACCCTCTTTTGACC

[0037] R.pGEf (SEQ ID NO.5): G GAATTC ATGCGTTATGCAACCCCGC

[0038] R.pGNr (SEQ ID NO.6): TT GCGGCCGC TTACCCTCTTTTGACCA

[0039] 1.2 Amplification of cDNA sequence of Mucor pusillus glucoamylase

[0040] Using Rhizomucor pusillus (purchased from the General Microbiology Center of the Chinese Microbial Culture Collection and Management Committee in Beijing, CGMCC, strain number 3.204) cDNA first strand as a template, PCR amplification was carried out with R.pGAf / R.pGTr primers , Cloned the glucoamylase (Gla) gene, its sequence is shown in SEQ ID NO.1. PCR reaction conditions: 94°C for 5min; 94°C for 30s, 60°C for 30s, 72°C for 2min, 20 cycles (annealing temperature decreased by 0.5°C for each cycle); 94°C for 30s, 54°C for 30s, 72°C for 2min, 15 cycles; 72℃...

Embodiment 2

[0055] Example 2. Construction and transformation of recombinant expression vector pPICZαAmy

[0056] 2.1 Primer design

[0057] R.pA-f (SEQ ID NO.7): ATGAAATTCAGCATCTCTCTCTCGG

[0058] R.pA-r (SEQ ID NO.8): TTAAGCAGAGGTGAAGATAGCGGA

[0059] R.pAEf (SEQ ID NO.9): GAATTC AGCCCTTTGCCCCAACAGCA

[0060] R.pANr (SEQ ID NO.10): TT GCGGCCGC TTAAGCAGAGGTGAAGATAG

[0061] 2.2 Amplification of Mucor pusillus α-amylase

[0062] Using the first strand cDNA of Mucor pusillus as a template, PCR amplification was carried out with primers R.pA-f / R.pA-r, and the α-amylase (Amy) gene was cloned. Its sequence is shown in SEQ ID NO.2. . PCR reaction conditions: 94°C for 5min; 94°C for 30s, 65°C for 30s, 72°C for 1min30s, 30 cycles (annealing temperature decreased by 0.5°C for each cycle); 94°C for 30s, 50°C for 30s, 72°C for 1min30s, 10 cycles; 72℃10min, store at 4℃. The PCR products were subjected to agarose gel electrophoresis, recovered and connected to pMD19-T Simple Vector, and transformed into E...

Embodiment 3

[0071] Example 3. Verification of amylase activity produced by recombinant Pichia pastoris transformants

[0072] (1) Pick the recombinant Pichia strains KM71 / 9KGla, KM71 / ZαAmy, KM71 / 9KGla-ZαAmy, and inoculate a single colony of BMMY plates containing 2% soluble starch. Place them in an incubator at 30°C, and grow them on the plate every 24h. Add 200ul filtered and sterilized absolute ethanol to the lower layer;

[0073] (2) Develop the color with iodine solution after 2-3 days, observe the size of the hydrolysis circle around the transformant, and judge whether the transformant successfully expresses Mucor pusillus amylase.

[0074] The result is figure 2 As shown, hydrolysis circles appear around the recombinant Pichia pastoris transformants, indicating that the recombinant Pichia pastoris strains KM71 / 9KGla, KM71 / ZαAmy, and KM71 / 9KGla-ZαAmy can secrete and express amylase.

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Abstract

The invention provides a recombined pichia pastoris strain for commonly expressing glucamylase and alpha-amylase and a construction method of the recombined pichia pastoris strain as well as a mixed enzyme preparation prepared from the recombined pichia pastoris strain. The recombined pichia pastoris strain contains glucamylase genes from tiny mold funli and alpha-amylase genes at the same time; and gene sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO.2. According to the mixed enzyme preparation prepared from the recombined strain, the saccharifying enzyme activity is improved by 79% when being compared with that of a strain which only carries the glucamylase genes, and the starch liquefacation enzyme activity is improved by 183% when being compared with that of a strain which only carries the alpha-amylase genes. The optimum operation temperatures and the optimum operation pH (Potential of Hydrogen) of the two enzymes are close; and the two enzymes have the synergistic promoting effect in a starch hydrolytic process, so that the application potential and advantages are great.

Description

Technical field [0001] The present invention relates to the field of biology, in particular to a recombinant Pichia yeast strain co-expressing glucoamylase and alpha-amylase, a construction method thereof, and a mixed enzyme preparation prepared from the recombinant Pichia yeast strain. technical background [0002] As an important storage substance for plants, starch is one of the most abundant substances in the world. Amylase refers to a type of glycosidase that can hydrolyze starch to produce low molecular weight polysaccharides or monosaccharides. Amylase is widely used in starch hydrolysis or its fermentation industry, such as ethanol production, starch sugar production, beer brewing, etc. The most important amylases in current industrial applications are glucoamylase, alpha-amylase, pullulanase, etc. [0003] Glucoamylase (EC3.2.1.3), also known as glucoamylase, is an exonuclease that acts on the non-reducing end of malto-oligosaccharides to cut α-1,4 glycosidic bonds to ge...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/34C12N9/30C12R1/84C12R1/645
Inventor 魏东芝高蓓何正贵
Owner EAST CHINA UNIV OF SCI & TECH
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