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Three kinds of recombinant glucoamylases and their preparation methods and applications

A technology of glucoamylase and enzyme activity, which is applied in the field of enzyme genetic engineering, can solve the problems of poor thermal stability and low specific activity of glucoamylase, and achieve the effect of good temperature resistance and high pH stability

Active Publication Date: 2020-06-09
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is aimed at two strains producing saccharification enzyme bacterial species Emerson Talaromyces emersonii (Talaromyces emersonii) and Aspergillus niger (Aspergillus niger), the former produces the higher specific activity of saccharification enzyme, but the thermostability of enzyme is relatively poor; The latter has better stability, but the existing problem that the specific activity of glucoamylase is not high, by recombining and mutating the structural domains of the two glucoamylase genes, at least three enzymes with better thermostability and higher enzyme activity are provided. High recombinant glucoamylase, preparation method and application

Method used

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  • Three kinds of recombinant glucoamylases and their preparation methods and applications
  • Three kinds of recombinant glucoamylases and their preparation methods and applications
  • Three kinds of recombinant glucoamylases and their preparation methods and applications

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Construction of gene cloning and expression vector

[0034] 1.1 Acquisition of glucoamylase recombinant gene

[0035] 1.1.1 Amplification of each domain of glucoamylase

[0036] According to the glycoamylase GATE (GenBank ID: AJ304803.1) and GAA1 (GenBank ID: HQ537427.1) sequences reported by NCBI, design primers P912GA1SF, P912GA1R, P912TESF, P912TER, GATE1F, GATE1R, GATE2F, GATE2R, GAA1F, GAA1R, GAA2F , GAA2R, according to the combined method shown in Table 1, the CD region, Linker region and SBD region of GAA1 and GATE were independently amplified (see Table 1 below), and each part of the product was cut and recovered to obtain the two glucoamylases Six domain fragments GAA1CD, GAA1L, GAA1SBD, GATECD, GATEL, GATESBD.

[0037] P912GA1SF: 5'-ATActcgagAAAAGGATGTCGTTCCGATCCTCTACTCGCCCTG-3'

[0038] P912GA1R: 5'-ATAAGAATgcggccgcTCAATGGTGATGGTGATGATGCACAGTGACATACCAGAGCGGGTCGC-3'

[0039] P912TESF: 5'-AAActcgagAAAAGGATGGCGTCCCTCGTTGCTGGCG-3'

[0040] P912...

Embodiment 2

[0067] Embodiment 2: Construction of Pichia pastoris engineering bacteria

[0068] The recombinant expression vectors pJ912-19-GAA1, pJ912-19-GATE, pJ912-19-GA1~pJ912-19-GA7 were electrotransformed into the host cell Pichia X-33, and positive transformants were obtained through colony PCR verification. Eight positive transformants of glucoamylase were fermented in 48-well plates, induced by methanol for 96 hours, enzyme activity was measured, and highly expressed recombinant glucoamylase GA1-GA6 and mutant glucoamylase GA7 Pichia engineering bacteria were screened.

Embodiment 3

[0069] Embodiment 3: Fermentation and enzyme activity assay

[0070] 3.1 Put the original glucoamylase GAA1 and GATE strains, the recombinant glucoamylase GA1~GA6 recombinant strains and the mutant glucoamylase GA7 strain on a shaker at 30°C, and use BMD1, BMM2 and BMM10 as the medium for methanol-induced fermentation. After induction for 96 hours, take fermentation The enzyme activity of the liquid supernatant was measured, and the enzyme activity of each glucoamylase was as follows: image 3 As shown, GA1 and GA2 recombinant glucoamylase strains and GA7 mutant glucoamylase strains with higher enzyme activity were screened out. The results showed that the enzyme activity of GAA1 was 8955.8U / mL, the activity of GATE was 27212.8U / mL, the activity of GA1, GA2, and GA7 were 22041.3U / mL, 17962.9U / mL, and 20163.8U / mL, respectively. Liveness increased by 146.1%, 100.6%, and 125.2%, respectively.

[0071] The medium composition is as follows:

[0072] BMD1: 0.2M potassium phosphat...

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Abstract

The invention provides at least three recombinant glucoamylases with good thermal stability and high enzymatic activity, a preparation method and application. The structural domains of genes of two glucoamylases, namely, GATE (GenBank ID: AJ304803.1) and GAA1 (GenBank ID: HQ537427.1) are recombined, and then mutation deletion is conducted to obtain the recombinant glucoamylases. The amino acid sequences of the three recombinant glucoamylases are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively. The invention further provides recombinant expression vectors for functional expression, recombinant strains containing the recombinant expression vectors and offspring of the recombinant strains. The recombinant expression vectors for functional expression are obtained by connecting nucleotide sequences encoding the glucoamylases with an expression vector.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of enzymes, and in particular relates to three recombinant saccharification enzymes obtained by recombination and mutation of two saccharification enzyme domains, and a preparation method and application thereof. Background technique [0002] Glucoamylase (EC.3.2.1.3.), also known as Glucoamylase (Glucoamylase, GA), is a single-chain acid glycoside hydrolase with exonuclease activity, and is the main enzyme that hydrolyzes starch to produce glucose. It can Hydrolyze starch from the non-reducing end of α-1,4 glucosidic bonds to produce glucose, and slowly hydrolyze α-1,6 glucosidic bonds to convert them into glucose, and also hydrolyze dextrin, releasing the non-reducing end of glycogen beta-D-glucose. There are two types of glucoamylase, type I (GAI) and type II (GAII). Most types lack the SBD region, and a few even lack the O-glycosylation linking domain. [0003] Glucoamylase is t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/34C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2428C12N15/815C12Y302/01003
Inventor 黎明袁颢瑜路福平
Owner TIANJIN UNIV OF SCI & TECH