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Alpha-glucosidase, gene thereof, preparation method, vector and host cell

A technology of glucosidase and recombinant host cells, applied in botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low enzyme activity, limited expression, limited application, etc., to achieve Effects of high-efficiency expression, strong sucrose hydrolysis function, high temperature resistance and thermal stability

Active Publication Date: 2013-04-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the codon preference of the gene promoter encoding the enzyme is different from that of bacteria, but similar to that of eukaryotes, the T7 strong promoter cannot be used for high expression in E. coli, which limits the expression of the enzyme quantity
In addition, enzymatic hydrolysis of sucrose is also a way to industrially produce glucose. Although the above-mentioned α-glucosidase of Sulfobus solfataricus also has the function of hydrolyzing sucrose, its enzyme activity is not high. Its application in catalyzing the hydrolysis of sucrose

Method used

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  • Alpha-glucosidase, gene thereof, preparation method, vector and host cell
  • Alpha-glucosidase, gene thereof, preparation method, vector and host cell

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Embodiment 1

[0038] Embodiment 1. Containing the construction of the recombinant cloning vector pGEM-TGlu plasmid of the sequence shown in SEQ ID: NO.1 and transformed strain DH5α (pGEM-TGlu)

[0039] 2L of water samples were collected from Hamazui Hot Spring (pH8, temperature 78°C) in Tengchong, Yunnan, and the bacteria in the water samples were collected by filtration through a 0.22 μm microporous membrane (Milipore Company), and washed with STE buffer (0.1M NaCl; 10mM Tris -HCl pH8.0; 1mM EDTA pH8.0) to wash the bacteria on the filter membrane, and centrifuge at 4000rpm for 10 minutes to obtain a fresh wet weight of about 0.5g of the bacteria. UltraClean for bacteria sediment TM The total DNA solution (100 ul, 0.4 μg / μl) was extracted by Soil DNAKit, and the absorbance ratio of the total DNA solution was determined: A260 / A280=1.947, A260 / A230=2.15. Take 0.2 μl of the above total DNA solution as a template, and design primers according to the upstream and downstream sequence information...

Embodiment 2

[0042] The construction of embodiment 2.α-glucosidase expression vector and transformed bacterial strain:

[0043] Both the pGEM-TGlu plasmid and the plasmid pET28a were digested with Nhe I and HindIII (50 μl digestion system contains 20 μg of plasmid, 20 U of each restriction enzyme, digested at 37°C for 4 hours), and the gel was cut by agarose electrophoresis After recovering the target insert and pET28a digested fragment (recovered in 20 μl deionized water respectively), take the two digestion products and connect (5 μl ligation system contains 1 μl pET28a digested fragment, 3 μl target insert fragment, 1 μl Promega T4 DNA ligation Enzyme, ligated overnight at 16°C) to construct a pETGlu recombinant expression vector, take 2 μl of the ligated product for electric shock (voltage 1.8KV, capacitance 25μF, electric shock time 3ms) to transform Escherichia coli BL21 (DE3) to obtain a recombinant expression vector containing SEQ ID: NO.1 The transformed strain BL21(DE3)(pETGlu) e...

Embodiment 3

[0044] Example 3. Preparation of α-glucosidase gene mutants and construction of transformed strains thereof

[0045]Using Stratagene's site-directed mutagenesis kit (QuikChange site-directed mutagenesis kit), using the expression vector pETGlu of Example 2 as a template, referring to the instructions, by introducing mutant bases into the primers and performing PCR amplification, SEQ ID: NO. 1 Perform the following 4 site-directed mutations: (1) mutation of A at position 211 to G; (2) mutation of G at position 272 to A; (3) mutation of C at position 1546 to T, and mutation of T at position 1571 to C (4) A mutation at position 211 to G, G at position 272 to A, C at position 1546 to T, and T at position 1571 to C. After the resulting mutant products were digested with Dpn I (20 μl system containing 15 μl PCR product and 5U Dpn I, digested at 37° C. for 2 hours), each 2 μl was electrotransformed (the same conditions as in Example 2) to E. coli XL1-Blue. state cells, obtain vector...

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Abstract

The invention discloses an Alpha-glucosaccharase which has amino acid sequences shown in SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6, SEQ ID: NO.8 and SEQ ID: NO.10. Or substitution, depletion or addition of one or more amino acids is carried out to the amino acid sequences shown in the SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6, SEQ ID: NO.8 or SEQ ID: NO.10 to obtain amino acid sequences of the Alpha-glucosaccharase with the same activity. The invention also provides a gene for coding the Alpha-glucosaccharase, a recombinant vector containing the gene and a host cell containing the recombinant vector as well as a preparation method of the Alpha-glucosaccharase. The Alpha-glucosaccharase provided by the invention can utilize a prokaryotic expression system for high-effective expression and has the advantages of good high-temperature resistance (the optimum temperature of 95 DEG C) and good thermal stability as well as comparatively powerful function of saccharose hydrolysis.

Description

technical field [0001] The invention relates to an α-glucosidase, its coding gene and preparation method, as well as a recombinant vector and a recombinant host cell containing the gene. Background technique [0002] α-glucosidase (α-glucosidase, EC 3.2.1.20) is a glycoside hydrolase that hydrolyzes the non-reducing terminal glucose unit connected by α-1,4-glucosidic bonds, and releases D-glucose at the same time, which can be used For converting maltose and maltodextrin into glucose, some α-glucosidases can also hydrolyze sucrose into a mixture of glucose and fructose, so α-glucosidase has important application value in the sugar industry, such as α-glucosidase Glucosidase contributes to the complete hydrolysis of starch to obtain high-yield glucose syrup, and can also be used to hydrolyze maltose produced by pullulanase [Sun et al., Plant Physiol. (Plant Physiol.) 94: 320-327, 1990; Biochemistry and Biology Advances in Physics (Arch Biochem Biophys.) 284:298-305, 1991], s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/26C12N15/56C12N1/21C12R1/19
Inventor 马延和周成薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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