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

A technology of glucosidase and recombinant host cells, which is applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low enzyme activity, limited application, limited expression, etc. High-efficiency expression, strong sucrose hydrolysis function, high temperature and thermal stability

Active Publication Date: 2009-03-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 Sulfolobus solfataricus also has the function of hydrolyzing sucrose, its enzymatic 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
  • Alpha-glucosidase, gene thereof, preparation method, vector and host cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0039] Collect 2L of water sample from Toad’s mouth hot spring (pH8, temperature 78℃) in Tengchong, Yunnan, filter through 0.22μm microporous membrane (Milipore) to collect the bacteria in the water sample, use STE buffer (0.1M NaCl; 10mM Tris) -HCl pH8.0; 1mM EDTA pH8.0) wash the bacteria on the filter membrane and centrifuge at 4000rpm for 10 minutes to obtain the fresh wet weight of the bacteria about 0.5g. UltraClean for bacterial pellet TM Soil DNAKit extracts the total DNA solution (100 μl, 0.4 μg / μl), and determines the absorbance ratio of the total DNA solution: A260 / A280=1.947, A260 / A230=2.15. Take 0.2μl of the above total DNA solution as a template, and design primers based on the upstream and downstream sequence information of the α-glycosidase in the Thermus thermophilus HB27 genome sequence in th...

Embodiment 2

[0040] Example 2. Construction of α-glucosidase expression vector and transformed strain:

[0041] Both pGEM-TGlu plasmid and plasmid pET28a were digested with Nhe I and HindIII (50μl digestion system containing 20μg plasmid, 20U each of the two restriction enzymes, digestion at 37°C for 4 hours), and cut the gel by agarose electrophoresis After recovering the target insert and pET28a digested fragments (recovered separately in 20μl deionized water), ligate the two digested products (5μl ligation system contains 1μl pET28a digested fragment, 3μl target insert, 1μl promega T4DNA ligase , Connect overnight at 16°C), construct a pETGlu recombinant expression vector, take 2μl of the ligation product and transform it into E. coli BL21 (DE3) by electric shock (voltage 1.8KV, capacitance 25μF, electric shock time 3ms) to obtain the recombinant expression containing SEQ ID: NO.1 The vector transformed strain BL21(DE3)(pETGlu).

Embodiment 3

[0042] Example 3. Preparation of mutants of α-glucosidase gene and construction of transformed strains

[0043] Using Stratagene's site-directed mutagenesis kit (QuikChange site-directedmutagenesis kit), using the expression vector pETGlu of Example 2 as a template, referring to the instructions, by introducing mutant bases in the primers and performing PCR amplification to SEQ ID: NO. 1 Carry out the following 4 kinds of site-directed mutations respectively: (1) A mutation at position 211 to G; (2) a mutation from G at position 272 to A; (3) a mutation from C at position 1546 to T, and a mutation from 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. The mutant products obtained were digested with Dpn I respectively (20μl system contains 15μl PCR product and 5U Dpn I, digested at 37°C for 2 hours), and 2μl each was electrotransformed (conditions are the same as in Example 2) E. coli XL1-Blue ...

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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 alpha-glucosidase and 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 (EC 3.2.1.20) is a glycoside hydrolase that hydrolyzes the terminal glucose unit of the non-reducing end connected by the α-1,4-glucosidase bond and releases D-glucose at the same time. It is used to convert maltose and maltodextrin into glucose, and some α-glucosidase can also hydrolyze sucrose into a mixture of glucose and fructose. Therefore, α-glucosidase has important application value in the sugar industry, such as α-glucosidase. Glucosidase helps the complete hydrolysis of starch to obtain high-yield glucose syrup, and can also be used to hydrolyze the maltose produced by pullulanase [Sun et al., Plant Physiol. 94:320-327, 1990; Biochemistry and Advances in Biophysics (Arch Biochem Biophys.) 284:298-305, 1991], some α-glucosidase enzymes c...

Claims

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Application Information

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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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