Neutral alpha-galactosidase M-GALC, and coding gene and application thereof

A technology of galactosidase and gene, applied in the field of neutral α-galactosidase M-GALC and its coding gene and application

Active Publication Date: 2013-09-25
TIANJIN BIOFEED TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chaetomium globosom (chaetomium globosom) belongs to Ascomycota, Discomycotina, Chaetomycetes, Chaetomaceae, Madura clade genus, currently there is no α-galactosidase gene derived from Chaetomium at home and abroad to report

Method used

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  • Neutral alpha-galactosidase M-GALC, and coding gene and application thereof
  • Neutral alpha-galactosidase M-GALC, and coding gene and application thereof
  • Neutral alpha-galactosidase M-GALC, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the acquisition of target protein M-GALC and its coding gene

[0040] A chaetomium globosom strain MK was screened from soil samples collected from a soybean product processing factory. Using the genomic DNA of the strain as a template, PCR amplification was performed with a pair of degenerate primers designed by ourselves, and a length of 0.8 The bp DNA fragment was used as a template, and Tail-PCR was performed with self-designed nested primers and degenerate primers to obtain a 3800bp gene fragment, the nucleotide sequence of which was shown in sequence 3 of the sequence listing. The sequence was analyzed to find an open reading frame of 2244bp, and its nucleotide sequence is shown in sequence 2 of the sequence listing.

[0041] The total RNA of the above-mentioned chaetomium globosom strain MK was extracted, and cDNA was obtained by reverse transcription; using the cDNA as a template, the following primers were used for PCR amplification:

[0042] QF:...

Embodiment 2

[0045] Embodiment 2, the preparation of target protein M-GALC

[0046] 1. Construction of recombinant expression vector

[0047] The total RNA of the above-mentioned chaetomium globosom strain MK was extracted, and cDNA was obtained by reverse transcription; using the cDNA as a template, the following primers were used for PCR amplification:

[0048] ZYF: 5'- GAATTC ATGAGTTTCGCTTCAATGTC-3' (ECOR I recognition sequence is underlined);

[0049] ZYR: 5'- GCGGCCGC CTACTGCCTCTCGAGCAGGA-3' (not I recognition sequence is underlined);

[0050]The PCR product was recovered and sequenced to obtain a DNA fragment connected to the 5' end of sequence listing sequence 2 with the ECOR I restriction recognition sequence and the 3' end with the Not I restriction recognition sequence. After the DNA fragment was double-digested with EcoR I and Not I, it was connected to the vector backbone fragment of the prokaryotic expression vector pET-30a(+) (purchased from Novegon) that had been cut w...

Embodiment 3

[0083] Example 3, Characterization of target protein M-GALC as α-galactosidase

[0084] In this example, the eluate obtained from the recombinant bacterium X in step 3 of Example 2 was used as the detection object.

[0085] 1. Optimum temperature and temperature stability

[0086] 1. Optimum temperature

[0087] Carry out according to the method in step 6 of Example 2, the difference lies in: the α-galactosidase enzyme activity is measured under the condition of temperature of 30, 37, 42, 45, 50, 55, 60 or 65°C.

[0088] When the temperature is 42°C, the measured enzyme activity is 0.35U / ml, which is recorded as 100%, and the percentage of the measured enzyme activity relative to 0.35U / ml under different temperature conditions is calculated, that is, the relative enzyme activity (%) . The experiment was repeated 3 times, and the results were averaged, such as image 3 shown.

[0089] image 3 Among them, when the reaction temperatures were 30, 37, 42, 45, 50, 55, and 60°...

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Abstract

The invention discloses a neutral alpha-galactosidase M-GALC, and a coding gene and application thereof. The protein M-GALC has alpha-galactosidase activity, is derived from chaetomium globosum, and is (a) or (b) as follows: (a) protein composed of amino acid sequence disclosed as Sequence 1 in the sequence table; or (b) protein derived from Sequence 1 in the sequence table with alpha-galactosidase activity, which is subjected to substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence disclosed as Sequence 1 in the sequence table. The specific activity of the protein M-GALC disclosed by the invention as the alpha-galactosidase is 1.91U / mg; the optimum pH value is 7.0; the optimum temperature is 42 DEG C; and the protein Z-GALC has high enzyme activity at the temperature of 37-50 DEG C under the pH value of 6.0-9.0. The invention provides a new way for preparing alpha-galactosidase, and has important application value.

Description

technical field [0001] The invention relates to a neutral α-galactosidase M-GALC, its coding gene and application. Background technique [0002] α-galactosidase (alpha-galactosidase, EC3.2.1.22), also known as melibiase, belongs to the class of exoglycosidases, which can specifically catalyze the hydrolysis of sugar chain ends in polysaccharides, glycolipids, and glycoproteins The α-1,6-galactosidic bond of the glucoside can not only hydrolyze oligosaccharides such as raffinose, stachyose and verbascose, but also hydrolyze heteropolysaccharides containing α-galactoside. As a new type of enzyme preparation, α-galactosidase has been widely used in the feed industry. [0003] Studies in recent years have shown that α-galactosidase, as a specific exogenous enzyme, is added to feed containing soybean meal, which can effectively promote the decomposition of α-galactoside substances in cake feed raw materials and improve feed quality. Improve the utilization of nutrients, elimina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/40C12N15/56C12N15/63C12N5/10C12N15/11C12N1/15C12N1/19C12N1/21C12P19/14C12P19/02
Inventor 段文娟杨禄良刘影王晓睿
Owner TIANJIN BIOFEED TECH CO LTD
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