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High activity cellobiase, coding gene and applications thereof

A cellobiase and encoding gene technology, applied in the field of genetic engineering, can solve the problems of high sugar tolerance and low cellobiose hydrolysis activity, and achieve the effects of high expression, high recovery and high catalytic activity

Inactive Publication Date: 2015-01-28
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reports have shown that some microbial cellobiases have higher glucose inhibition constants and high glucose tolerance (Lu, J., et al., Expression and characterization of a novel highly glucose-tolerant beta-glucosidase from a soil metagenome.Acta Biochimica Et Biophysica Sinica, 2013.45(8):p.664-673.), but the hydrolysis activity of these enzymes to cellobiose is quite low

Method used

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  • High activity cellobiase, coding gene and applications thereof
  • High activity cellobiase, coding gene and applications thereof
  • High activity cellobiase, coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of Cellobiase Expression Strain

[0030] 1. Amplification of the Cellobiase Gene

[0031] According to the cellobiase gene sequence (GenBank: CP003184.1) of the highly homologous Thermoanaerobacterium saccharolyticum JW / SL-YS485 used in the present invention with the bacterial strain Thermoanaerobacterium aotearoense SCUT27 as a template, PCR primers were designed:

[0032] P1: 5'-TAGCCCCATATGGCTAATTTTCCAAAAGGT-3' (SEQ ID No: 3)

[0033]P2: 5'-TCCGTCTCGAGAAAAACAATTGAAGCTCTATTTAT-3' (SEQ ID No: 4)

[0034] NdeI and XhoI restriction sites were introduced at the 5' of the primers respectively, and a 6×His tag was introduced at the 3' end of the cellobiase gene, so that the expressed cellobiase had a histidine tag, and the nickel column was used to In the affinity chromatography method, the expressed cellobiase can be purified by affinity chromatography, so as to facilitate rapid and efficient acquisition of pure cellobiase.

[0035] Using Thermoanaerobacter...

Embodiment 2

[0046] Cellobiase expression and protein purification

[0047] The overnight cultured recombinant bacteria E.coli BL21(DE3) / pET-30a-bgl / pGro7 containing the cellobiase gene were inoculated at a ratio of 1:50 in the culture medium containing 20 μg / ml chloramphenicol, 50 μg / ml kanamycin Placed in fresh LB liquid medium for culture at 37°C and 250rpm. When the cell density OD 600 About 0.3-0.5, add the final concentration of 1mg / ml L-arabinose to induce the expression of chaperone protein and continue to culture at 37°C; add IPTG with a final concentration of 1mM to the OD of about 0.6-1.0 to induce the expression of cellobiase, continue at The culture was continued for 6 hours at 25° C. and 200 rpm. Such as image 3 As shown, the recombinant cellobiase protein accounts for about 15-25% of the total protein amount, reaching 12.29 mg / L fermentation broth.

[0048] Collect the cells by centrifugation at 4°C and 8000g, add 1ml of buffer A (20mM phosphate buffer, 500mM sodium chl...

Embodiment 3

[0050] Activity Determination of Recombinant Cellobiase

[0051] Enzyme Activity Determination The cellobiase activity obtained in Example 2 was measured using p-nitrophenylglucoside (pNPGlu) as a substrate.

[0052] Cellobiase enzyme activity assay method is: in 170μl citric acid (100mM) - disodium hydrogen phosphate

[0053] (50mM) pH 6.0 buffer solution, add 20μl 25mM pNPGlu, add 10μL enzyme solution, react at 60℃ for 5min, quickly add 600μL 1M Na 2 CO 3 Stop the reaction. Measure the absorbance value at 405nm, by querying p-nitrophenol (pNP) and A 405 Between the standard curve, the amount of pNP generated by catalysis was obtained.

[0054] The unit of cellobiase activity was defined as the amount of enzyme needed to catalyze the release of 1 μmol pNP in (U)1min.

[0055] The cellobiase specific enzyme activity obtained in embodiment 2 is 180.02U / mg, and the corresponding Michaelis constant K m is 0.69mM.

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Abstract

The invention discloses high activity cellobiase, a coding gene and applications thereof. The cellobiase is a (a) protein or a (b) protein. The (a) protein is composed of amino acid sequences represented by the SEQ ID No.2 of the sequence table; and the (b) protein is derived from the (a) protein by substituting and / or losing and / or adding one or several amino acid residues to the amino acid sequences represented by the SEQ ID No.2 of the sequence table, and has the high cellobiase activity. The cellobiase has a strong activity on decomposing cellobiose, and the maximal reaction speed can reach 740.39 + / - 10.23 U / mg. In the aspect of product glucose inhibition, the cellobiase has a very good tolerant performance, and the inhibition constant (Ki) is as high as 800 mM. Researches show that the provided cellobiase has the advantages of high catalytic activity, high tolerance, and heat resistance, and has a great potential in production, application, and promotion.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a cellobiase capable of efficiently decomposing cellobiose at a relatively high temperature, and the coding gene and application of the cellobiase. Background technique [0002] Cellobiase (EC 3.2.1.21, also known as β-glucosidase β-glucosidase) belongs to the class of exohydrolases. Its characteristic is that it can hydrolyze the non-reducing β-D-glycosidic bond bound to the terminal, and release β-D-glucose and the corresponding ligand at the same time; in addition, it can weakly hydrolyze p-nitrophenyl-β-D-semi Lactose and beta-D-xyloside. Cellobiase is not only an important component of cellulase, a key enzyme for degrading cellulose, but also often used as a flavor enzyme to improve the flavor of beer and fruit juice. [0003] At present, looking for cellobiases with high sugar tolerance has become a hotspot in the research of cellobiases, because this enzyme can increas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12N5/10C12P19/14C12R1/19
CPCC12N9/2445C12Y302/01021
Inventor 李爽阳芳
Owner SOUTH CHINA UNIV OF TECH
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