Thermo philic alkali beta glucosidase and its coding gene

A glucosidase, alkaline technology, applied in genetic engineering, plant genetic improvement, enzymes, etc., can solve the problem of β-glucosidase not seen

Inactive Publication Date: 2006-06-14
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there are no related reports on β-glucosidases that can still have

Method used

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  • Thermo philic alkali beta glucosidase and its coding gene
  • Thermo philic alkali beta glucosidase and its coding gene
  • Thermo philic alkali beta glucosidase and its coding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Extraction of total DNA from Bacillus thermodenitrophilus NG80-2 (CGMCC No.1228)

[0037] Studies have shown that the gene encoding β-glucosidase can be isolated from the genome of Geobacillus thermodenitrificans NG80-2, which is especially suitable for degrading cellobiose under high temperature conditions. It maintains the high activity of the enzyme in a high temperature environment, which is different from the characteristics of the known β-glucosidase, and is a new type of β-glucosidase. Therefore, in this example, the thermophilic denitrification bacillus NG80-2 obtained from the oil well formation water separation of Guan 69-8 Block, Dagang Oilfield, Tianjin, China was used, and 3 ml of the fresh culture cultured overnight was used to collect the bacteria by centrifugation. , the cells were suspended in 250 microliters of 50mM Tris buffer (pH8.0), added 10 microliters of 0.4M EDTA (pH8.0), mixed well and incubated at 37°C for 20min, then added 30 microliters o...

Embodiment 2

[0048] Embodiment two Purification and properties of recombinant β-glucosidase of the present invention

[0049] Insert the above-mentioned recombinant strain E. Coli BL21GLUB monoclonal into 20ml of LB medium containing 50μg / ml Kan, culture at 37°C, 180rpm / min for 12 hours, and then insert the culture at 1% (V / V) inoculum 200ml of LB medium containing 50μg / ml Kan (total 10 shake flasks), 37°C, 220rpm / min culture A600 is 0.6, add IPTG to a final concentration of 0.2mM, 37°C, 150rpm / min induction for 4 hours. The cells were collected by centrifugation, suspended in 50 mM Tris-Cl (pH 7.5) buffer, and the cells were disrupted by ultrasonic waves. The centrifuged supernatant was the crude extract of recombinant β-glucosidase. The supernatant was purified by chelating Sepharose nickel affinity column chromatography, and the obtained enzyme preparation showed a band on SDS-PAGE. The basic properties of this recombinant β-glucosidase were determined using standard methods known ...

Embodiment 3

[0050] Embodiment three Recombinant β-glucosidase of the present invention hydrolyzes cellobiose

[0051] In the 2% cellobiose solution (prepared with pH7.5, 0.2M sodium phosphate buffer), add the purified enzyme solution prepared in the above-mentioned embodiment 2, and the addition amount is 2% of the volume of the cellobiose solution. One-tenth, heat up to 70°C for six hours. The above-mentioned recombinant thermophilic alkaline β-glucosidase hydrolyzes cellobiose to produce glucose as measured by high pressure liquid chromatography.

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Abstract

The invention relates to a beta-glucosaccharase and its coding gene. The enzyme can keep high enzyme activity at high temperature and alkalinity condition. Its optimum reaction temperature is about 70centigrade degree; its pH value is about 8.0. It can catalyze and hydrolyze beta-glucose glycoside link to form glucose. It has important application value in foods, medicine, weaving, energy source, and so on.

Description

technical field [0001] The invention relates to an enzyme and its coding gene, in particular to a thermophilic alkaline β-glucosidase and its coding gene. Background technique [0002] β-glucosidase (β-glucosidase, EC 3.2.1.21), also known as β-D-glucoside hydrolase, is an enzyme that can catalyze the hydrolysis of various β-glucosidic bonds. It is an important class of enzymes and has important application value in food, medicine, textile, energy and other aspects. The cellulose degrading enzyme system is roughly composed of three types of hydrolytic enzymes, namely cellulase (EC 3.2.1.4), exocellulase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21). In the process of cellulose degradation, the synergistic action of the above three enzymes is required to decompose cellulose into glucose. β-glucosidase (EC3.2.1.21) can hydrolyze cellobiose to glucose (Machida.M et al., Appl Environ Microbiol, 4:31-47, 1988), which is the rate-limiting step in the process of cellulose hydroly...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N1/21C12N9/42C12N15/63C12N15/70C12R1/19
Inventor 冯露王磊袁刚王威高春绪
Owner NANKAI UNIV
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