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Bacillus thermophilus capable of producing high-temperature and alkali resistant xylanase and application of bacillus thermophilus

A thermophilic bacillus, xylanase technology, applied in the direction of enzymes, bacteria, and microorganism-based methods, can solve the problem of xylanase impurity, alkali resistance and high temperature resistance range, and it is difficult to achieve high temperature resistance Alkaline xylanase research and development objectives and requirements and other issues

Active Publication Date: 2014-03-26
HUNAN HONGYING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] From this point of view, many international and domestic experts, scholars and scientific research staff have spared no expense in money, manpower and time to increase the research and development of high temperature and alkali resistant xylanase, aiming to apply biotechnology to papermaking, food , Feed and other fields to a higher, newer, stronger new era, whether it is the acquisition of new strains, or the use of genetic recombination technology, although such a large amount of financial resources and energy have been spent, the results are not satisfactory , it is difficult to achieve the research and development goals and requirements of high temperature resistance and alkali resistance xylanase, either temperature resistance is not alkali resistance; or alkali resistance is not temperature resistance; or the range of alkali resistance and high temperature resistance is not wide; and have cellulase activity; or the substrate is unsuitable, etc., it is necessary to isolate and breed a good strain that produces high-temperature-resistant alkaline xylanase

Method used

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  • Bacillus thermophilus capable of producing high-temperature and alkali resistant xylanase and application of bacillus thermophilus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Screening of departure strains

[0022] (1) Collect 2g soil sample from waste pulp from Xueli Paper Mill in Hunan Province, add it to a small triangular flask containing 50mL of cooled sterile water, shake at 200r / min for 20min, and then put it in a water bath at 80°C , water bath for 10min, shake the triangular flask to mix the soil sample, let stand for 5min, draw 100μL of supernatant, and dilute to 10 -1 ~10 -9 concentration, choose 10 -3 , 10 -4 , 10 -5 , 10 -6 Concentration coated beef extract peptone plate, incubated at 37°C for 24h, and continued to be incubated at 30°C for 24h.

[0023] (2) According to the characteristics of the shape, size, surface structure, edge structure, texture, luster, transparency, color, and soluble pigments produced by the microbial colony, use an inoculation loop to pick out an isolated single colony and move it to a screening culture. On the base plate, numbered and incubated at 34°C for 48h.

[0024] The screening...

Embodiment 2

[0030] Mutagenesis Screening of Example 2 Strain

[0031] (1) Ultraviolet Mutagenesis

[0032] Draw the bacterial liquid of the starting strain HYX0021 for 10-12 hours and inject it into a flat dish, and the amount of bacterial liquid in each flat dish is 10 mL. Place the plate containing the bacterial liquid on the magnetic stirrer under the ultraviolet lamp in the mutagenesis box, and the vertical distance between the plate and the ultraviolet lamp is 30cm. Turn on the UV lamp and preheat for 30min to stabilize the light wave. Adjust the rotation speed of the stirrer. After the rotation speed is stable, open the lid of the plate for irradiation and start timing. The irradiation time of the plate is 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 minutes respectively. Take 0.1 mL of bacterial suspension from each plate and make appropriate dilution to obtain bacterial suspensions of different dilutions. Draw 0.3 mL of the diluted bacterial suspension to coat the separation medium plate,...

Embodiment 3

[0046] Example 3: Strain identification

[0047] Physiological and biochemical identification of strains: carbon source utilization test, catalase test, hydrogen sulfide test, indole test, nitrate and nitrite reduction test, etc. ; Morphological identification: The strains to be identified were seeded on a solid plate of beef extract peptone medium, cultured at 37°C for 2 days, and the culture characteristics of the colony and the morphological characteristics of the bacteria were described and recorded. Physiological and biochemical identification characteristics are shown in Table 2. The results in the table are consistent with the species characteristics of Bacillus thermophilus according to the "Berger's Manual of Bacterial Identification". Molecular biological identification: The extraction of bacterial genomic DNA was carried out according to the method of "Molecular Cloning Experiment Guide" (3rd Edition). Primers were designed based on the most conserved sequence in ...

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Abstract

The invention discloses a bacillus thermophilus 701 capable of producing high-temperature and alkali resistant xylanase and belongs to the technical field of microbial fermentation. The bacillus thermophilus is obtained through the steps that a soil sample is collected from a paper mill, a bacillus thermophilus is separated from the soil sample, and ultraviolet mutagenesis and nitrosoguanidine mutagenesis, and screening are performed repeatedly. The xylanase produced by the bacillus thermophilus has the characteristics of strong high-temperature resistance and alkali resistance. The bacillus thermophilus is preserved in the China Center for Type Culture Collection on October 12, 2013, the preservation No. is CCTCC M2013537. The high-temperature and alkali resistant xylanase prepared from the bacillus thermophilus 701 is 350-420 IU / ml in specific activity, 40-75 DEG C in applicable temperature range, 65 DEG C in optimum reaction temperature, completely stable in enzyme activity at the temperature of 75 DEG C, 5.0-11.0 in applicable reaction pH value, completely stable in enzyme activity at the pH value of 11.0, and is 10.0 in optimum reaction pH value.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, in particular to a thermophilic bacillus producing high temperature resistant alkaline xylanase and its application. Background technique [0002] Cellulose, hemicellulose and lignin are the main components of plant hemicellulose. They work together to form the supporting skeleton of plant cell walls. Among the three, hemicellulose accounts for more than 35% of the dry weight of plants. It is also the general term for a variety of complex polysaccharides, the main component of which is xylan, which is a complex polypentacarbon sugar. The main chain of xylan is connected by β-1-4 glycosidic bonds with multiple xylopyranosyl groups, and the side chains are connected with different substituents, such as acetyl group, glucuronyl group, L-arabinosyl group et al (Collins et al., 2005). Therefore, the complete degradation of xylan requires the joint participation of multiple enzymes. X...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/42C12R1/07
Inventor 李洪兵朱永明辛盛朱思铭辛钢雷敏
Owner HUNAN HONGYING BIOTECHNOLOGY CO LTD
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