CN102719379A - Bacillus tequilensis and application thereof - Google Patents

Abstract

The invention discloses bacillus tequilensis CGX5-1 which is preserved with the number CGMCC No.5935 in China General Microbiological Culture Collection Center on March 26, 2012. The bacillus tequilensis has high capability of producing beta-1, 3-1, 4-glucanase, and the bacillus tequilensis and good genes thereof are applied to the field of production of industrial enzyme preparations and have good prospects in reduction of cost for producing the beta-1, 3-1, 4-glucanase and improvement on enzyme preparation quality.

Description

A Strain of Bacillus Tequila and Its Application

technical field

The invention belongs to the technical field of bioengineering, and relates to a strain of Bacillus tequila and its application.

technical background

There are many kinds of Bacillus, which are widely distributed, and mainly exist in soil, water, air and the intestinal tract of animals. And extracellular enzymes are abundant, which has a good research prospect. Due to the complexity of the growth environment, Bacillus will also secrete and produce cellulase, such as β-glucanase. β-glucanase is widely used in beer brewing: using β-glucanase in the saccharification process can reduce the viscosity of wort, increase the filtration speed, and reduce energy consumption; in addition, in wort Adding β-glucanase can also reduce the glucan content and prevent beer turbidity, thereby improving the abiotic stability of beer. Due to the high content of β-glucan in wheat feed, it hinders the digestion and absorption of active ingredients in the feed in the animal intestines, becomes an anti-nutritional factor, and provides rich nutrients for the colonization and reproduction of pathogenic bacteria, which will cause Adding an appropriate amount of β-glucanase to the feed will effectively solve the above problems and improve the utilization rate of the feed.

my country is a big country of beer production. In 2011, the beer production reached 46.1 million kiloliters. The continuous increase of beer production means that the demand for β-glucanase is also increasing day by day. At present, the enzyme preparations used in China are domestically produced except for a small amount of feed enzyme preparations. Most of the enzyme preparations rely on imported products. Since the production technology is a commercial secret, this greatly limits the development of the beer and feed industry. Industrial enzyme preparation technology with independent intellectual property rights is of great significance. Isolation and screening of microorganisms with high production of β-glucanase is the basis of enzyme technology. my country has a vast territory and complex environmental conditions, which has created abundant microbial resources and provided many possibilities for the development of isolation and screening work. Using an isolated β-glucanase-producing wild bacterium as a resource, combined with breeding technology, an industrial production strain with high β-glucanase production was obtained, and finally provided new ideas and resources for the production of industrial enzyme preparations.

Contents of the invention

The invention provides a strain of Bacillus tequilensis CGX5-1.

The Bacillus tequilensis CGX5-1 provided by the present invention has been preserved in the China Center for Type Culture Collection on March 26, 2012, and the preservation number is CGMCCNo.5935.

The invention provides an application of the Bacillus tequila, which is to extract the β-1,3-1,4-glucanase gene, acidic, neutral and alkaline Genetic engineering bacteria for constructing industrial enzyme preparations through exogenous expression of sex protease genes.

The Tequila bacillus was screened from wasp nest fragments, and its storage conditions were as follows: take 1 ring of bacillus from a well-grown plate and transfer it to LB medium, culture it in a shaker flask at 37°C for 15 hours, and transfer 0.75 mL into a container containing Put 0.25mL sterile 40% glycerol in a glycerol tube and store in a -70°C refrigerator.

The LB medium is: Tryptone 10g L ^-1 , Yeast Extract 5g L ^-1 , Nacl 10g L ^-1 , pH 7.0.

The fermentation medium is: corn flour 40g L ^-1 , bean cake flour 61.1g L ^-1 , barley flour 68.4g L ^-1 , dipotassium hydrogen phosphate 1g L ^-1 , calcium chloride 0.1g L ^-1 , magnesium sulfate 1g L ^-1 , high temperature amylase 16μL/L.

The culture condition of Bacillus Tequila is as follows: the Bacillus preserved in a glycerol tube is inserted into LB liquid medium, and cultured in a shaking flask at 37° C. and 200 rpm for 15 hours to the mid-late logarithm, as a seed solution. Transfer the seed solution to the fermentation medium with 2% inoculation amount, culture at 37°C and 200rpm, and take sampling points at 12, 24, 36, 48, and 60 hours to determine the β-1,3-1,4-glucose in the samples. Glycanase activity.

biological material deposit

The Bacillus tequilensis CGX5-1 provided by the present invention has been preserved in the China Center for Type Culture Collection on March 26, 2012, and the preservation number is CGMCCNo.5935.

Detailed ways

Embodiment 1 Bacillus tequila screening and enzyme activity assay method

Take a certain sample from the fragments of wasp nests and add it to 30mL sterile saline, break up the glass beads to make a suspension, and treat at 80°C for 10min. The suspension was serially diluted, spread on an MRS plate supplemented with 2% Congo red, and incubated in a 37°C incubator for 24h. Pick a single colony conspicuous by the Congo red discoloration circle. Streak separation on the plate was repeated three times to obtain a purified single colony for identification in the next step.

Identification is mainly through: 1) Observation of colony characteristics: directly observe with the naked eye, and select bacterial colonies with a diameter of 1-1.5mm, round shape, smooth surface with slightly wrinkled, milky white, etc. that meet the characteristics of Bacillus colonies. 2) Observation of individual morphology: Observation by Gram staining with an optical microscope. Pick Gram-positive, spore-forming strains. Strains meeting the identification conditions were picked out of glycerol tubes for preservation.

Inoculate the glycerol stock solution at -70°C with 2% inoculum in LB medium, and incubate in shake flasks at 37°C and 200 rpm for 15 hours. Take the shake flask culture at 200 rpm at 37°C with a 2% inoculum amount, transfer it to the fermentation medium and culture it in a shake flask at 200 rpm at 37°C for 36 hours. The fermentation broth was centrifuged for 5min (8000rpm, 4°C) to obtain the supernatant. Dilute the supernatant with double-distilled water to an appropriate multiple, take 0.1mL of the diluted solution, and add the blue dextran substrate preheated at 40°C (mix with 0.2M pH5.5 acetate buffer in equal volume before use) , react at 40°C for 10min, add 3.0mL reaction stop solution to each reaction sample, shake well and centrifuge at 8000rpm for 5min, measure the absorbance at 590nm in a 1cm cuvette, replace the enzyme solution with 0.1mL double distilled water Do a blank control. By measuring the activity of β-1,3-1,4-glucanase in the fermentation broth, a strain with strong ability to produce β-1,3-1,4-glucanase was screened, and the 16SrDNA Sequence analysis shows that the strain is Bacillus tequila, which was deposited in the China Center for General Microbiology on March 26, 2012, with the preservation number CGMCCNo.5935.

Example 2 Process for producing β-1,3-1,4-glucanase by fermentation

The culture medium used for producing β-1,3-1,4-glucanase by Bacillus tequila fermentation is divided into seed medium and fermentation medium. The seed medium is LB medium: Tryptone 10g L ^-1 , Yeast Extract 5g L ^-1 , Nacl 10g L ^-1 , pH 7.0. The main components of the fermentation medium are: corn flour 40g L ^-1 , bean cake flour 61.1g L ^-1 , barley flour 68.4g L ^-1 , dipotassium hydrogen phosphate 1g L ^-1 , calcium chloride 0.1g L ^-1 , magnesium sulfate 1g L ^-1 , high temperature amylase 16μL/L. The filling volume is 30mL per 250mL Erlenmeyer flask. The medium sterilization condition is 121°C, 20min.

Pick a single colony of Bacillus tequila from the plate and activate it on the slant for 8 hours, pick a ring of bacteria from the slant and inoculate it into LB medium, and culture it in a shaker flask at 200 rpm at 37°C for 15 hours to the middle and late logarithmic stages, as seeds liquid. Transfer the seed solution to the fermentation medium with 2% inoculum amount, culture at 37°C and 200rpm, and take sampling points at 12, 24, 36, 48, 52, 56, and 60 hours to measure the β-1,3-1 , 4-glucanase activity. The results showed that the activity of β-1,3-1,4-glucanase in the fermentation liquid reached the highest value, which was 191.96 U/mL, after the bacteria grew in the fermentation medium for 52 hours.

Embodiment 3 is applied to the construction of genetically engineered bacteria

According to the bglS design in NCBI in Bacillus amyloliquefaciens LL3 (NC_017190.1), primers were cloned and the β-1,3-1,4-glucanase gene was used to construct an expression plasmid, which can be expressed in Escherichia coli or Pichia pastoris A series for efficient fermentative expression and production of β-1,3-1,4-glucanase.

It can be understood that those skilled in the art can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention, and all these changes or replacements should belong to the protection scope of the appended claims of the present invention.

Claims (5)

1. A strain of Bacillus tequilensis CGX5-1 was deposited in the General Microbiology Center of China Committee for the Collection of Microorganisms on March 26, 2012, with the preservation number CGMCC No.5935. 2. the method for cultivating the bacillus tequila described in claim 1 is to cultivate the bacillus tequila in LB medium or nutrient broth medium in shake flasks. 3. The cultivation method according to claim 2, characterized in that the preferred pH is 7.0; the preferred cultivation temperature is 37°C. 4. The Bacillus tequila described in claim 1 is applied to clone the β-1,3-1,4-glucanase gene and construct the genetically engineered bacterium of industrial enzyme preparation. 5. The Bacillus tequila described in claim 1 is applied to fermentation to produce β-1,3-1,4-glucanase.