CN109628340B - A kind of Bacillus circulans strain producing high activity β-galactosidase and its breeding method - Google Patents

Abstract

The invention discloses a Bacillus circulans strain for producing high-activity beta-galactosidase and a breeding method thereof, wherein the Bacillus circulans strain for producing high-activity beta-galactosidase is Bacillus circulans QHT-310-M481(Bacillus circulans QHT-310-M481), which is preserved in China center for type culture collection in 2018, 10 and 22 months, and the preservation number is CCTCC NO: m2018699, the preservation address is Wuhan university in Wuhan City, China. The bacillus circulans strain for producing the beta-galactosidase with high activity is obtained by carrying out mutagenesis treatment on a bacillus circulans starting strain by using a 1-methyl-3-nitro-1-nitrosoguanidine mutagen, can produce beta-galactosidase liquid with high fermentation activity, and is used for industrially catalyzing galactosyl transfer reaction to produce galactooligosaccharides.

Description

A kind of Bacillus circulans strain producing high activity β-galactosidase and its breeding method

technical field

The invention belongs to the fields of fermentation engineering and microbial breeding, and in particular relates to a Bacillus circulans strain producing high-activity β-galactosidase and a breeding method thereof.

Background technique

Galactooligosaccharides (GOS) are functional oligosaccharides with natural properties. Galacto-oligosaccharide is a low-energy sugar that occurs naturally in animal and human milk. Galacto-oligosaccharide is an excellent source of nutrients and an effective proliferation factor for beneficial bacteria such as Bifidobacterium and Lactobacillus acidophilus in the human intestinal tract, which can improve the digestion and absorption function of the human intestinal tract. In addition, galactooligosaccharides can also improve lipid metabolism, reduce serum cholesterol concentration, promote mineral element absorption, improve lactose intolerance, and improve immunity.

Galacto-oligosaccharides are widely used in the food industry and are used as prebiotic ingredients in dairy products, bakery products, candy processing and functional foods. With the approval of the regulations, galacto-oligosaccharides prepared by β-galactosidase have been approved as a new variety of food additives, which can be used in infant formula and cereal complementary food for infants and young children, alone or in combination. With the full effect of the comprehensive two-child policy, the fertility level has been moderately improved. Infant milk powder, other dairy products and health care products, as the main research and application fields of galactooligosaccharides, have ushered in a new development peak.

In industrial production, galacto-oligosaccharides are produced by using lactose as raw material and catalyzing the galactosyl transfer reaction by microbial beta-galactosidase. The molecular structure of galacto-oligosaccharides is through β-(1→3) galactosidic bond, β-(1→4) galactosidic bond or β-(1→6) galactoside on galactose or glucose molecule Bonds link 1-7 galactosyl groups.

β-Galactosidase widely exists in animals, plants and microorganisms. Its main function is to catalyze the hydrolysis of lactose to generate glucose and galactose.

In industrial production, β-galactosidase is basically derived from microorganisms, and has the characteristics of easy mass preparation and good stability. However, the strains directly isolated from nature generally have low enzymatic fermentation activity and cannot meet the requirements of industrial production. Therefore, the strains should be improved according to the morphology and physiological characteristics of the strains. The mutation of microorganisms can be carried out spontaneously under natural conditions, but the mutation rate of spontaneous mutation is very low, and the probability of obtaining mutant strains that meet the requirements is even lower, and the probability is only 10 ^-6 to 10 ^-10 . In order to make the enzyme more suitable for practical application, physical and chemical factors are used to improve the mutation rate of the strain, so that the possibility of screening mutant strains with favorable characters is greatly increased. Chemical mutagens mainly interfere with DNA replication by interacting with nucleic acid bases, acting as base analogs or frameshift mutagenic agents, so that their genetic material changes and mutates. The mutagen 1-methyl-3-nitro-1-nitrosoguanidine is a bifunctional alkylating agent that can interact with nucleic acid bases. It can interact with many parts of the DNA molecule and easily replace the The active hydrogen atom causes the base and accounting part of the DNA molecule to be alkylated, which leads to base pairing errors during DNA replication and causes mutations. In addition, it can also form covalent bonds between the DNA double strands, hindering the DNA replication process. The unwinding of the double-strand, thereby causing mutation, can greatly increase the mutation frequency.

Current related research reports prove that Bacillus circulans is an important source of β-galactosidase, and the produced β-galactosidase has an ideal transformation effect on lactose. The use of Bacillus circulans to produce β-galactosidase has significant application value.

Therefore, it is necessary to study strains of Bacillus circulans capable of producing high-activity β-galactosidase and their breeding methods.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the object of the present invention is to provide a Bacillus circulans strain for producing high-activity β-galactosidase, which can produce high-activity β-galactosidase for industrial catalysis Galactosylation reaction to produce galactooligosaccharides. The invention also provides a breeding method for Bacillus circulans strain producing high activity β-galactosidase. The breeding method uses 1-methyl-3-nitro-1-nitrosoguanidine as a mutagen , After multiple rounds of mutagenesis, the β-galactosidase enzyme activity of the mutant strains was detected to obtain a Bacillus circulans strain that produced high-activity β-galactosidase. The breeding method is simple and feasible, and the mutation screening High success rate.

The technical scheme adopted in the present invention is: a strain of Bacillus circulans for producing high activity β-galactosidase, and the Bacillus circulans strain for producing high activity β-galactosidase is Bacillus circulans QHT- 310-M481 (Bacillus circulans QHT-310-M481), was deposited in the China Center for Type Culture Collection on October 22, 2018, the deposit number is CCTCC NO: M 2018699, and the deposit address is Wuhan University, Wuhan, China.

Specifically, the Bacillus circus strain producing high-activity β-galactosidase is obtained from a Bacillus circus origin strain that undergoes mutagenesis treatment with a mutagen, and the originating Bacillus circus strain is a cyclic Bacillus circus strain Bacillus circulars, purchased from the American Type Culture Collection (ATCC), No. 31382.

Preferably, the mutagen is a 1-methyl-3-nitro-1-nitrosoguanidine solution, and the 1-methyl-3-nitro-1-nitrosoguanidine solution has a concentration of 0.2-1.0 mg/mL solution of 1-methyl-3-nitro-1-nitrosoguanidine acetone.

Preferably, during the mutagenesis process, the amount of 0.2-1.0 mg/mL 1-methyl-3-nitro-1-nitrosoguanidine acetone solution is 0.5-1.5 mL.

Preferably, the time of the mutagenesis treatment is 15-60 min.

The method for selecting and breeding Bacillus circulans strains for producing high-activity β-galactosidase comprises the following steps:

1) Insert the starting strain of Bacillus circulans into the seed medium, cultivate at 37°C for 4-6 hours, collect the bacteria by centrifugation, add sterile phosphate buffer solution to wash and filter, and adjust the concentration of the bacterial suspension to 10 ⁵ -10 ⁷ pcs/mL;

2) Add mutagen to the bacterial suspension in step 1), carry out mutagenesis treatment to obtain a mutagenic bacterial liquid, coat the mutagenic bacterial liquid on a solid medium plate, cultivate at 37° C. for 1-2 days, wait for After the colonies grow, select the colonies with good growth and large colonies, inoculate them in a 96-well plate containing fermentation medium, and cultivate at 37°C for 36-48 hours to obtain the M1 mutant strain, and detect the β-galactogen of the M1 mutant strain. The enzyme activity of glycosidase was calculated, and the mutagenic effect of the M1 mutant strain was calculated;

3) Carry out genetic stability analysis on the M1 mutant strain whose β-galactosidase activity in step 2) has increased by more than 30%, subculture for 7 consecutive times, and detect the β-galactosidase activity of the mutant strain , select the M1 mutagenic strain with the best genetic stability as the mutagenic parent;

4) Carry out 1-5 rounds of mutagenesis treatment on the mutagenized parent obtained in step 3), detect the β-galactosidase enzyme activity of the mutant strain, and select the mutant strain with the highest β-galactosidase enzyme activity as the A strain of Bacillus circus that produces a highly active beta-galactosidase.

Preferably, a method for breeding Bacillus circulans strains that produce high activity β-galactosidase, comprising the following steps:

1) Insert the starting strain of Bacillus circulans into the seed medium, cultivate at 37°C for 4-6 hours, collect the bacteria by centrifugation, add sterile phosphate buffer solution to wash and filter, and adjust the concentration of the bacterial suspension to 10 ⁵ -10 ⁷ pcs/mL;

2) Add 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with a concentration of 0.2-1.0 mg/mL to 1 mL of the bacterial suspension in step 1), and perform mutagenesis for 30 min to obtain a mutagenic solution. 100 μL of mutagenic bacterial solution Ⅰ was coated on solid medium plate, cultured at 37°C for 1-2 days, colony count was carried out, lethality Ⅰ was calculated, and the optimal mutagenic concentration was determined;

Add 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the optimal mutagenesis concentration determined in step 2) to 1 mL of the bacterial suspension in step 1), and carry out mutagenesis treatment for 15-60 min. To obtain the mutagenic bacterial solution II, take 100 μL of the mutagenic bacterial solution II to coat the solid medium plate, cultivate at 37°C for 1-2 days, count the colonies, calculate the lethality II, and determine the optimal mutagenesis time;

The obtained mutagenic bacteria solution II with the best mutagenesis time was coated on a solid medium plate, and after the colonies had grown, the colonies with good growth and large colonies were selected and inoculated in a 96-well plate containing fermentation medium. Cultivated at ℃ for 36-48h to obtain the M1 mutant strain, detect the β-galactosidase enzyme activity of the mutant strain, and calculate the mutagenesis rate of the M1 mutant strain;

3) Carry out genetic stability analysis on the M1 mutant strain whose β-galactosidase activity in step 2) has increased by more than 30%, subculture for 7 consecutive times, and detect the β-galactosidase activity of the mutant strain , select the M1 mutagenic strain with the best genetic stability as the mutagenic parent;

4) Carry out 1-5 rounds of mutagenesis treatment on the mutagenized parent obtained in step 3), detect the β-galactosidase enzyme activity of the mutant strain, and select the mutant strain with the highest β-galactosidase enzyme activity as the A strain of Bacillus circus that produces a highly active beta-galactosidase.

Preferably, in step 1), the seed culture medium is prepared by the following method: adding 1000 mL of distilled water to 15-20 g of prefabricated nutrient broth dry powder, heating to dissolve, the pH value does not need to be adjusted separately, and cooling to obtain the seed culture medium.

More preferably, in step 1), the seed medium is prepared by the following method: adding 1000 mL of distilled water to 18 g of prefabricated nutrient broth dry powder, heating and dissolving, the pH value does not need to be adjusted separately, and cooling to obtain the seed medium.

Preferably, in step 1), the sterile phosphate buffer solution is a 0.01 mol/L phosphate buffer solution with a pH value of 6.

Preferably, in step 2), the solid medium plate is prepared by the following method: adding 1000 mL of distilled water to 30-35 g of prefabricated nutrient agar dry powder, heating to dissolve, the pH value does not need to be adjusted separately, poured into a petri dish, cooled, and obtained Solid medium plates.

More preferably, in step 2), the solid medium plate is prepared by the following method: add 1000 mL of distilled water to 33 g of prefabricated nutrient agar dry powder, heat to dissolve, the pH value does not need to be adjusted separately, poured into a petri dish, cooled to obtain a solid medium plate.

Preferably, in step 2), the fermentation medium comprises the following components by weight: 0.6-1.6% soy peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate , 0.12-0.55% magnesium sulfate, 1.0-1.5% lactose and 95.5-97.5% water, the pH value of the fermentation medium is 6.0-6.5.

In industrial production applications, our aim is to maximize the activity unit of total enzyme activity produced by fermentation per unit volume. The enzymatic activity changes of β-galactosidase in Bacillus circulans are controlled by different levels of non-linear regulatory networks such as DNA, RNA and protein in bacteria. The strain variation caused by each round of mutagenesis only affects the local area of the regulatory network, for example, the β-galactosidase gene expression regulatory element is enhanced, the stability of the β-galactosidase gene transcript is significantly improved, the β-galactosidase gene - Positive changes in the gene sequence corresponding to the key amino acids of the enzymatic pocket of galactosidase. A breeding process for a Bacillus circulans strain producing high-activity β-galactosidase is guided by a significant increase in the total enzyme activity of a fermentation broth, according to the β-galactosidase of the strains of each round of mutagenesis According to the actual situation that the total enzyme activity is improved, the screening criteria of each round of strains are flexibly set, and a currently optimized strain is finally obtained.

Compared with the prior art, the present invention has the following beneficial effects:

1. A kind of Bacillus circulans strain of producing high activity β-galactosidase of the present invention, can produce the β-galactosidase liquid of high fermentation activity, is used for industrially catalyzed galactosyl transfer reaction to produce low Polygalactose.

2. The breeding method of the Bacillus circulans strain for producing high-activity β-galactosidase of the present invention, using 1-methyl-3-nitro-1-nitrosoguanidine as a mutagen, after several rounds of Mutagenesis treatment to obtain a strain of Bacillus circulans that produces high activity β-galactosidase, 1-methyl-3-nitro-1-nitrosoguanidine mutagen is a mutagen that can interact with nucleic acid bases Bifunctional alkylating agent can interact with many parts of DNA molecules, easily replace active hydrogen atoms in DNA molecules, and make the bases and nucleic acid parts of DNA molecules alkylated, resulting in base pairing errors during DNA replication. Mutation, in addition, it can also form a covalent bond between the DNA double strands, which hinders the unwinding of the double strands during DNA replication, thereby causing mutations, which can greatly increase the mutation frequency.

3. The breeding method is oriented to the significant improvement of the total enzyme activity of the fermentation broth, and according to the actual situation of the increase of the total enzyme activity of β-galactosidase of the strains of each round of mutagenesis, flexibly set the total enzyme activity of each round of strains. Screening criteria, and finally obtain a currently optimized strain, the method is flexible and pragmatic, simple and easy to implement, and the success rate of mutagenesis screening is high, which is more conducive to obtaining positive mutagenesis results and obtaining a ring that produces high activity β-galactosidase. Bacillus strains.

Description of drawings

FIG. 1 is a graph showing the results of the detection of the β-galactosidase enzyme activity of the M4 mutant strain of Example 1. FIG.

FIG. 2 is a microscopic view of the microbial cells of the M4-81 mutant strain of Example 1. FIG.

FIG. 3 is a diagram of the colony morphology of the M4-81 mutant strain of Example 1. FIG.

Detailed ways

The present invention will be further described below in conjunction with the embodiments.

Calculation method of β-galactosidase enzyme activity:

1. Reagent preparation

(1) Z-buffer

Dissolve 16.1g disodium hydrogen phosphate, 5.5g sodium dihydrogen phosphate, 0.75g potassium chloride, 0.246g magnesium sulfate and 2.7mL 2-mercaptoethanol in 800mL water, add 2mol/L sodium hydroxide solution, adjust pH to 6.0 ±0.05, detected by pH meter. Transfer the solution to a 1000mL volumetric flask, make up to volume with water, and mix.

(2) o-nitrophenyl-β-D-galactopyranoside (ONPG) solution

Dissolve 250.0 mg of ONPG with 75 mL of Z-buffer, transfer the solution to a 100-mL volumetric flask, and make up to volume with Z-buffer as a substrate.

(3) Stop solution

Using water as solvent, dissolve 10g of sodium carbonate, transfer to a 100mL volumetric flask and dilute to volume.

(4) Test sample preparation

Prepare test enzyme samples such that 0.05-0.25 units of β-galactosidase are contained per mL of final solution.

2. Detection steps

A series of glass test tubes with a size of 20*150mm were placed in a water bath at 50±0.1°C, and then 0.25mL of ONPG solution was sucked into the glass test tubes with a pipette, and the water bath was kept at a constant temperature.

Quickly add 0.25mL of the sample to be tested with a pipette gun (add 0.25mL of water to the control test tube), then start the shaking, press the stopwatch to start timing; after shaking for 10min, suck out 0.5mL of the reaction solution from each test tube, add into a tube containing 0.5 mL of stop solution, then stir to mix.

The samples were transferred to the ELISA plate, the detection wavelength was 405 nm, and the solution in the control test tube was used as a control to detect the absorbance of each sample.

3. Drawing of the standard curve

Transfer 139.0 mg of o-nitrophenol (ONP) into a 1000 mL volumetric flask, dissolve with 95% alcohol before transferring, make up to volume with water, and mix well. Aspirate 2.5, 5, 12.5 and 25 mL of the solution into a 100 mL volumetric flask with a pipette respectively, make up to the volume with 10 wt% sodium carbonate solution, and mix well. These solutions contained 0.025, 0.05, 0.125, 0.25 micromoles of ONP per mL, respectively.

A 1cm quartz tube was used to install the standard solution, and the absorbance was measured at a wavelength of 405nm. Taking water as the control, the concentration of ONP was the abscissa, and the absorbance of each concentration standard was the ordinate, and a straight line passing through the origin could be obtained.

4. Calculation

The standard curve is obtained by calculation and analysis: Y=4.0688X+0.0152, R ² =0.9991

(Y is the light absorption value at 405nm, X is the ONP concentration)

The β-galactosidase activity assay method is as follows: one lactase unit (LacU) is defined as the amount of enzyme activity required to release 1 micromol of ONP per minute under the conditions of this method.

Enzyme activity (U/mL)=4*DF*X/10

5. Convert the enzyme activity in 0.25mL diluted enzyme solution to 1mL enzyme activity;

DF: dilution factor;

X: ONP concentration (μmol);

10: reaction time, 10 min.

The seed culture medium used in the examples was prepared by the following method: adding 1000 mL of distilled water to 15-20 g of prefabricated nutrient broth dry powder, heating to dissolve, the pH value does not need to be adjusted separately, and cooling to obtain the seed culture medium.

The sterile phosphate buffer solution is a 0.01 mol/L phosphate buffer solution with a pH value of 6.

The solid medium plate is prepared by the following method: add 1000 mL of distilled water to 30-35 g of prefabricated nutrient agar dry powder, heat to dissolve, the pH value does not need to be adjusted separately, pour it into a petri dish, and cool to obtain a solid medium plate.

The fermentation medium includes the following components by weight: 0.6-1.6% soy peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate, 0.12-0.55% magnesium sulfate, 1.0-1.5% lactose and 95.5-97.5% water, fermentation medium pH 6.0-6.5.

Example 1

1. Preliminary screening of Bacillus circulans producing high activity β-galactosidase, including the following steps:

1) Activation of the starting strain: Bacillus circulars (Bacillus circulars, purchased from the American Culture Collection Center (ATCC, No. 31382) was used as the starting strain for streak culture. , inoculated in 100mL fermentation medium (the composition of fermentation medium is: 1.2wt% soybean peptone, 0.65wt% yeast extract, 0.35wt% disodium hydrogen phosphate, 0.25wt% sodium carbonate, 0.55wt% of magnesium sulfate, 1.5wt% lactose and 95.50wt% water, pH 6.0-6.5), incubate the flask at 37°C for 48h, centrifuge, take the supernatant, and measure the enzyme activity of β-galactosidase. The measurement results are shown in Table 1:

Table 1 β-galactosidase enzyme activity of Bacillus circulans starting strain

2) Pick a ring of slanted bacteria in the seed medium (prefabricated nutrient broth medium), culture at 37°C for 6 hours, centrifuge at 10,000 rpm for 10 minutes, collect the bacterial cells, resuspend in pH 6.0 phosphate buffer solution, and pass After filtration with absorbent cotton, it was placed in a small triangular flask equipped with glass beads, shaken evenly, and prepared into a single-cell bacterial suspension with a concentration of 10 ⁵ to 10 ⁷ cells/mL.

3) Take 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with concentrations of 0.25, 0.5, 0.75, and 1.0 mg/mL, add them to 1 mL of bacterial suspension, and perform mutagenesis for 30 min , get the mutagenic bacterial solution I, take 100 μL of the mutagenic bacterial solution I to coat the solid medium plate, incubate at 37 ℃ for 48 h, count the colonies, calculate the lethality I, and determine the optimal mutagenic concentration to be 0.5 mg/mL. The results are shown in Table 2.

Table 2 Mutagenic concentration of 1-methyl-3-nitro-1-nitrosoguanidine

Mutagenic concentration (mg/mL) 0.25 0.5 0.75 1 Lethality I (%) 82.6 99.0 99.9 99.9

4) Take 1 mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with a concentration of 0.5 mg/mL, add it to 1 mL of bacterial suspension, and shake for 15, 30, and 45 minutes at 37°C, respectively. , 60min, to get the mutagenic bacteria solution II, take 100 μL of the mutagenic bacteria solution II to coat the solid medium plate, cultivate at 37 ℃ for 48h, calculate the lethality II, and determine the optimal mutagenesis time as 30min. The results are shown in Table 3.

Table 3 Mutagenesis time of 1-methyl-3-nitro-1-nitrosoguanidine

Mutagenesis time (min) 15 30 45 60 Lethality II (%) 80.3 99.0 99.9 99.9

5) Take 100 μL of the mutagenized bacterial solution II treated for 30 min in step 4), spread it on a solid medium plate, and cultivate at 37° C. for 48 h.

6) After the colony is grown, select the colony with good growth and large colony, and use a sterile toothpick to seed the labeled colonies one by one in a 96-well plate (the composition of the fermentation medium is the same as that of step 1). 2mL deep-well plate, the volume of each well is 800uL), place the 96-well plate in a shaker and cultivate it at 37°C, 80% humidity, 300r/min for 48h to obtain the M1 mutant strain, and use a microplate reader to detect M1 The β-galactosidase enzyme activity of the mutant strain was calculated, and the mutagenic effect of the M1 mutant strain was calculated.

7) Re-examine the M1 mutant strains whose β-galactosidase enzyme activity increased by more than 30% picked out in step 6), transplanted to a solid medium plate, cultured for 48 hours, and then transplanted to 96 wells The β-galactosidase enzyme activity was measured, and the enzyme activities of M1-38 and M1-63 were most significantly improved, which were higher than the starting strains respectively. 30.35% and 30.22%;

2. Genetic stability analysis of Bacillus circulans producing high activity β-galactosidase

The M1-38 and M1-63 mutant strains were inserted into the slanted solid medium, cultured at 37°C for 48 hours, 9 mL of sterile phosphate buffer solution was added, eluted, diluted and coated on a solid medium plate, and after the colonies grew, A single colony with good growth and large colony was selected and subcultured for 7 consecutive times, and the β-galactosidase enzyme activity of the strain was detected to determine the genetic stability of the strain in producing high activity β-galactosidase. Among them, M1-38 has good genetic stability, and the M1-38 mutant strain was selected as the mutagenic parent. The specific data are shown in Table 4.

Table 4 M1 generation genetic stability verification

Enzyme activity (U/mL) original 1st generation 2nd generation 3rd lowest 4th generation 5th generation 6th generation 7th generation M1-38 19.94 19.92 19.98 19.89 19.95 19.94 19.96 19.91 M1-63 18.82 19.45 17.88 15.71 15.24 14.95 15.36 14.81

3. Multiple rounds of mutagenesis of Bacillus circulans producing high activity β-galactosidase

Select the genetically stable M1-38 mutagenic strain as the mutagenic parent, pick a ring of slanted strains in fresh seed medium, cultivate at 37°C for 6 hours, centrifuge at 10,000 rpm for 10 minutes, collect the bacteria, and resuspend in pH 6 .0 phosphate buffer solution, filtered with absorbent cotton, placed in a small triangular flask with glass beads, shaken evenly, and prepared into a single-cell bacterial suspension with a concentration of 10 ⁵ to 10 ⁷ /mL, taking 1 mL of the concentration of 0.5 mg /mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution was added to 1mL of bacterial suspension, treated for 30min, the mutagenized bacterial solution was coated with solid medium for plate culture, and good growth was selected. A single colony was fermented and cultured using a 96-well plate culture system and the biomass and β-galactosidase activity of the strain were detected. Repeat the mutagenesis step 3 times to obtain progeny mutagenized strains labeled as M2, M3, and M4, respectively. After 4 rounds of 1-methyl-3-nitro-1-nitrosoguanidine mutagen treatment, the M4-81 mutant strain was finally screened out as a Bacillus circus strain that produces high activity β-galactosidase , the activity of the enzyme produced by the M4 generation is shown in Figure 1, the horizontal axis is the strain number, and the vertical axis is the enzyme activity.

It can be seen from Figure 1 that the enzyme activity of the fourth-generation mutant M4-81 is 39.86 U/mL, which is 2.60 times that of the enzyme produced by the starting strain (Bacillus circulans ATCC, No. 31382), and is currently the most optimized. strains. The strain was named Bacillus circulans QHT-310-M481 (Bacillus circulans QHT-310-M481), and it was deposited in the China Center for Type Culture Collection on October 22, 2018, and the deposit number is CCTCC NO: M 2018699. The address is Wuhan University, Wuhan, China.

The morphology of the described Bacillus circulans strain producing high activity β-galactosidase is shown in Figure 2 and Figure 3, the colony is bright yellow, the shape is regular, the surface is moist, and the edges are neat; Single arrangement, spore terminal, cyst not inflated, Gram-positive.

Claims (1)

1. A Bacillus circulans strain QHT-310-M481, which was deposited in the China Center for Type Culture Collection on October 22, 2018, with the deposit number CCTCC NO: M 2018699, and the deposit address is Wuhan, China Wuhan University.

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