CN109628340B - Bacillus circulans strain for producing high-activity beta-galactosidase and breeding method thereof - Google Patents
Bacillus circulans strain for producing high-activity beta-galactosidase and breeding method thereof Download PDFInfo
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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
Technical Field
The invention belongs to the field of fermentation engineering and microorganism breeding, and particularly relates to a bacillus circulans strain for producing high-activity beta-galactosidase and a breeding method thereof.
Background
Galactooligosaccharides (GOS) are functional oligosaccharides with natural properties. Galactooligosaccharides are low-energy sugars that occur naturally in animal and human milk. The galactooligosaccharide is an excellent nutrient source and an effective proliferation factor of beneficial bacteria such as bifidobacterium, lactobacillus acidophilus and the like in human intestinal tracts, and can improve the digestion and absorption functions of the human intestinal tracts. In addition, the galacto-oligosaccharide can improve lipid metabolism, reduce serum cholesterol concentration, promote mineral element absorption, improve lactose intolerance, improve body immunity and the like.
The galacto-oligosaccharide is widely applied in the food industry, and is used as a prebiotic component in the fields of dairy products, baked foods, candy processing, functional foods and the like. With regulatory approval, β -galactosidase produced galactooligosaccharides are approved as a new food additive variety for infant formulas and infant cereal adjuncts, either alone or in combination. Along with the full play of the policy effect of the second child, the fertility level is moderately improved. The field of main research and application of infant milk powder, other dairy products and health care products as galactooligosaccharides is met with a new development peak.
In industrial production, galactooligosaccharides are produced by using lactose as a raw material and using microbial beta-galactosidase to catalyze galactosyl transfer reaction. The molecular structure of galactooligosaccharide is 1-7 galactosyl groups linked to galactose or glucose molecules through beta- (1 → 3) galactoside linkage, beta- (1 → 4) galactoside linkage or beta- (1 → 6) galactoside linkage.
Beta-galactosidase is widely present in animals, plants and microorganisms, has the main function of catalyzing lactose hydrolysis to generate glucose and galactose, and has the function of catalyzing galactosyl transfer activity to generate galacto-oligosaccharide.
In industrial production, beta-galactosidase is basically derived from microorganisms, and has the characteristics of easiness in mass preparation, good stability and the like. However, the strains isolated directly from nature generally have relatively low enzyme fermentation activity and cannot meet the requirements of industrial production, so the strains are improved according to the morphological and physiological characteristics of the strains. The mutation of the microorganism can be spontaneously carried out under natural conditions, but the mutation rate of the spontaneous mutation is very low, the probability of obtaining a mutant strain meeting the requirement is lower, and the probability is only 10-6～10-10. In order to make the enzyme more suitable for practical application, the mutation rate of the strain is improved by means of physicochemical factors and the like, so that the possibility of screening mutant strains with favorable properties is greatly increased. Chemical mutagens are mutated by altering their genetic material, primarily by interacting with nucleic acid bases, acting as base analogs or frame-shift mutagens to interfere with DNA replication. The mutagen 1-methyl-3-nitro-1-nitrosoguanidine is a double capable of acting with nucleic acid base The functional alkylating agent can act with several parts of DNA molecule to replace active hydrogen atom in DNA molecule and to alkylate the base and accounting part of DNA molecule and result in base pairing error to cause mutation during DNA replication.
Related research reports at present prove that the bacillus circulans is an important source of beta-galactosidase, and the produced beta-galactosidase has ideal transformation effect on lactose. The beta-galactosidase produced by the bacillus circulans has obvious application value.
Therefore, there is a need to develop a bacillus circulans strain capable of producing beta-galactosidase with high activity and a breeding method thereof.
Disclosure of Invention
In view of the deficiencies of the prior art, the present invention aims to provide a bacillus circulans strain capable of producing beta-galactosidase with high activity, which can be used for industrially catalyzing galactosyltransferase reaction to produce galactooligosaccharides. The invention also provides a breeding method of the bacillus circulans strain for producing the high-activity beta-galactosidase, the breeding method takes 1-methyl-3-nitro-1-nitrosoguanidine as a mutagen, and detects the activity of the beta-galactosidase enzyme of the mutagenized strain through multiple rounds of mutagenesis treatment so as to obtain the bacillus circulans strain for producing the high-activity beta-galactosidase, the breeding method is simple and easy to implement, and the mutagenesis screening success rate is high.
The technical scheme adopted by the invention is as follows: the Bacillus circulans strain for producing the beta-galactosidase with high activity is Bacillus circulans QHT-310-M481(Bacillus circulans QHT-310-M481) which is preserved in China center for type culture collection (CCTCC NO) in 2018, 10 and 22 days: m2018699, the preservation address is Wuhan university in Wuhan City, China.
Specifically, the Bacillus circulans strain for producing the high-activity beta-galactosidase is obtained by mutagenizing a Bacillus circulans starting strain by a mutagen, wherein the Bacillus circulans starting strain is Bacillus circulans (ATCC) with the number of 31382.
Preferably, the mutagen is 1-methyl-3-nitro-1-nitrosoguanidine solution, and the 1-methyl-3-nitro-1-nitrosoguanidine solution is 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the concentration of 0.2-1.0 mg/mL.
Preferably, 0.2 to 1.0mg/mL of 1-methyl-3-nitro-1-nitrosoguanidineacetone solution is used in an amount of 0.5 to 1.5mL during the mutagenesis treatment.
Preferably, the time for the mutagenesis treatment is 15 to 60 min.
The breeding method of the bacillus circulans strain for producing the high-activity beta-galactosidase comprises the following steps:
1) inoculating Bacillus circulans strain into seed culture medium, culturing at 37 deg.C for 4-6 hr, centrifuging to collect thallus, adding sterile phosphoric acid buffer solution, cleaning, filtering, and adjusting bacterial suspension concentration to 105-107Per mL;
2) adding a mutagen into the bacterial suspension obtained in the step 1), carrying out mutagenesis treatment to obtain a mutagenic bacterial liquid, coating the mutagenic bacterial liquid on a solid culture medium flat plate, culturing for 1-2 days at 37 ℃, after bacterial colonies grow out, selecting bacterial colonies with good growth vigor and large bacterial colonies, inoculating the bacterial colonies into a 96-well plate containing a fermentation culture medium, culturing for 36-48 hours at 37 ℃ to obtain an M1 mutagenic strain, detecting the beta-galactosidase activity of the M1 mutagenic strain, and calculating the mutagenesis effect of the M1 mutagenic strain;
3) performing genetic stability analysis on the M1 mutant strain with the beta-galactosidase enzyme activity improving range larger than 30% in the step 2), continuously performing subculture for 7 times, detecting the beta-galactosidase enzyme activity of the mutant strain, and selecting the M1 mutant strain with the best genetic stability as a mutant female parent;
4) carrying out 1-5 rounds of mutagenesis treatment on the mutagenic female parent obtained in the step 3), detecting the activity of beta-galactosidase of the mutagenic strain, and selecting the mutagenic strain with the highest activity of the beta-galactosidase as a bacillus circulans strain for producing high-activity beta-galactosidase.
Preferably, the breeding method of the bacillus circulans strain for producing the beta-galactosidase with high activity comprises the following steps:
1) inoculating Bacillus circulans starting strain into seed culture medium, culturing at 37 deg.C for 4-6 hr, centrifuging to collect thallus, adding sterile phosphoric acid buffer solution, cleaning, filtering, and adjusting suspension concentration to 105-107Per mL;
2) adding 1mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the concentration of 0.2-1.0mg/mL into 1mL of the bacterial suspension in the step 1), carrying out mutagenesis treatment for 30min to obtain a mutagenic bacterial liquid I, coating 100 mu L of the mutagenic bacterial liquid I on a solid culture medium flat plate, culturing at 37 ℃ for 1-2 days, carrying out colony counting, calculating the lethality rate I, and determining the optimal mutagenesis concentration;
adding 1mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the optimal mutagenesis concentration determined in the step 2) into 1mL of the bacterial suspension in the step 1), carrying out mutagenesis treatment for 15-60min to obtain a mutagenesis bacterial liquid II, coating 100 mu L of the mutagenesis bacterial liquid II on a solid culture medium flat plate, culturing at 37 ℃ for 1-2 days, carrying out colony counting, calculating the lethality rate II, and determining the optimal mutagenesis time;
coating the obtained mutagenic bacterium liquid II with the optimal mutagenesis time on a solid culture medium flat plate, after the bacterium grows out, selecting a bacterium colony with good growth vigor and large bacterium colony, inoculating the bacterium colony into a 96-well plate containing a fermentation culture medium, culturing for 36-48h at 37 ℃ to obtain an M1 mutagenic strain, detecting the beta-galactosidase activity of the mutagenic strain, and calculating the mutagenesis rate of the M1 mutagenic strain;
3) Performing genetic stability analysis on the M1 mutant strain with the beta-galactosidase enzyme activity improving range larger than 30% in the step 2), continuously performing subculture for 7 times, detecting the beta-galactosidase enzyme activity of the mutant strain, and selecting the M1 mutant strain with the best genetic stability as a mutant female parent;
4) carrying out 1-5 rounds of mutagenesis treatment on the mutagenic female parent obtained in the step 3), detecting the activity of beta-galactosidase of the mutagenic strain, and selecting the mutagenic strain with the highest activity of the beta-galactosidase as a bacillus circulans strain for producing high-activity beta-galactosidase.
Preferably, in step 1), the seed culture medium is prepared by the following method: adding 1000mL of distilled water into 15-20g of the prepared nutrient broth dry powder, heating for dissolving, and cooling to obtain the seed culture medium, wherein the pH value does not need to be adjusted separately.
More preferably, in step 1), the seed culture medium is prepared by the following method: adding 1000mL distilled water into 18g of the prepared nutrient broth dry powder, heating for dissolving, and cooling to obtain the seed culture medium, wherein the pH value does not need to be adjusted.
Preferably, in step 1), the sterile phosphate buffer solution is 0.01mol/L phosphate buffer solution with pH value of 6.
Preferably, in step 2), the solid medium plate is prepared by the following method: adding 1000mL of distilled water into 30-35g of the prefabricated nutrient agar dry powder, heating to dissolve, pouring into a culture dish without adjusting the pH value, and cooling to obtain a solid culture medium plate.
More preferably, in step 2), the solid medium plate is prepared by the following method: adding 1000mL of distilled water into 33g of the prepared nutrient agar dry powder, heating to dissolve, pouring the solution into a culture dish without adjusting the pH value, and cooling to obtain a solid culture medium plate.
Preferably, in step 2), the fermentation medium comprises the following components in percentage by weight: 0.6-1.6% of soybean peptone, 0.35-0.65% of yeast extract, 0.2-0.5% of disodium hydrogen phosphate, 0.12-0.25% of sodium carbonate, 0.12-0.55% of magnesium sulfate, 1.0-1.5% of lactose and 95.5-97.5% of water, wherein the pH value of the fermentation medium is 6.0-6.5.
In industrial production applications, our goal is to maximize the unit of activity of the total enzyme activity produced by fermentation per unit volume. The enzymatic activity change of the beta-galactosidase of the bacillus circulans is controlled by nonlinear control networks of different levels of DNA, RNA, protein and the like in a bacterial body. The strain variation caused by each round of mutagenesis only affects local regions of the regulatory network, for example, a beta-galactosidase gene expression regulatory element is enhanced, the stability of a transcription product of the beta-galactosidase gene is obviously improved, and a gene sequence corresponding to key amino acids of an enzymatic activity pocket of the beta-galactosidase is changed in a positive direction. A breeding process of a bacillus circulans strain for producing high-activity beta-galactosidase is guided by the remarkable improvement of the total enzyme activity of fermentation liquor, and according to the actual situation that the total enzyme activity of the beta-galactosidase of each round of mutagenized strains is improved, the screening standard of each round of strains is flexibly set, and finally a currently optimized strain is obtained.
Compared with the prior art, the invention has the following beneficial effects:
1. the bacillus circulans strain for producing high-activity beta-galactosidase can produce high-fermentation-activity beta-galactosidase liquid, and is used for industrially catalyzing galactosyl transfer reaction to produce galactooligosaccharides.
2. The invention relates to a breeding method of a bacillus circulans strain for producing high-activity beta-galactosidase, 1-methyl-3-nitro-1-nitrosoguanidine is used as a mutagen, and is subjected to multiple rounds of mutagenesis treatment to obtain a bacillus circulans strain for producing high-activity beta-galactosidase, wherein the 1-methyl-3-nitro-1-nitrosoguanidine mutagen is a bifunctional alkylating agent capable of acting with nucleic acid bases, can react with a plurality of parts of DNA molecules, is easy to replace active hydrogen atoms in the DNA molecules, leads bases and nucleic acid parts on the DNA molecules to be alkylated, leads to base pairing errors when the DNA is copied, causes mutation, and in addition, it can also form covalent bond between DNA double strands to prevent the double strands from being untied in the process of DNA replication, thereby causing mutation and greatly improving mutation frequency.
3. The breeding method is guided by the remarkable improvement of the total enzyme activity of the fermentation liquor, the screening standard of each round of strains is flexibly set according to the actual situation of the improvement of the total enzyme activity of the beta-galactosidase of each round of mutagenized strains, and finally the currently optimized strain is obtained.
Drawings
FIG. 1 is a graph showing the results of the detection of the activity of M4-mutagenized strain beta-galactosidase in example 1.
FIG. 2 is a bacteriological microscopic photograph of the M4-81 mutant strain of example 1.
FIG. 3 is a colony morphology map of the M4-81 mutant strain of example 1.
Detailed Description
The present invention is further illustrated by the following examples.
The method for calculating the activity of the beta-galactosidase comprises the following steps:
1. reagent preparation
(1) Z-buffer
16.1g of disodium hydrogenphosphate, 5.5g of sodium dihydrogenphosphate, 0.75g of potassium chloride, 0.246g of magnesium sulfate and 2.7mL of 2-mercaptoethanol were dissolved in 800mL of water, and a 2mol/L sodium hydroxide solution was added thereto to adjust the pH to 6.0. + -. 0.05 and the pH was measured by a pH meter. The solution was transferred to a 1000mL volumetric flask, made to volume with water and mixed well.
(2) O-Nitrobenzene-beta-D-galactopyranoside (ONPG) solution
250.0mg of ONPG was dissolved in 75mL of Z-buffer, and the solution was transferred to a 100mL volumetric flask and the volume was determined using Z-buffer as a substrate.
(3) Stop solution
Dissolving 10g of sodium carbonate in water as a solvent, and transferring the solution into a 100mL volumetric flask for constant volume.
(4) Preparation of test specimens
The test enzyme samples were prepared so that each mL of the final solution contained 0.05-0.25 units of beta-galactosidase.
2. Detection step
A series of 20X 150mm glass test tubes were placed in a 50. + -. 0.1 ℃ water bath, 0.25mL of ONPG solution was pipetted into the test tubes, and the temperature was maintained in the water bath.
Quickly adding 0.25mL of sample to be detected (0.25 mL of water is added into a control test tube) by using a pipette gun, starting oscillation, and pressing a stopwatch to start timing; after shaking for 10min, 0.5mL of the reaction solution was aspirated from each tube, added to a tube containing 0.5mL of the stop solution, and then stirred well.
The samples were transferred to an ELISA plate and the detection wavelength was 405nm, and the absorbance of each sample was measured using the solution in the control tube as a control.
3. Drawing of standard curve
Transferring 139.0mg of o-nitrophenol (ONP) into a 1000mL volumetric flask, dissolving with 95% alcohol before transferring, adding water to constant volume, and mixing. Using a pipette to suck 2.5, 5, 12.5 and 25mL of solution into a volumetric flask of 100mL respectively, using 10 wt% of sodium carbonate solution to fix the volume, and mixing uniformly. These solutions contained 0.025, 0.05, 0.125, 0.25 micromoles of ONP per mL, respectively.
A1 cm quartz tube was filled with the standard solution, and absorbance was measured at a wavelength of 405nm, using water as a control, the ONP concentration as an abscissa, and the absorbance of each concentration standard as an ordinate, to obtain a straight line passing through the origin.
4. Computing
And calculating and analyzing to obtain a standard curve: 4.0688X +0.0152, R2＝0.9991
(Y is the light absorption at 405nm and X is the concentration of ONP)
The method for measuring the activity of the beta-galactosidase comprises the following steps: one lactase unit (LacU) is defined as the amount of enzyme activity required to release 1 micromole of ONP per minute under the conditions of the present method.
Enzyme activity (U/mL) ═ 4 DF X/10
5. Converting the enzyme activity in 0.25mL of diluted enzyme solution into the enzyme activity of 1 mL;
DF: dilution times;
x: ONP concentration (μmol);
10: reaction time, 10 min.
The seed medium used in the examples was prepared as follows: adding 1000mL of distilled water into 15-20g of the prepared nutrient broth dry powder, heating for dissolving, and cooling to obtain the seed culture medium, wherein the pH value does not need to be adjusted separately.
The sterile phosphoric acid buffer solution is 0.01mol/L phosphoric acid buffer solution with the pH value of 6.
The solid medium plate is prepared by the following method: adding 1000mL of distilled water into 30-35g of the prefabricated nutrient agar dry powder, heating to dissolve, pouring into a culture dish without adjusting the pH value, and cooling to obtain a solid culture medium plate.
The fermentation medium comprises the following components in percentage by weight: 0.6-1.6% of soybean peptone, 0.35-0.65% of yeast extract, 0.2-0.5% of disodium hydrogen phosphate, 0.12-0.25% of sodium carbonate, 0.12-0.55% of magnesium sulfate, 1.0-1.5% of lactose and 95.5-97.5% of water, wherein the pH value of the fermentation medium is 6.0-6.5.
Example 1
1. The preliminary screening of bacillus circulans for producing beta-galactosidase with high activity comprises the following steps:
1) activating a starting strain: bacillus circulans (purchased from American type culture Collection (ATCC, number 31382)) is taken as an initial strain, streak culture is carried out, the initial strain is inoculated in 100mL of fermentation medium (the composition of the fermentation medium is 1.2 wt% of soybean peptone, 0.65 wt% of yeast extract, 0.35 wt% of disodium hydrogen phosphate, 0.25 wt% of sodium carbonate, 0.55 wt% of magnesium sulfate, 1.5 wt% of lactose and 95.50 wt% of water, pH is 6.0-6.5) after being subjected to activation culture by using a three-time slant nutrient agar medium, centrifugation is carried out after shaking culture at 37 ℃ for 48h, supernatant is taken, and beta-galactosidase enzyme activity measurement is carried out, wherein the measurement results are shown in Table 1:
TABLE 1 Bacillus circulans starting Strain beta-galactosidase enzyme Activity
2) Selecting a ring of slant strain, culturing in seed culture medium (prefabricated nutrient broth culture medium) at 37 deg.C for 6 hr, centrifuging at 10000rpm for 10min, collecting thallus, resuspending in phosphoric acid buffer solution with pH of 6.0, filtering with absorbent cotton, placing in small triangular flask containing glass beads, and shaking to obtain unicellular bacteria suspension with concentration of 10 5～107One per mL.
3) Taking 1mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the concentration of 0.25, 0.5, 0.75 and 1.0mg/mL, respectively adding the solution into 1mL of bacterial suspension, carrying out mutagenesis treatment for 30min to obtain mutagenic bacteria solution I, taking 100 mu L of mutagenic bacteria solution I to coat a solid culture medium plate, culturing for 48h at 37 ℃, carrying out colony counting, calculating the lethality rate I, and determining the optimal mutagenesis concentration to be 0.5 mg/mL. The results are shown in Table 2.
TABLE 21 mutagenesis concentration of methyl-3-nitro-1-nitrosoguanidine
| Concentration of mutagenesis (mg/mL) | 0.25 | 0.5 | 0.75 | 1 |
| Lethality I (%) | 82.6 | 99.0 | 99.9 | 99.9 |
4) Adding 1mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the concentration of 0.5mg/mL into 1mL of bacterial suspension, oscillating for 15 min, 30min, 45 min and 60min at 37 ℃ respectively to obtain mutagenic bacteria liquid II, coating 100 mu L of mutagenic bacteria liquid II on a solid culture medium plate, culturing for 48h at 37 ℃, calculating the lethality rate II, and determining the optimal mutagenesis time to be 30 min. The results are shown in Table 3.
TABLE 31 mutagenesis time of methyl-3-nitro-1-nitrosoguanidine
| Mutagenesis time (min) | 15 | 30 | 45 | 60 |
| Lethality II (%) | 80.3 | 99.0 | 99.9 | 99.9 |
5) Taking 100 mu L of mutagenic bacterium liquid II subjected to mutagenic treatment in the step 4) for 30min, coating a solid medium plate, and culturing at 37 ℃ for 48 h.
6) After the colonies grow out, colonies with good growth vigor and large colonies are selected, the colonies marked by one-by-one point seeding are placed on a 96-well plate (2mL deep-well plate, liquid filling amount of each well is 800uL) containing a fermentation culture medium (the composition of the fermentation culture medium is the same as that in the step 1), the 96-well plate is placed in a shaking table for cultivation, the cultivation is carried out for 48 hours at 37 ℃, the humidity is 80% and 300r/min, M1 mutant strains are obtained, the beta-galactosidase enzyme activity of the M1 mutant strains is detected by an enzyme labeling instrument, and the mutation effect of the M1 mutant strains is calculated.
7) Rechecking the M1 mutant strain which is selected from the step 6) and has the beta-galactosidase enzyme activity improving amplitude of more than 30%, transplanting the mutant strain to a solid medium flat plate, culturing for 48h, then transplanting the culture strain to a 96-pore plate for fermentation culture, performing 5 parallel verifications on each strain, culturing for 48h at 37 ℃, and determining the beta-galactosidase enzyme activity, wherein the enzyme activities of M1-38 and M1-63 are improved most obviously and are respectively improved by 30.35% and 30.22% compared with the original strain;
2. genetic stability analysis of Bacillus circulans for production of high Activity beta-galactosidase
Inoculating M1-38 and M1-63 mutant strains into a slant solid culture medium, culturing for 48h at 37 ℃, adding 9mL sterile phosphoric acid buffer solution, eluting, diluting and coating a solid culture medium flat plate, selecting a single colony with good growth vigor and large colony after the colony grows out, carrying out continuous 7-time subculture, detecting the beta-galactosidase enzyme activity of the strains, and determining the genetic stability of the strains in the aspect of producing high-activity beta-galactosidase. Wherein M1-38 has good genetic stability, M1-38 mutant strain is selected as the mutant parent, and the specific data are shown in Table 4.
TABLE 4M 1 generation genetic stability validation
| Enzyme activity (U/mL) | Primary generation of | Generation 1 | Generation 2 | 3 rd is low | Generation 4 | 5 th generation | 6 th generation | 7 th 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 for production of high-activity beta-galactosidase
Selecting M1-38 mutagenic strain with stable heredity as mutagenic female parent, selecting a ring of slant strain in fresh seed culture medium, culturing at 37 deg.C for 6 hr, centrifuging at 10000rpm for 10min, collecting thallus, resuspending in phosphoric acid buffer solution with pH of 6.0, filtering with absorbent cotton, placing in small triangular flask with glass beads, shaking to obtain unicellular strain suspension with concentration of 105～107Taking 1mL of 1-methyl-3-nitro-1-nitrosoguanidine acetone solution with the concentration of 0.5mg/mL, adding the solution into 1mL of bacterial suspension, treating for 30min, coating the mutagenic bacterium solution on a solid culture medium for plate culture, selecting a single colony with good growth vigor, and performing fermentation culture and detecting the biomass of the strain and the activity of beta-galactosidase by using a 96-pore plate culture system. The mutagenesis step was repeated 3 times to obtain progeny mutagenized strains labeled M2, M3, M4, respectively. After 4 rounds of treatment with 1-methyl-3-nitro-1-nitrosoguanidine mutagen, M4-81 mutant strain is finally screened out to be a bacillus circulans strain for producing high-activity beta-galactosidase, the activity of the enzyme produced in M4 generation is shown in figure 1, the horizontal axis is the strain number, and the vertical axis is the enzyme activity.
As can be seen from FIG. 1, the fourth generation mutant strain M4-81 has an enzyme activity of 39.86U/mL, which is 2.60 times the activity of the enzyme produced by the original strain (Bacillus circulans ATCC No.31382), and is the currently optimized strain. The strain is named as Bacillus circulans QHT-310-M481(Bacillus circulans QHT-310-M481), and is preserved in China center for type culture collection in 2018, 10 months and 22 days, with the preservation number of CCTCC NO: m2018699, the preservation address is Wuhan university in Wuhan City, China.
The form of the bacillus circulans strain for producing the high-activity beta-galactosidase is shown in fig. 2 and fig. 3, the bacterial colony is bright yellow, the shape is regular, the surface is wet, and the edge is neat; the thallus is rod-shaped, is arranged singly, has terminal spores, does not expand and is gram-positive.
Claims (1)
1. Bacillus circulans (A)Bacillus circulans) The strain QHT-310-M481 is preserved in the China center for type culture Collection in 2018, 10 months and 22 days, and the preservation number is CCTCC NO: m2018699, the preservation address is Wuhan university in Wuhan City, China.
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