CN104371994A - Method for high-throughput screening of recombinase high-yield bacillus subtilis host in combination with normal pressure room temperature plasma mutation mode - Google Patents

Abstract

The invention describes a novel method for combining mutagenesis and high-throughput screening of high-yielding recombinant enzyme strains, and belongs to the technical fields of genetic engineering, enzymology and fermentation. The host of Bacillus subtilis WB600 was mutagenized by ARTP mutagenesis technology, and a mutant strain with high yield of recombinase was obtained by means of screening plate primary screening, 96-well plate high-throughput secondary screening, and shaking flask fermentation screening verification . Using alkaline amylase as a screening model enzyme, high-throughput screening produced a mutant strain B.subtilis WB600-mutl2 with high enzyme activity, and the enzyme activity obtained by fermentation was 186.4U/mL, which was 130.3% of the original strain. After heterologous expression of catalase in B. subtilis WB600-mut12, the enzyme activity increased to 135.0% of the original host enzyme activity. The invention has important practical and economic value.

Description

A high-throughput screening method for high-throughput screening of recombinase-producing Bacillus subtilis hosts combined with atmospheric pressure and room temperature plasma mutagenesis

technical field

The invention relates to the fields of genetic engineering, enzymology and fermentation technology, in particular to a method for combining ARTP mutagenesis technology and high-throughput screening of a high-yield recombinant enzyme B. subtilis strain. the

Background technique

Bacillus subtilis is a simple Gram-positive bacterium that produces spores, and its genetic characteristics are relatively clear. In addition, due to the advantages of non-pathogenicity, easy fermentation, and strong protein secretion and production capacity, it is an ideal host for secreting and expressing foreign proteins in the current prokaryotic expression system. the

As the center of biocatalysis, industrial enzymes play an important role in the production of bulk biochemicals. Commonly used industrial enzyme preparations can be basically divided into two categories: one is hydrolytic enzymes, including amylase, cellulase, protease, lipase, pectinase, lactase, etc., accounting for more than 75% of market sales. More than 60% of the enzyme preparations have been produced with genetically modified strains; the second category is hydrolytic enzymes, which account for about 10% of the market sales and have a tendency to increase year by year, mainly for analytical reagent enzymes and enzymes for the pharmaceutical industry. In the food supply station, starch processing enzymes still account for the largest proportion, accounting for 15%, followed by the dairy industry, accounting for 14%. Industrial enzymes are widely used in food, textile, leather and other industries and environmental protection, so the increase in the production of industrial enzyme preparations will help achieve greater economic benefits. the

In recent years, the technology of using ARTP mutagenesis to screen mutant strains has developed rapidly and has become a very active interdisciplinary research field. Compared with traditional mutagenesis technology and other plasma technologies, ARTP biological mutagenesis has many advantages: under normal pressure, it can produce a variety of uniformly distributed highly active particles, which can instantaneously act on microorganisms to cause teratogenic mutations ; Low-voltage radio frequency is used, which has high safety; the temperature of the generated plasma is close to room temperature, which can avoid thermal damage to microorganisms caused by excessive temperature; in addition, ARTP equipment is simple, easy to operate, and low operating cost. At present, the methods for obtaining high-yielding strains of recombinant enzymes mainly include: screening from nature, using traditional physical and chemical methods for mutagenesis, optimizing the fermentation process, and constructing recombinant bacteria. Therefore, we can try to combine ARTP mutagenesis technology and 96-well plate high-throughput screening to obtain high-yield strains of recombinant enzymes, so as to solve the current situation of low production of industrial enzymes. the

Contents of the invention

The purpose of the present invention is to provide a method based on ARTP mutagenesis technology and high-throughput screening of high-yield recombinant protein B.subtilis hosts, which can effectively improve the activity of recombinase (this patent uses alkaline amylase as a screening model enzyme, and peroxidase Hydrogenase is the host verification enzyme). the

The technical scheme of the present invention is: after the starting strain B.subtilis WB600 is subjected to plasma mutagenesis, the alkaline amylase recombinant plasmid is transformed into the B.subtilis WB600 mutation library, and the starch-trypan blue nutrient plate is used for screening, based on the transparent circle The size ratio was used for primary screening, and then 96-well plates were used for high-throughput re-screening. The strains that obtained high-yielding recombinant alkaline amylase were cultured in shake flasks and then re-screened for verification. Finally, high-yielding recombinant alkaline amylase strains were obtained. the

The specific steps are as follows:

1) Activation of the starting strain: Streak culture of B. subtilis WB600, pick a single colony in fresh LB liquid medium, culture at 37°C for 8-12 hours, then transfer to fresh LB medium for synchronous culture;

2) ARTP mutagenesis treatment: wash the cells with sterile normal saline, add normal saline to resuspend the cells, dilute the cell concentration to 10 ^6-8 cells/mL, suck 5-15 μL and apply it evenly on the slide, place in the treatment Under the source, carry out mutagenesis treatment;

3) Post-cultivation: put the mutagenized bacterial solution (together with slides) into fresh LB medium immediately, cultivate after 2-3 hours, dilute the bacterial cell concentration to 10 ^5-7 cells/mL, Spread on fresh LB plates for culture;

4) Screening of recombinase high-yield strains: transform the recombinant plasmid containing the recombinase gene into the mutant library obtained by mutagenesis in step 3) to obtain a B.subtilis WB600 mutant library containing the recombinase plasmid; transform the mutant in the library into Connect on starch-trypan blue nutrient plate, observe the size of the transparent circle after cultivation, and screen mutants with small colony and large transparent circle; further carry out high-throughput screening on 96-well plate, and finally pass shake flask fermentation verification. the

The bacterial cells mentioned above were washed 2 to 6 times with sterile normal saline, and then resuspended in normal saline to obtain a bacterial suspension (OD ₆₀₀ is about 1.5 to 4.5); properly dilute the OD ₆₀₀ between 1.0 and 3.0 In order to avoid overlapping effects affecting the uniformity of mutagenesis. The parameters of the ARTP mutagenesis treatment in step 2) are set as follows: air flow 6-14 slm, radio frequency power 60-140 W, time 20-40 s, distance from the treatment source 2 mm. After the plasma mutagenesis, culture for 2-3 hours, the DNA damage can be mutated in SOS repair, and the mutation can be more stable.

In the screening method described above, a mutant library is obtained by post-culturing plated plates. Transform the mutant library with the recombinant plasmid containing the alkaline amylase gene to obtain the B. subtilis WB600 mutant library containing the recombinant plasmid of alkaline amylase. Transfer the mutants to the starch-trypan blue nutrient plate for screening, screen high-yielding recombinant enzyme mutants by the size of the transparent circle, and then perform high-throughput re-screening on a 96-well plate to obtain a small number (<20) of high-yielding mutants, and finally High-yielding strains were verified by shake flask fermentation. The composition of starch-trypan blue nutrient medium used is: soluble starch 0.5-1.5%, trypan blue 0.02-0.08%, NaCl 0.5-1.5%, Yeast Extract 0.2-0.8%, Tryptone 0.5-2.0%, agar 1.0- 2.5%. the

The seed medium is LB medium, and after culturing at 37°C for 8-12 hours, it is inoculated into the fermentation medium at an inoculum amount of 0.5-3.0%. High-throughput screening uses a 96-well plate, and the volume of each well is 500-1000 μL. The composition and preparation steps of the fermentation medium used are: first prepare phosphate buffer, weigh 1.5-3.0g KH ₂ PO ₄ and 10.0-15.0g K ₂ HPO ₄ , dilute to 100mL; Yeast Extract 18.0～30.0g, Tryptone 8.0～16.0g, glycerin 2.0～6.0mL, dissolve in 900mL water; sterilize, when the phosphate buffer is cooled to about 60℃, add 900mL Medium, shake well, set aside. The ingredients of the shake flask fermentation medium are: soluble starch 0.2-0.8%, soybean peptone 0.5-2.5%, NaCl 0.5-1.5%, bean cake powder 1.0-2.0%, liquid volume 15-80mL/250mL, fermentation temperature 37℃ , The fermentation culture time is 48～72h. The enzyme activity of alkaline amylase was determined by DNS method.

Description of drawings

Figure 1 Starch-Trypan Blue Nutrient Plate Primary Screening Transparent Circle

Figure 2 96-well plate high-throughput screening results

Figure 3 Shake flask verification results of No. 12 and No. 20 mutant strains

Detailed ways

In order to have a deeper understanding of the present invention, the present invention will be further described in conjunction with specific application examples. the

Example 1: Mutagenesis of B. subtilis WB600 by plasma mutagenesis at room temperature at atmospheric pressure

The starting strain B.subtilis WB600 was activated and cultured on LB plates, and a single colony was picked and placed in fresh LB liquid medium. The culture temperature was set at 37°C, and the liquid volume was 20mL medium in a 250mL triangular flask, and kanamycin The working concentration is 50μg/mL, the rotation speed is 200rpm, and the incubation time is 10h; 50% of the inoculum amount is transferred to fresh LB medium. After 2-3h, when the bacteria are in a state of synchronous logarithmic growth, absorb 1mL of the bacterial liquid, and use it after centrifugation. Wash with sterile normal saline for 3 to 5 times, and dilute appropriately to cell concentration OD ₆₀₀ = 1.5 to 2.0; draw 10 μL of bacterial liquid and apply it evenly on the sterilized and cooled slide; use He as the working gas, the radio frequency power is 100W, and the gas flow rate is 100W. 10slm, processing time 30s.

Perform post-cultivation operation on the bacteria after plasma mutagenesis, and immediately put the treated bacteria (together with the slide) into a 5mL centrifuge tube filled with fresh LB liquid medium, the liquid volume is 1mL, and the sealing film is sealed. , fully shaken at 37°C, 200rpm, and cultured for 2-3 hours, so that the DNA can generate a variety of mutation sites in SOS repair, and make the mutation stable inheritance. the

Example 2: Screening of alkaline amylase high-yield strains after mutagenesis

(1) Preliminary screening of starch-trypan blue nutrition plate

Dilute the post-cultivated bacterial solution to 10 ^-4 , apply two LB plates for each gradient, and incubate at 37°C for 12 hours to obtain the mutant library, transform the recombinant plasmid containing the alkaline amylase gene into the mutant library, and obtain the mutant library containing For the B. subtilis WB600 library of the alkaline amylase recombinant plasmid, use a sterile toothpick to spot the starch-trypan blue nutrient plate one by one, culture at 37°C for 8-10 hours, and select colonies with transparent circles larger than the starting bacteria (Figure 1).

(2) 96-well plate high-throughput screening

The colonies selected in the previous step were respectively inoculated into the seed medium for high-throughput screening on a 96-well plate. Each strain was subjected to three parallel experiments, and fermented at 37°C for 48 hours to measure the enzyme activity. The results of the enzyme activity assay were as follows: figure 2. Among them, No. 12 and No. 20 had the most significant increase in enzyme activity, which increased to 130.0% and 121.0% of the starting strains, respectively. the

(3) Shake flask fermentation screening verification

The No. 12 and No. 20 bacteria obtained by high-throughput screening were subjected to shake flask fermentation, 200rpm, cultured for 12h, and transferred to the fermentation medium according to the inoculum size of 2%. 200rpm, after fermentation for 60h, after sampling and centrifugation, enzyme activity was measured by DNS method. The results showed that the shake flask results were similar to those of the 96-well plate re-screening. The enzyme activity of the No. 12 mutant strain (12#) increased to 130.3% of that of the starting strain (ck), and the enzyme activity reached 186.4U/mL (Figure 3). the

Example 3: Overexpression of catalase in the mutagenic host B.subtilis WB600-mut12

The B. subtilis WB600-mut12 host containing the alkaline amylase recombinant plasmid was eliminated by adding SDS to the medium to a final concentration of 0.002-0.005% to obtain a B. subtilis WB600-mut12 host without the recombinant plasmid . Transform the constructed catalase recombinant plasmid into the B. subtilis WB600-mut12 host that has eliminated the plasmid, and obtain recombinant bacteria that can express recombinant catalase. The enzyme activity was measured after fermentation, and the results showed that the catalase-producing ability of B. subtilis WB600-mut12 after mutagenesis increased to 135.0% compared with the original host. the

Claims (5)

1., in conjunction with a method for atmospheric pressure at room plasma (ARTP) mutagenesis method high flux screening high yield recombinase (as alkali starch enzyme) B.subtilis mutant strain, it is characterized by:

1) bacterium that sets out activates: undertaken streak culture by B.subtilis WB600, choose single bacterium colony in fresh LB liquid nutrient medium, and after 37 DEG C of cultivation 8 ~ 20h, switching fresh LB, carries out synchronized culture;

2) ARTP mutagenic treatment: with resuspended thalline again, suitably dilution cell concentration to 10 after stroke-physiological saline solution washing thalline ^{6 ~ 8}individual/mL, inhales 5 ~ 15 μ L and is evenly applied on slide glass, under being placed in process source, carry out mutagenic treatment;

3) cultivate afterwards: the bacterium liquid that mutagenic treatment is crossed, put into fresh LB substratum immediately, after rear cultivation 2 ~ 3h, suitably dilution cell concentration is 10 ^{5 ~ 7}individual/mL, coats on fresh LB flat board and cultivates;

4) recombinase Screening of strain with high productivity: the recombinant plasmid transformed step 3 by containing recombinase gene) in mutagenic obtained mutant library, obtain the B.subtilis WB600 mutant library containing recombinase recombinant plasmid; By the mutant in library one by one dibbling in screening, dull and stereotyped (host that suddenlys change in for this patent screens the recombinant basic amylase of employing, its screening flat board is that starch-trypan blue nutrition is dull and stereotyped) on, observe transparent circle size after cultivating 8 ~ 12h, what the little transparent circle of bacterium colony was large may be the strain of high yield alkali starch enzyme mutant; After reducing screening scope, carry out 96 orifice plate high flux screenings, finally carry out shake flask fermentation checking superior strain.

2. the method in conjunction with ARTP mutagenesis and high flux screening recombinase high yield B.subtilis mutant strain according to claim 1, is characterized in that described 2) in stroke-physiological saline solution washing 2 ~ 6 times, resuspendedly prepare bacteria suspension (OD ₆₀₀be about 15 ~ 45); Suitable dilution OD ₆₀₀between 1.0 ~ 3.0, unlikely eclipsing effect affects the homogeneity of mutagenic treatment; Processing parameter is set to: airshed 6 ~ 14slm, radio frequency power 60 ~ 140W, time 20 ~ 40s, distance process spacing are from 2 ~ 3mm.

3. the method in conjunction with ARTP mutagenesis and high flux screening recombinase high yield B.subtilis mutant strain according to claim 1, it is characterized in that: described 3), cultivate after carrying out 2 ~ 3h, the DNA damage supervened is obtained and repairs, and make sudden change more stable in sudden change.

4. the method in conjunction with ARTP mutagenesis and high flux screening recombinase high yield B.subtilis mutant strain according to claim 1, the host that suddenlys change in this patent screens and adopts recombinant basic amylase as pattern enzyme, it is characterized in that: transform the B.subtilis WB600 mutant library obtained containing alkali starch enzyme recombinant plasmid, high productive mutant is obtained according to transparent circle size around starch-trypan blue nutrition flat board, medium component used is: Zulkovsky starch 0.5 ~ 1.5%, trypan blue 0.02 ~ 0.08%, NaCl 0.5 ~ 1.5%, Yeast Extract 0.2 ~ 0.8%, Tryptone0.5 ~ 2.0%, agar 1.0 ~ 2.5%, enzyme activity determination method selection DNS method.

5. the method in conjunction with ARTP mutagenesis and high flux screening recombinase high yield B.subtilis mutant strain according to claim 1, it is characterized in that: utilize 96 orifice plates to carry out the high flux screening of High yield Mutant in library, fermentation medium components used and preparation steps are: first join phosphoric acid buffer, take 1.5 ~ 3.0g KH respectively ₂pO ₄with 10.0 ~ 15.0g K ₂hPO ₄, be settled to 100mL; Yeast Extract 18.0 ~ 30.0g, Tryptone 8.0 ~ 16.0g, glycerine 2.0 ~ 6.0mL, is dissolved in 900mL water; Sterilizing, when phosphoric acid buffer is cooled to about 60 DEG C, adds in 900mL substratum, shakes up, stand-by.