CN116286575B - A method for efficiently expressing raw starch α-amylase using Bacillus subtilis - Google Patents

Abstract

The invention discloses a method for efficiently expressing raw starch α-amylase using Bacillus subtilis, and belongs to the fields of genetic engineering and bioengineering. The recombinant Bacillus subtilis strain of the present invention uses pBHVY as the vector skeleton, uses the starch-derived α-amylase gene derived from Pontibacillus sp.ZY as the target gene, uses Bacillus subtilis WB600 as the expression host, transforms the promoter, and screens the signal peptide , optimized the RBS sequence, and optimized the 3-L tank fermentation fed-feed process for the obtained recombinant bacteria. The recombinant strain of Bacillus subtilis of the present invention can be used to efficiently express raw starch α-amylase. It is used as a production strain and fermented in a shake flask for 48 hours. The enzyme activity of raw starch α-amylase in the fermentation supernatant is 4824.2U/mL. After fermentation and culture in 3-L tank, the enzyme activity of extracellular starch α-amylase can reach as high as 49082U/mL, which is the highest level reported in the literature.

Description

A method for efficiently expressing raw starch α-amylase using Bacillus subtilis

Technical field

The invention relates to a method for efficiently expressing raw starch α-amylase using Bacillus subtilis, and belongs to the technical fields of genetic engineering and microbial engineering.

Background technique

α-amylase (α-amylase, EC 3.2.1.1) is an important class of industrial enzymes among glycoside hydrolases. It comes from a wide range of sources and is distributed in animals, plants and microorganisms. α-Amylase acts on the α-1,4-glucosidic bonds within starch, polysaccharides and oligosaccharides to produce maltose and glucosaccharides with retained α-anomeric configuration. Therefore, α-amylase is widely used in food processing, sewage treatment, pharmaceutical industry, brewing industry, and new bioenergy applications.

Among the α-amylases that have been discovered, less than 10% have the ability to degrade raw starch. This type of enzyme directly acts on raw starch without gelatinization, which can effectively simplify the preliminary processing of raw starch in the modern fermentation industry and reduce energy consumption and costs. Raw starch is also known as granular starch and does not require cooking, low temperature, sub-gelatinization temperature or unconventional starch hydrolysis. It is considered a major breakthrough in the starch processing industry. Therefore, the development of recombinant strains with the ability to efficiently express raw starch α-amylase Show greater application prospects.

At present, most raw starch α-amylases have been cloned and heterologously expressed, and the main expression hosts are Escherichia coli and Bacillus. Although the expression level of raw starch α-amylase in E. coli is relatively good, it mainly appears in the form of inclusion bodies, which is not conducive to the isolation and purification of downstream enzyme proteins. In addition, E. coli hosts have food safety issues, which limits their industrial application as fermentation hosts; while the yield of raw starch α-amylase in Bacillus subtilis is very low and cannot meet industrial needs. In the previous study, Li et al. used signal peptide screening, translation efficiency and recombinant bacterial fermentation optimization to optimize the shake flask fermentation supernatant and 3-L tank fermentation supernatant of raw starch α-amylase in Bacillus subtilis (Bacillus subtilis). The activity increased to 3915.2 and 25070U/mL, which are the highest levels currently reported in literature on raw starch-degrading α-amylase in Bacillus subtilis (Ref: He Li, et al. Enhanced extracellular raw starch-degradingα-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization. Biochemical Engineering Journal, 2022,189). Due to the relatively low enzymatic activity of extracellular starch α-amylase recombinantly produced by Bacillus subtilis, the industrial production and application of raw starch α-amylase are limited. Therefore, improving the recombinant expression level of raw starch α-amylase in Bacillus subtilis and realizing the efficient expression of raw starch α-amylase using Bacillus subtilis is of great significance to the industrial production and application of raw starch α-amylase.

Contents of the invention

The present invention uses pBHVY as the vector skeleton and Bacillus subtilis WB600 as the expression host. A single promoter screening was used to obtain the best single promoter P _ylB , and a tandem promoter strategy was used to obtain the best dual promoter P _veg - _{P ylB} to improve the transcription level; a signal peptide screening strategy was used, and by The screening method of adding different signal peptides upstream of the target gene obtained the best signal peptide SP _nucB to promote the secretion of the target protein; through the RBS Calculator, the P _veg -P _ylB sequence was added to the RBS region as a constant upstream sequence, and the downstream sequence was from the signal peptide SP _nucB to the TAA of AmyZ1, the host selected Bacillus subtilis subspecies, the RBS mutant library range was set to a maximum of 8000, a mutant library containing 8000 mutants was constructed, and the above sequences were compared according to the translation initiation rate and Gibbs free The optimal RBS sequence was sequenced and integrated into the plasmid to enhance translation efficiency; on the basis of the above, the obtained Bacillus subtilis recombinant strain was optimized for a 3-L tank fermentation feeding process to achieve raw starch α- Highly efficient expression of amylase.

The invention provides a recombinant strain of Bacillus subtilis WB600/pBHVY-G3-amyZ1, which is deposited in the China Type Culture Collection Center with the deposit number CCTCC NO: M2023021. The preservation date is January 4, 2023.

The invention also provides a method for efficiently expressing raw starch α-amylase using Bacillus subtilis, which has at least two improvements in the following (a) to (c):

(a) Use the tandem promoter P _veg -P _ylB to increase transcription levels:

(b) Use the signal peptide SP _nucB to promote the secretion of the target protein;

(c) Use RBS library calculation to obtain sequences that can improve translation efficiency.

In one embodiment of the present invention, the amino acid sequence of the raw starch α-amylase gene AmyZ1 is shown in SEQ ID NO.1.

In one embodiment of the present invention, the nucleotide sequence encoding the raw starch α-amylase gene AmyZ1 is shown in SEQ ID NO. 2.

In one embodiment of the present invention, the signal peptide gene is inserted upstream of the target gene.

In one embodiment of the present invention, the Bacillus subtilis WB600 was purchased from the China General Microbial Culture Collection Center (CGMCC), address: Chaoyang District, Beijing, China, and the collection number is CGMCC NO: 1.821.

In one embodiment of the invention, the recombinant plasmids pBHVY-SP _nucB -amyZ1, pBHVY-SP _ypuA -amyZ1, pBHVY-SP _lipB -amyZ1, pBHVY-SP _pel -amyZ1, pBHVY-SP _ydbk -amyZ1, pBHVY -SP _yndA -amyZ1, pBHVY-SP _ywmC -amyZ1, pBHVY-SP _yjcN -amyZ1.

The present invention also provides the use of the above recombinant Bacillus subtilis WB600/pBHVY-G3-amyZ1 in increasing the expression of raw starch α-amylase.

The invention also provides a method for producing raw starch α-amylase, which method is prepared by fermentation using the recombinant Bacillus subtilis WB600/pBHVY-G3-amyZ1.

In one embodiment of the present invention, the amino acid sequence of the raw starch α-amylase is shown in SEQ ID NO. 1.

In one embodiment of the present invention, the method is to inoculate the Bacillus subtilis recombinant strain WB600/pBHVY-G3-amyZ1 into a seed culture medium and culture it at 35-38°C and 180-220 rpm for 8 -12h, obtain the seed liquid; then inoculate the seed liquid into the fermentation medium for fermentation culture, and obtain the fermentation liquid containing raw starch α-amylase.

In one embodiment of the present invention, the Bacillus subtilis recombinant strain is first inoculated into a seed culture medium and cultured at 30-37°C and 180-220 rpm for 45-50 hours to obtain a seed liquid.

In one embodiment of the present invention, the seed culture medium contains 8 to 12 g/L peptone, 4 to 6 g/L yeast powder, and 8 to 12 g/L sodium chloride.

In one embodiment of the present invention, the fermentation culture is shake flask fermentation culture. The seed liquid is inoculated into the shake flask fermentation medium at an inoculation amount of 1% to 3%, and the fermentation culture is carried out at 30 to 37°C. Cultivate for 45-50 hours at 180-220 rpm; the shake flask fermentation medium contains: yeast powder 8-12g/L, peptone 14-18g/L, 4-6g/L sodium chloride, CaCl ₂ 8-12mM, calcium The initial pH of the fermentation medium containing 20 to 40 mg/L of namycin is 6 to 8.

In one embodiment of the present invention, the seed liquid is inoculated into a shake flask fermentation medium and cultured at 30-37°C and 180-220 rpm for 45-50 hours.

In one embodiment of the present invention, the fermentation culture is an upper tank fermentation culture. The seed liquid is inoculated into the seed culture medium according to an inoculation amount of 1% to 3%, and the fermentation culture is performed at 35-38°C and 180-38%. Cultivate for 8-12 hours at 220 rpm to prepare a secondary seed liquid. Inoculate the prepared secondary seed liquid into the upper tank fermentation medium at an inoculation amount of 8% to 10%, with a pH of 6.5-7.5, 30-37°C, Carry out fermentation culture at 20-40% dissolved oxygen; when the dissolved oxygen increases by more than 20%, add feed medium, and the initial feed flow rate is 14-18 mL/L·h.

In one embodiment of the present invention, the secondary seed liquid is inoculated into the upper tank fermentation medium, and 3-L tank fermentation is carried out with pH 6.5-7.5, 30-37°C, and dissolved oxygen 20-40%.

In one embodiment of the invention, the upper tank fermentation medium contains: molasses 4-6g/L, industrial yeast extract 23.5-27.5g/L, soy peptone 6.5-10.5g/L, 4-6g/L L of sodium chloride, 2 to 4 mL/L of ionic liquid, CaCl ₂ 2 to 4 mM, kanamycin 20 to 40 mg/L, and the initial pH is 6 to 8.

The ionic liquid of the upper tank medium is: 0.01% MnSO ₄ ·H ₂ O, 0.018% ZnSO ₄ ·7H ₂ O, 0.05% CaCl ₂ , 0.835% FeCl ₃ , 0.016% CuSO ₄ ·5H ₂ O, 1.005% Na ₂ -EDTA, 0.018% CoCl ₂ ·6H ₂ O.

In one embodiment of the invention, the feed culture medium contains: molasses 350-370g/L, industrial yeast extract 80-100g/L, soy peptone 20-40g/L, 4-6g/L chlorine Sodium chloride, 20-40mL/L ionic liquid, CaCl ₂ 2-4mM, kanamycin 20-40mg/L.

The ionic liquid of the feeding medium is: 0.01% MnSO ₄ ·H ₂ O, 0.018% ZnSO ₄ ·7H ₂ O, 0.05% CaCl ₂ , 0.835% FeCl ₃ , 0.016% CuSO ₄ ·5H ₂ O, 1.005% Na ₂ -EDTA, 0.018% CoCl ₂ ·6H ₂ O.

The present invention also provides the application of the above recombinant Bacillus subtilis WB600/pBHVY-G3-amyZ1 in the preparation of food, washing, papermaking, textile, alcohol and medicine containing raw starch α-amylase.

beneficial effects

(1) The present invention has successfully achieved high-efficiency expression of raw starch α-amylase AmyZ1 in Bacillus subtilis, using the technical solution of the present invention, using Bacillus subtilis WB600 (Bacillus subtilis WB600) as the expression host and pBHVY as the vector skeleton, By optimizing the promoter, signal peptide and RBS sequences, the transcription, secretion and translation efficiency of the starch-producing α-amylase AmyZ1 in Bacillus subtilis were optimized, and the recombinant Bacillus subtilis strain Bacillus subtilis WB600/pBHVY-RBS ₁ - was constructed. SP _nucB -amyZ1 (Bacillus subtilis WB600/pBHVY-G3-amyZ1).

(2) Using Bacillus subtilis WB600/pBHVY-G3-amyZ1 constructed in the present invention, after 48 hours of shake flask fermentation, the enzyme activity of raw starch α-amylase in the shake flask fermentation supernatant can be increased to 4824.2U/mL. Top-tank fermentation was carried out in a 3-L fermentation tank. The enzyme activity of extracellular starch α-amylase from the recombinant Bacillus subtilis strain was as high as 49082 U/mL, which greatly improved the enzyme activity of α-amylase. According to current literature reports, The highest shake flask fermentation enzyme activity (3915.2U/mL) and 3-L tank fermentation enzyme activity (25070U/mL) are 1.23 times and 1.96 times (Ref: He Li, et al. Enhanced extracellular rawstarch-degradingα-amylase production in Bacillus subtilis through signalpeptide and translation efficiency optimization. Biochemical Engineering Journal, 2022, 189), which has great application prospects in industrial production.

Preservation of biological materials

A recombinant strain of Bacillus subtilis, WB600/pBHVY-G3-amyZ1, is taxonomically named Bacillussubtilis WB600/pBHVY-G3-amyZ1. It was deposited at the China Type Culture Collection Center on January 4, 2023, with the deposit number CCTCC NO. :M 2023021, deposited at Wuhan University, Wuhan, China.

Description of the drawings

Figure 1 is a logical schematic diagram of vector construction for promoter replacement of the present invention.

Figure 2 is a logical schematic diagram of vector construction for signal peptide replacement of the present invention.

Figure 3 is a logical schematic diagram of vector construction for RBS replacement in the present invention.

Figure 4 shows the SDS-PAGE electrophoresis diagram of the recombinant strain B. subtilis WB600/pBHVY-RBS1-SP _nucB -amyZ1 shake flask fermentation.

Figure 5 shows the fermentation enzyme activity curves of different Bacillus subtilis recombinants in 3-L tanks.

Figure 6 shows the OD600 curves of 3-L tank fermentation of different Bacillus subtilis recombinant strains.

Figure 7 shows the fermentation enzyme activity curve of the recombinant strain B. subtilis WB600/pBHVY-G3-amyZ1 in a 3-L tank with different carbon to nitrogen ratio fed media.

Figure 8 shows the OD600 fermentation curve of the recombinant strain B. subtilis WB600/pBHVY-G3-amyZ1 in a 3-L tank fermentation medium with different carbon to nitrogen ratios.

Detailed ways

The implementation methods in the following examples are all conventional methods unless otherwise specified.

The detection methods involved in the following examples are as follows:

Alpha-amylase enzyme activity detection method:

Mix 0.3 mL of 2% raw rice starch solution and 0.27 mL of 50 mM, pH 7.0 phosphate buffer thoroughly, preheat at 40°C for 10 min, add 0.03 mL of crude enzyme solution, shake and mix, react for 10 min and then add 0.3mL DNS, shake, boil for 15 minutes and then cool quickly, centrifuge at 12000g for 1 minute, and measure the photometry at 540nm (using the inactivated enzyme solution as a control).

Under the above conditions, the amount of enzyme required to produce 1 μmol maltose per unit time is defined as 1U.

The culture media involved in the following examples are as follows:

Seed culture medium: 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride.

Shake flask fermentation medium: The initial pH of the fermentation medium consisting of 10g/L yeast powder, 16g/L peptone, 5g/L sodium chloride, 10mM CaCl ₂ and 30mg/L kanamycin is 7.0.

LB solid medium: 10g/L peptone, 5g/L yeast extract powder, 10g/L NaCl, 0.2g/L agar powder.

LB liquid medium: 10g/L peptone, 5g/L yeast extract powder, 10g/L NaCl.

Salt solution (T-base): 2g/L (NH ₄ ) ₂ SO ₄ , 18.3g/L K ₂ HPO ₄ ·3H ₂ O, 6g/L KH ₂ PO ₄ , 1g/L sodium citrate ·2H ₂ O.

GMI culture medium: 20 mL of salt solution (T base), 0.2 mL of 50% (w/v) glucose, 0.2 mL of 2% (w/v) MgSO ₄ , 0.2 mL of 10% (w/v) yeast extract, 1% (w/v) casein hydrolyzate 0.4mL, 2mg/mL tryptophan solution 0.5mL.

GMII culture medium: 10 mL of salt solution (T base), 0.1 mL of 50% (w/v) glucose, 0.1 mL of 2% (w/v) MgSO ₄ , 0.04 mL of 10% (w/v) yeast extract, 1% (w/v) casein hydrolyzate 0.02 mL, 6% (w/v) CaCl ₂ 0.01 mL, 10% (w/v) MgCl ₂ 0.05 mL, 2 mg/mL tryptophan solution 0.25 mL.

Note: Among the components of GMI and GMII culture media, except for the salt solution, the remaining solutions must be sterilized separately, tryptophan must be filtered off, and all components must be mixed before use.

The basic culture medium A of the upper tank culture medium is: glycerol 10g/L, peptone 24g/L, yeast extract 15g/L, sodium chloride 7.5g/L, CaCl ₂ 3mM, kanamycin 30mg/L. The initial pH of the fermentation medium is 7.0.

The feed medium A of the upper tank medium is: glycerol 400g/L, peptone 60g/L, yeast extract 20g/L, sodium chloride 7.5g/L, CaCl ₂ 3mM, and kanamycin 30mg/L. The initial pH of the fermentation medium was 7.0.

The basic medium B of the upper tank culture medium is: molasses 5g/L, industrial yeast extract 25.5g/L, soy peptone 8.5g/L, 5g/L sodium chloride, 3mL/L ionic liquid, CaCl ₂ 3mM , the initial pH of the fermentation medium containing 30 mg/L kanamycin is 7.0; the ionic liquid is: 0.01% MnSO ₄ ·H ₂ O, 0.018% ZnSO ₄ ·7H ₂ O, 0.05% CaCl ₂ , 0.835% FeCl ₃ , 0.016% CuSO ₄ ·5H ₂ O, 1.005% Na ₂ -EDTA, 0.018% CoCl ₂ ·6H ₂ O.

The feed medium B of the upper tank culture medium is: 360g/L molasses, 90g/L industrial yeast extract, 30g/L soy peptone, 5g/L sodium chloride, 30mL/L ionic liquid, CaCl ₂ 3mM, Kanamycin 30mg/L; ionic liquid: 0.01% MnSO ₄ ·H ₂ O, 0.018% ZnSO ₄ ·7H ₂ O, 0.05% CaCl ₂ , 0.835% FeCl ₃ , 0.016% CuSO ₄ ·5H ₂ O, 1.005 %Na ₂ -EDTA, 0.018% CoCl ₂ ·6H ₂ O.

Example 1: Construction of recombinant Bacillus subtilis bacteria containing different single promoters and their shake flask fermentation

Specific steps are as follows:

(1) Obtaining the carrier skeleton

The vector backbone pBH-amyZ1 fragment was obtained from pBHYCO6 using primers P1/P2 (the construction method of the pBHYCO6 vector was disclosed in He Li, et al. Enhanced extracellular raw starch-degradingα-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization.Microb Cell FactJournal.2022.21(1).127); the nucleotide sequence of the α-amylase amyZ1 is shown in SEQ ID NO.2; the primer sequence is shown in Table 1.

Table 1: Primer sequences

primer Sequence(5’-3’) P1 ACTTCTCAAAGATCCCATGTGCT P2 GGTACCCCCATAGATGAATCCGAA

The PCR amplification program was as follows: pre-denaturation at 94°C, 5 min; denaturation at 94°C, 10 s, annealing at 60°C, 10 s, extension at 72°C, 4 min, 30 cycles; 72°C, 10 min. PCR products were recovered by 1% agarose electrophoresis.

The vector backbone pBH-amyZ1 fragment was prepared.

(2) Obtaining promoter

The Bacillus subtilis 168 genome was used as the template, and P3/P4, P5/P6, P7/P8, P9/P10, P11/P12, P13/P14 were used as primers to amplify the single promoter P _veg and P _ylB , P _srfA , P _spoVG , P _hag and P _hapll , the primer sequences are shown in Table 2:

Table 2: Primer sequences

The PCR amplification program is as follows: pre-denaturation at 94°C, 5 min; denaturation at 94°C, 10 s, annealing at 55°C, 10 s, extension at 72°C, 20 s, 30 cycles; 72°C, 10 min. PCR products were recovered by 1.5% agarose electrophoresis.

Single promoter P _veg , P _ylB , P _srfA , P _spoVG , P _hag and P _hapll fragments were prepared respectively.

(3) Obtaining recombinant bacteria containing different single promoters

Use the POE-PCR method to connect the vector backbone pBH-amyZ1 fragment obtained in step (1) and the single promoter fragment obtained in step (2). The POE-PCR reaction is shown in Table 3:

Table 3: POE-PCR reaction system

The PCR amplification program was as follows: pre-denaturation at 94°C, 5 min; denaturation at 94°C, 10 s, annealing at 55°C, 10 s, extension at 72°C, 15 min, 30 cycles; 72°C, 20 min.

Transform the PCR product into Bacillus subtilis WB600 competent cells; transform the recombinant Bacillus subtilis, the specific steps are as follows:

Streak Bacillus subtilis WB600 on an LB solid plate containing 1% starch, and culture it at 37°C overnight; use an inoculation loop to pick a single clone and graft it into 5 mL of GMI solution, and culture it at 37°C with shaking at 200 rpm for 10-12 hours. The next day, take 2 mL of fresh culture solution and transfer it to 18 mL of GMI solution. Incubate at 37°C with shaking at 200 rpm for 4.5 hours. Then take 10 mL of the above culture solution and transfer it into 90 mL of GMII. Incubate at 37°C with shaking at 200 rpm for 1.5 hours. Competent cells. Dispense the competent cells into 500 μL into a 2 mL sterilized centrifuge tube for later use. It is best to prepare the competent cells for immediate use; during transformation, add an appropriate amount of DNA (~1 μg) to 500 μL of the competent cells. Incubate for 2.5 hours at 37°C with slow shaking (120 rpm), then spread on a plate containing 30 mg/L Kan and 1% soluble starch, and culture at 37°C overnight; pick a single colony with an obvious transparent starch ring the next day, extract the plasmid and send it for sequencing. The correct sequence analysis is the recombinant Bacillus subtilis corresponding to the single promoter.

Recombinant bacteria were prepared respectively: B.subtilis WB600/pBH-P _veg -amyZ1, B.subtilis WB600/pBH-P _ylB -amyZ1, B.subtilis WB600/pBH-P _srfA -amyZ1, B.subtilis WB600/pBH-P _spoVG -amyZ1, B.subtilis WB600/pBH-P _hag -amyZ1, B.subtilis WB600/pBH-P _hapll -amyZ1.

(4) Shake flask fermentation of recombinant strains of Bacillus subtilis containing different single promoters

The recombinant Bacillus subtilis obtained in step (3) was first inoculated into the seed culture medium, and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. The seed liquid was inoculated into the shake flask at an inoculum volume of 2% (v/v). In the fermentation medium, the fermentation liquid was obtained by culturing for 48 h at 30°C and 200 rpm.

The obtained fermentation broth of the recombinant Bacillus subtilis was centrifuged at 4°C, 8000 rpm for 10 minutes, and the supernatant after centrifugation was the crude enzyme solution obtained from the fermentation. The crude enzyme liquid obtained was tested for raw starch α-amylase activity, and the results are shown in Table 4.

Table 4: Shake flask fermentation enzyme activities of different recombinant bacteria

strains Enzyme activity (U/mL) B.subtilis WB600/pBH- _Pveg -amyZ1 1655.9 B.subtilis WB600/pBH- _PylB -amyZ1 1836.2 B.subtilis WB600/pBH-P _srfA -amyZ1 395.6 B.subtilis WB600/pBH-P _spoVG -amyZ1 1466.9 B.subtilis WB600/pBH- _Phag -amyZ1 1445.2 B.subtilis WB600/pBH- _Phapll -amyZ1 1390.8

The results showed that the recombinant bacteria using promoters P _veg and P _ylB had the best enzyme activity.

Example 2: Construction of recombinant Bacillus subtilis with tandem promoters and shake flask fermentation

Specific steps are as follows:

(1) Obtaining tandem promoters

The above two promoters P _veg and P _ylB were connected using the Overlap method to form a dual promoter P _veg -P _ylB . P _veg and P _ylB were added to the reaction system at a molar ratio of 1:1. The specific reaction system is shown in Table 5:

Table 5: Overlap reaction system

The PCR amplification program was as follows: pre-denaturation at 94°C, 5 min; denaturation at 94°C, 10 s, annealing at 55°C, 10 s, extension at 72°C, 40 s, 30 cycles; 72°C, 10 min. PCR products were recovered by 1.5% agarose electrophoresis.

(2) Prepare the vector backbone pBH-amyZ1 fragment according to the method of Example 1.

(3) Obtaining recombinant bacteria containing tandem promoters

Use the POE-PCR method to connect the vector skeleton pBH-amyZ1 obtained in step (2) with the dual promoter P _veg -P _ylB obtained in step (1), and then transform the PCR product into Bacillus subtilis WB600 competent cells. , pick the positive clones, extract the plasmid and conduct sequence analysis. The correct one is the dual promoter expression vector pBHVY-amyZ1 (as shown in Figure 1). At the same time, the recombinant strain B. subtilis WB600/pBHVY-amyZ1 was prepared. The POE-PCR reactions are shown in Table 6.

Table 6: POE-PCR reaction system

The PCR amplification program was as follows: pre-denaturation at 95°C, 5 min; denaturation at 94°C, 10 s, annealing at 55°C, 10 s, extension at 72°C, 15 min, 30 cycles; 72°C, 10 min.

(4) Perform shake flask fermentation on the correctly sequenced strains.

The shake flask fermentation method was the same as the single promoter shake flask fermentation in step (4) of Example 1 above, and a crude enzyme liquid was prepared. The crude enzyme activity of the fermentation supernatant was detected, and the results are shown in Table 7.

Table 7: Shake flask fermentation enzyme activity of recombinant bacteria

strains Shake flask enzyme activity (U/mL) B.subtilis WB600/pBHVY-amyZ1 3687.7

Example 3: Construction and fermentation of recombinant Bacillus subtilis bacteria containing different signal peptides

(1) Obtaining backbone plasmid and signal peptide

The plasmid pBHVY-amyZ1 constructed in Example 2 was used as the template, and P15/P16 was used as the primer to amplify the backbone plasmid pBHVY-SP-amyZ1. The Bacillus subtilis 168 genome was used as the template, and P17/P18, P19/P20, P21/P22, P23/P24, P25/P26, P27/P28, P29/P30 and P31/P32 were used as primers to amplify the signal peptide. SP _nucB , SP _ypuA , SP _lipB , SP _pel , SP _ydbk , SP _yndA , SP _ywmC and SP _yjcN .

The primer sequences are shown in Table 8:

Table 8: Primer sequences

The PCR amplification program is as follows: pre-denaturation at 94°C, 5min; denaturation at 94°C, 10s, annealing at 55°C, 10s, extension at 72°C, 4min (backbone plasmid)/20s (signal peptide SP _nucB ), 30 cycles; 72°C, 10 minutes. PCR products were recovered by 1% agarose electrophoresis (Figure 2).

(2) Obtaining recombinant bacteria containing different signal peptides

The backbone plasmid pBHVY-SP-amyZ1 was connected to the signal peptide sequence using the POE-PCR method, and then transformed into Bacillus subtilis WB600 competent cells. Positive clones were picked, the plasmid was extracted and sequence analyzed to obtain the correct plasmid. , at the same time, recombinant bacteria containing different signal peptides B.subtilis WB600/pBHVY-SP _nucB -amyZ1, B.subtilis WB600/pBHVY-SP _ypuA -amyZ1, B.subtilis WB600/pBHVY-SP _lipB -amyZ1, B. subtilis WB600/pBHVY-SP _pel -amyZ1, B.subtilis WB600/pBHVY-SP _ydbk -amyZ1, B.subtilis WB600/pBHVY-SP _yndA -amyZ1, B.subtilis WB600/pBHVY-SP _ywmC -amyZ1, B.subtilis WB600 /pBHVY- _SPyjcN -amyZ1. The POE-PCR reactions are shown in Table 9.

Table 9: POE-PCR reaction system

The PCR amplification program is as follows: pre-denaturation at 95°C, 5 min; denaturation at 94°C, 10 s, annealing at 55°C, 10 s, extension at 72°C, 15 min, 30 cycles; 72°C, 20 min.

(3) Perform shake flask fermentation on the correctly sequenced strains.

The crude enzyme activity of the fermentation supernatant was detected respectively, and the results are shown in Table 10: The shake flask fermentation method was the same as step (4) of Example 1.

Table 10: Shake flask fermentation enzyme activities of different recombinant bacteria

strains Shake flask enzyme activity (U/mL) B.subtilis WB600/pBHVY-SP _nucB -amyZ1 4199.1 B.subtilis WB600/pBHVY- _SPypuA -amyZ1 3687.7 B.subtilis WB600/pBHVY-SP _lipB -amyZ1 3160.5 B.subtilis WB600/pBHVY- _SPpel -amyZ1 3409.2 B.subtilis WB600/pBHVY- _SPydbk -amyZ1 3442.6 B.subtilis WB600/pBHVY-SPyndA- _amyZ1 2922.1 B.subtilis WB600/pBHVY- _SPywmC -amyZ1 3370.8 B.subtilis WB600/pBHVY- _SPyjcN -amyZ1 3009.2

It can be seen that the alpha-amylase activity of shake flask fermentation raw starch of the recombinant Bacillus subtilis strain B. subtilis WB600/pBHVY-SP _nucB -amyZ1 is the highest.

Example 4: Construction of recombinant Bacillus subtilis strains containing different RBS sequences and shake flask fermentation

(1) Obtaining different RBS sequences

The tandem promoter P _veg -P _ylB sequence to the RBS region is used as a constant upstream sequence through the RBS Calculator. The downstream sequence is from the signal peptide SP _nucB to the TAA of AmyZ1. The host is selected as Bacillus subtilis subspecies. The RBS mutant library range is set to a maximum of 8000. , a mutant library containing 8000 mutants was constructed. The above sequences were sorted according to translation initiation rate and Gibbs free energy to obtain the best 10 RBS sequences (see Table 11), which were integrated into the plasmid and completed. Transformation.

Table 11: Different RBS sequences

Sequence sorting RBS sequence translation initiation rate au Total Gibbs free energy change (kcal/mol RBS-1 AAAGGAGGTTTTGGA 1868068.013 -16.27145146 RBS-2 AAAGGAGGTGTTAGA 1760246.408 -16.13935146 RBS-3 AAAGGAGGTTTTACA 1705887.779 -16.06965146 RBS-4 AAAGGAGGTTTGGGA 1695631.205 -16.05625146 RBS-5 AAAGGAGGTTATAGA 1651200.015 -15.99725123 RBS-6 AAAGGAGGTTTGAGA 1549676.089 -15.85625117 RBS-7 AAAGGAGGTTAGGGA 1539111.619 -15.84105146 RBS-8 AAAGGAGGTTATACA 1514649.072 -15.80545146 RBS-9 AAAGGAGGTGTGAGT 1470916.815 -15.74035155 RBS-10 AAAGGAGGTGAGAGT 1453674.735 -15.71415142

Use P36/P35, P37/P35, P38/P35, P39/P35, P40/P35, P41/P35, P42/P35, P43/P35, P44/P35 and P45/P35 as primers to amplify RBS ₁ and RBS ₂ respectively. , RBS ₃ , RBS ₄ , RBS ₅ , RBS ₆ , RBS ₇ , RBS ₈ , RBS ₉ , RBS ₁₀ sequence. The primer sequences are shown in Table 12.

Table 12 Primer sequences

primer Sequence(5’-3’) P33 GTTTAATGGTTCCGCAACAGATCCAAGGCGCATCTTCGGGATC P34 CCATTTTTTCATTGATCCTTCCTCCTTTAATTGGGCTAATAG P35 GCAAGAAACAGGCCTGCCATCCATTTTTTCATTTCCAAAACCTCCTTT P36 CTATTAGCCCAATTAAAGGAGGTTTTGGAAATGAAAAAATGG P37 CTATTAGCCCAATTAAAGGAGGTGTTAGAAATGAAAAAATGG P38 CTATTAGCCCAATTAAAGGAGGTTTTACAAATGAAAAAATGG P39 CTATTAGCCCAATTAAAGGAGGTTTGGGAAATGAAAAAATGG P40 CTATTAGCCCAATTAAAGGAGGTTATAGAAATGAAAAAATGG P41 CTATTAGCCCAATTAAAGGAGGTTTGAGAAATGAAAAAATGG P42 CTATTAGCCCAATTAAAGGAGGTTAGGGAAATGAAAAAATGG P43 CTATTAGCCCAATTAAAGGAGGTTATACAAATGAAAAAATGG P44 CTATTAGCCCAATTAAAGGAGGTGTGAGTAATGAAAAAATGG P45 CTATTAGCCCAATTAAAGGAGGTGAGAGTAATGAAAAAATGG

The PCR amplification program is as follows: pre-denaturation at 94°C, 5 min; denaturation at 94°C, 10 s, annealing at 55°C, 10 s, extension at 72°C, 2 min (backbone plasmid)/2 min (RBS sequence), 30 cycles; 72°C, 10 min. PCR products were recovered by 1% agarose electrophoresis (Figure 3).

(2) Obtaining recombinant bacteria containing different RBS sequences

The plasmid pBHVY-SP _nucB -amyZ1 constructed in the above Example 3 was used as a template, and P33 and P34 were used as primers to amplify the backbone plasmid pBHVY-SP _nucB -amyZ1.

The backbone plasmid pBHVY-SP _nucB -amyZ1 was connected to the RBS ₁ to RBS ₁₀ sequences using the POE-PCR method. The POE-PCR reaction is shown in Table 13:

Table 13: POE-PCR reaction system

Then the ligation product was transformed into Bacillus subtilis WB600 competent cells, positive clones were picked, plasmids were extracted and sequence analyzed. The correct plasmid was pBHVY-RBS _1～10 -SP _nucB -amyZ1. At the same time, recombinant bacteria containing different RBS sequences were prepared: B. subtilis WB600/pBHVY-RBS _1～10 -SP _nucB -amyZ1.

(3) Shake flask fermentation of recombinant Bacillus subtilis strains with different RBS sequences:

The obtained recombinant strain of Bacillus subtilis was first inoculated into the seed medium, and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. The seed liquid was inoculated into the shake flask fermentation medium at an inoculum volume of 2% (v/v). The fermentation broth was obtained by culturing for 48 hours at 30°C and 200 rpm.

The obtained fermentation broth of the recombinant Bacillus subtilis was centrifuged at 4°C, 8000 rpm for 10 minutes, and the supernatant after centrifugation was the crude enzyme solution obtained from the fermentation. Among them, the SDS-PAGE electrophoresis diagram of the shake flask fermentation of the recombinant strain B. subtilis WB600/pBHVY-RBS ₁ -SP _nucB -amyZ1 is shown in Figure 4. The crude enzyme solution obtained was tested for raw starch α-amylase activity. The results As shown in Table 14.

Table 14: Shake flask fermentation enzyme activities of different recombinant bacteria

strains Enzyme activity (U/mL) B.subtilis WB600/pBHVY-RBS ₁ -SP _nucB -amyZ1 4824.2 B.subtilis WB600/pBHVY-RBS ₂ -SP _nucB -amyZ1 2639.1 B.subtilis WB600/pBHVY-RBS ₃ -SP _nucB -amyZ1 4251.0 B.subtilis WB600/pBHVY-RBS ₄ -SP _nucB -amyZ1 2644.5 B.subtilis WB600/pBHVY-RBS ₅ -SP _nucB -amyZ1 3287.7 B.subtilis WB600/pBHVY-RBS ₆ -SP _nucB -amyZ1 2575.4 B.subtilis WB600/pBHVY-RBS ₇ -SP _nucB -amyZ1 3603.4 B.subtilis WB600/pBHVY-RBS ₈ -SP _nucB -amyZ1 3210.9 B.subtilis WB600/pBHVY-RBS ₉ -SP _nucB -amyZ1 2999.2 B.subtilis WB600/pBHVY-RBS ₁₀ -SP _nucB -amyZ1 2255.4

It can be seen that the starch α-amylase activity in the fermentation supernatant of the recombinant B. subtilis WB600/pBHVY-RBS ₁ -SP _nucB -amyZ1 can be as high as 4824.2U/mL, which is the highest level of the recombinant B. subtilis WB600/pBHVY-RBS 1 -SP nucB -amyZ1. 1.14 times that of pBHVY-SP _nucB -amyZ1 (4199.1), and the Bacillus subtilis recombinant strain B. subtilis WB600/pBHVY-RBS ₁ -SP _nucB -amyZ1 was named Bacillus subtilis WB600/pBHVY-G3-amyZ1.

Example 5: 3-L tank fermentation of different Bacillus subtilis recombinant strains

Specific steps are as follows:

(1) The activated recombinant B. subtilis WB600/pBH- _PylB -amyZ1 (Example 1), B.subtilis WB600/pBHVY-amyZ1 (Example 2), and B.subtilis WB600/pBHVY- SP _nucB -amyZ1 (Example 3) and B. subtilis WB600/pBHVY-G3-amyZ1 (Example 4) were inoculated into 5 ml seed culture medium containing 30 mg/L kanamycin and placed at 37°C and 200 rpm. Cultivate for 10-12 hours, and obtain the first-level seed liquid prepared separately;

(2) Inoculate the first-level seed liquid into a shake flask containing 100 mL of seed culture medium containing 30 μg/mL kan at an inoculum volume of 2% (v/v), and culture on a 37°C shaker at 200 rpm for 8-10 hours. As a secondary seed liquid;

(3) Inoculate the secondary seed liquid into a 3-L fermenter containing 1100 mL of basal medium A containing 30 μg/mL kan and 3 mM CaCl ₂ at an inoculum volume of 8-10% (v/v). 3-L tank fermentation was carried out at pH 7.5, 30°C, and dissolved oxygen 20 to 40%. When the dissolved oxygen rise exceeds 20%, feed (feed medium A) is added, and the initial feed flow rate is 16 mL/L·h. Samples were taken every 4 hours until the enzyme activity no longer increased, which was considered the fermentation end point. Samples were taken at different fermentation times and the biomass and fermentation enzyme activity of the samples were measured.

The fermentation broth of the recombinant Bacillus subtilis obtained by sampling was centrifuged at 4°C, 8000 rpm for 10 minutes, and the supernatant after centrifugation was the crude enzyme solution obtained from the fermentation. The crude enzyme liquid obtained was tested for raw starch α-amylase activity respectively, among which B.subtilis WB600/pBH- _PylB -amyZ1 (Example 1), B.subtilis WB600/pBHVY-amyZ1 (Example 2), B .subtilis WB600/pBHVY-SP _nucB -amyZ1 (Example 3) and B.subtilis WB600/pBHVY-G3-amyZ1 (Example 4) obtained the maximum enzyme activity at 60, 68, 64 and 68h respectively (Figure 5). The results As shown in Table 15, and the OD600 curves of the 3-L tank fermentation of the above-mentioned different Bacillus subtilis recombinant strains are shown in Figure 6.

Table 15: 3-L tank enzyme activity of recombinant bacteria

strains 3-L tank enzyme activity (U/mL) B.subtilis WB600/pBH- _PylB -amyZ1 16280.8 B.subtilis WB600/pBHVY-amyZ1 32179.5 B.subtilis WB600/pBHVY-SP _nucB -amyZ1 35824.4 Bacillus subtilis WB600/pBHVY-G3-amyZ1 41251.3

It can be seen that the raw starch α-amylase activity in the fermentation supernatant of the recombinant Bacillus subtilis B. subtilis WB600/pBHVY-G3-amyZ1 can be as high as 41251.3U/mL, which is the highest level of the recombinant Bacillus subtilis B. subtilis WB600/pBH-P _ylB -2.55 times that of amyZ1.

Example 6: Optimization of 3-L tank horizontal fermentation of recombinant strain Bacillus subtilis WB600/pBHVY-G3-amyZ1

Adjust the carbon-to-nitrogen ratio in feed medium B and set the total carbon-nitrogen source to 480g/L, with molasses as the carbon source, industrial yeast extract and soy peptone as the compound nitrogen source, and among the compound nitrogen sources, industrial yeast extract The ratio of soybean peptone and soybean peptone is 3:1. Adjust the carbon-nitrogen ratio in the culture medium, that is, adjust the ratio of carbon source and compound nitrogen source. The adjustment results are shown in Table 16. The specific steps are as follows:

(1) Plate the recombinant Bacillus subtilis WB600/pBHVY-G3-amyZ1 glycerol tube stored in a -80°C refrigerator on LB solid medium containing kanamycin sulfate resistance (30 mg/L) and 1% soluble starch. Line, culture the streaked LB solid culture medium at 37°C for 12 hours, pick single clones from the LB solid culture medium into the LB liquid culture medium, and culture it at 37°C and 200 rpm for 12 hours to obtain the seed liquid.

(2) Inoculate the seed liquid obtained in step (1) into 3-L of the basal medium B of the upper tank medium containing 30 μg/mL kan and 3mM CaCl ₂ at an inoculation volume of 8 to 10% (v/v) In the fermentation tank, culture at 30°C and pH 7.5; inoculate the bacterial cells onto the basal medium of the upper tank and culture for 6 to 8 hours. When the dissolved oxygen rises by more than 20%, start to flow into the basal medium of the upper tank and add water from the tank. Feeding medium B, the initial feeding flow rate is 16mL/L·h;

Among them, adjust the carbon-nitrogen ratio in the feeding medium (set the total carbon-nitrogen source to 480g/L, in which molasses is the carbon source, industrial yeast extract and soy peptone are the compound nitrogen source, and among the compound nitrogen sources, industrial yeast The ratio of extract to soy peptone is 3:1, and the carbon-nitrogen ratio in the culture medium (that is, adjusting the ratio of carbon source and compound nitrogen source) is adjusted to 7:1, 5:1, 3:1, 5:1, respectively. 1:1 and 1:2 (as shown in Table 16) and by coupling the stirring speed (200~800rpm) and the pure oxygen content in the ventilation (0~30%), the dissolved oxygen content in the fermentation broth during the fermentation process Control it between 20-40%; in addition, use dilute hydrochloric acid (7-8%, sterile water) to regulate the pH of the fermentation broth during the fermentation process and maintain it at around 7.0 to facilitate the stable expression of raw starch α-amylase.

Table 16: Component B of feed medium with different carbon to nitrogen ratios

The results showed (Figure 7) that when the carbon-to-nitrogen ratio was 3:1, the extracellular starch α-amylase activity of the recombinant Bacillus subtilis fermentation was the highest at 49082 U/mL for 68 hours, which was the highest level reported in the previous literature (25070 U/mL). mL) (described in HeLi, et al. Enhanced extracellular raw starch-degradingα-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization. Biochemical Engineering Journal, 2022, 189). The OD600 curves of the 3-L tank fermentation of the recombinant strain B.subtilisWB600/pBHVY-G3-amyZ1 with different carbon-nitrogen ratio fed media are shown in Figure 8.

Comparative example 1:

The specific implementation is the same as Example 3, except that the recombinant strain is adjusted to B. subtilis WB600/pBHSS _142- C1-amyZ1 (BZYACO6): the plasmid is adjusted, the adaptive signal peptide SP _ypuA is screened and optimized Shine-Dalgarno (SD) sequence, construct double 5' untranslated regions (double UTRs), optimize the UTR spacer, and then transfer the recombinant expression vector pBHSS ₁₄₂ -SP _ypuA -amyZ1 to the Bacillus subtilis WB600 competent state through chemical transformation In the process, the following was constructed: Bacillus subtilis recombinant strain B. subtilis WB600/pBHSS ₁₄₂ -C1-amyZ1 (BZYACO6) (the construction method of BZYACO6 is described in He Li, et al. Enhanced extracellular raw starch-degradingα-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization. Biochemical Engineering Journal, 2022,189).

The recombinant Bacillus subtilis strain BZYACO6 was cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. The seed liquid was inoculated into the shake flask fermentation medium at an inoculum volume of 2% (v/v) and cultured at 30°C and 200 rpm for 48 hours. fermentation broth. After optimization of shake flask fermentation, the enzyme activity of extracellular starch α-amylase from the recombinant Bacillus subtilis strain BZYACO6 was 3915.2U/mL.

In Example 4, the recombinant strain B. subtilis WB600/pBHVY-RBS ₁ -SP _nucB -amyZ1 in shake flask fermentation extracellular starch α-amylase activity was as high as 4824.2U/mL, which was higher than the recombinant strain BZYACO6 extracellular starch α-amylase. Enzyme activity increased by 1.23 times.

Therefore, it can be seen that by optimizing the promoter, signal peptide and RBS sequence of plasmid pBHE-amyZ1 and optimizing the feeding process of the recombinant bacteria 3-L tank fermentation, the finally obtained recombinant bacteria Bacillus subtilis WB600/pBHVY-G3-amyZ1 is indeed more BZYACO6 is more suitable for recombinant expression of raw starch α-amylase.

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (9)

1. Bacillus subtilis recombinant strain WB600/pBHVY-G3-amyZ1The culture medium is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m2023021, the preservation date is 2023, 01, 04.

2. A process for producing a raw starch alpha-amylase, which comprises using the recombinant Bacillus subtilis WB600/pBHVY-G3 according to claim 1amyZ1Fermenting to obtain; the amino acid sequence of the raw starch alpha-amylase is shown as SEQ ID NO. 1.

3. The process according to claim 2, wherein the recombinant Bacillus subtilis WB600/pBHVY-G3 is preparedamyZ1Firstly, inoculating the strain into a seed culture medium, and culturing for 8-12 hours at the temperature of 35-38 ℃ and at the speed of 180-220 rpm to obtain seed liquid; and then inoculating the seed solution into a fermentation medium for fermentation culture to obtain fermentation liquor containing raw starch alpha-amylase.

4. The method according to claim 3, wherein the seed medium comprises 8 to 12g/L peptone, 4 to 6g/L yeast powder and 8 to 12g/L sodium chloride.

5. The production method according to claim 4, wherein the fermentation culture is shake flask fermentation culture, the seed liquid is inoculated into a shake flask fermentation culture medium according to an inoculum size of 1% -3%, and the culture is carried out for 45-50 h at 30-37 ℃ and 180-220 rpm; the shake flask fermentation medium comprises: 8-12 g/L yeast powder, 14-18 g/L peptone, 4-6 g/L, caCl sodium chloride ₂ 8-12 mM, 20-40 mg/L kanamycin; the initial pH of the fermentation medium is 6-8.

6. The production method according to claim 4, wherein the fermentation culture is an upper tank fermentation culture, the seed liquid is inoculated into a seed culture medium according to an inoculum size of 1% -3%, the seed liquid is cultured for 8-12 hours at 35-38 ℃ and 180-220 rpm, the prepared secondary seed liquid is prepared, the prepared secondary seed liquid is inoculated into an upper tank fermentation culture medium according to an inoculum size of 8% -10%, and the fermentation culture is carried out at a pH of 6.5-7.5, 30-37 ℃ and dissolved oxygen of 20-40%; and when the rising amplitude of the dissolved oxygen exceeds 20%, adding a feed culture medium, wherein the initial feed flow rate is 14-18 mL/L.h.

7. The method of claim 6, wherein the upper tank fermentation medium comprises: 4-6 g/L molasses, 23.5-27.5 g/L industrial yeast extract, 6.5-10.5 g/L soybean peptone, 4-6 g/L sodium chloride, 2-4 mL/L ionic liquid and CaCl ₂ 2-4 mM, 20-40 mg/L kanamycin, and an initial pH of 6-8; the ionic liquid comprises the following components: 0.01% MnSO ₄ ·H ₂ O，0.018% ZnSO ₄ ·7H ₂ O，0.05% CaCl ₂ ，0.835% FeCl ₃ ，0.016% CuSO ₄ ·5H ₂ O，1.005% Na ₂ -EDTA，0.018% CoCl ₂ ·6H ₂ O, and the rest is water.

8. The method according to claim 7, wherein the feed medium is formedThe method comprises the following steps: 350-370 g/L molasses, 80-100 g/L industrial yeast extract, 20-40 g/L soybean peptone, 4-6 g/L sodium chloride, 20-40 mL/L ionic liquid and CaCl ₂ 2-4 mM, kanamycin 20-40 mg/L; the ionic liquid comprises the following components: 0.01% MnSO ₄ ·H ₂ O，0.018% ZnSO ₄ ·7H ₂ O，0.05% CaCl ₂ ，0.835% FeCl ₃ ，0.016% CuSO ₄ ·5H ₂ O，1.005% Na ₂ -EDTA，0.018% CoCl ₂ ·6H ₂ O, and the rest is water.

9. The recombinant Bacillus subtilis WB600/pBHVY-G3 of claim 1amyZ1The application in preparing food, washing products, paper products, textile products and medical products containing raw starch alpha-amylase; the amino acid sequence of the raw starch alpha-amylase is shown as SEQ ID NO. 1.