CN116286575B - Method for efficiently expressing raw starch alpha-amylase by using bacillus subtilis - Google Patents
Method for efficiently expressing raw starch alpha-amylase by using bacillus subtilis Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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Abstract
The invention discloses a method for efficiently expressing raw starch alpha-amylase by using bacillus subtilis, belonging to the fields of genetic engineering and biological engineering. The recombinant bacillus subtilis uses pBHVY as a vector skeleton, uses a raw starch alpha-amylase gene derived from Pontibacillus ussp.ZY as a target gene, uses bacillus subtilis WB600 as an expression host, reforms a promoter, screens signal peptide, optimizes RBS sequence, and optimizes the 3-L tank fermentation feed supplement process of the obtained recombinant bacillus. The bacillus subtilis recombinant strain can be used for efficiently expressing the raw starch alpha-amylase, the bacillus subtilis recombinant strain is taken as a production strain, the bacillus subtilis recombinant strain is subjected to shake flask fermentation for 48 hours, the enzyme activity of the raw starch alpha-amylase in a fermentation supernatant is 4824.2U/mL, and the enzyme activity of the extracellular raw starch alpha-amylase after fermentation culture in a 3-L tank can reach 49082U/mL, so that the bacillus subtilis recombinant strain is the highest level reported in the current literature.
Description
Technical Field
The invention relates to a method for efficiently expressing raw starch alpha-amylase by using bacillus subtilis, belonging to the technical fields of genetic engineering and microbial engineering.
Background
Alpha-amylase (EC 3.2.1.1) is an important class of industrial enzymes in the class of glycoside hydrolases, which are widely available and distributed in animals, plants and microorganisms. Alpha-amylase acts on the alpha-1, 4-glucosidic linkages within starch, polysaccharides and oligosaccharides, producing oligomeric maltose and glucose with retained alpha-anomer configuration. Therefore, the alpha-amylase is widely applied to the aspects of food processing, sewage treatment, pharmaceutical industry, brewing industry and novel biological energy source application.
Of the alpha-amylases found, only less than 10% of the enzymes have the ability to degrade raw starch. The enzyme directly acts on raw starch without gelatinization, so that the pretreatment process of raw starch in the modern fermentation industry can be effectively simplified, and the energy consumption and the cost are reduced. Raw starch is also known as granular starch, and no need for cooking, low temperature, sub-gelatinization temperatures or non-conventional starch hydrolysis is considered a major breakthrough in the starch processing industry, and therefore development of recombinant strains capable of efficiently expressing raw starch alpha-amylase has shown greater application prospects.
Currently, most raw starch alpha-amylase has been cloned and heterologously expressed, with E.coli and Bacillus being the primary expression hosts. Although the expression level of raw starch alpha-amylase in Escherichia coli is good, the raw starch alpha-amylase mainly appears in the form of inclusion bodies, which is not beneficial to the separation and purification of downstream enzyme proteins. In addition, E.coli hosts have food safety issues that limit their industrial use as fermentation hosts; the yield of raw starch alpha-amylase in bacillus subtilis (Bacillus subtilis) is very low, and the industrial requirement cannot be met. In earlier studies, li et al increased the enzyme activities of the shake flask fermentation supernatant and the 3-L tank fermentation supernatant of raw starch alpha-amylase in Bacillus subtilis (Bacillus subtilis) to 3915.2 and 25070U/mL, respectively, by signal peptide screening and translational efficiency and recombinant fermentation optimization, which are the highest levels currently reported in the literature for raw starch alpha-amylase in Bacillus subtilis (Bacillus subtilis) (Ref: he Li, et al enhanced extracellular raw starch-degradation alpha-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization. Biochemical Engineering Journal,2022,189). The current bacillus subtilis recombinant production extracellular starch alpha-amylase has lower enzyme activity, so that the industrial production and application of the starch alpha-amylase are limited. Therefore, the recombinant expression level of the raw starch alpha-amylase in the bacillus subtilis is improved, and the efficient expression of the raw starch alpha-amylase by using the bacillus subtilis is realized, so that the method has important significance for the industrial production and application of the raw starch alpha-amylase.
Disclosure of Invention
The invention uses pBHVY as a vector framework and bacillus subtilis WB600 (Bacillus subtilis WB 600) as an expression host. Single promoter screening was used to obtain the best single promoter P ylB And an optimal dual promoter P is obtained by using a tandem promoter strategy veg -P ylB Elevating the transcription level; the optimal signal peptide SP is obtained by a screening method in which different signal peptides are added upstream of the target gene using a signal peptide screening strategy nucB Promote secretion of target protein; through RBS Calculator, P veg -P ylB Sequence to RBS region as constant upstream sequence, downstream sequence from signal peptide SP nucB TAA to AmyZ1, host selection Bacillus subtilis subspecies, RBS mutant library range set to be 8000 max, constructing a mutant library containing 8000 mutants, obtaining the best RBS sequence according to translation initiation rate and Gibbs free energy sequence and integrating the sequence on plasmid to enhance translation efficiency; on the basis, the obtained bacillus subtilis recombinant bacteria are subjected to 3-L tank fermentation feed supplement process optimization so as to realize the efficient expression of the raw starch alpha-amylase.
The invention provides a bacillus subtilis recombinant strain WB600/pBHVY-G3-amyZ1, wherein the bacillus subtilis recombinant strain WB600/pBHVY-G3-amyZ1 is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023021, the preservation date is 2023, 01, 04.
The invention also provides a method for efficiently expressing raw starch alpha-amylase by using bacillus subtilis, which has at least two improvements of the following (a) to (c):
(a) With tandem promoter P veg -P ylB Elevation of transcript levels:
(b) With signal peptide SP nucB Promote secretion of target protein;
(c) Sequences that can improve translation efficiency are obtained using RBS library calculations.
In one embodiment of the invention, the amino acid sequence of the raw starch alpha-amylase gene AmyZ1 is shown in SEQ id No. 1.
In one embodiment of the invention, the nucleotide sequence encoding the raw starch alpha-amylase gene AmyZ1 is shown in SEQ ID No. 2.
In one embodiment of the invention, the signal peptide gene is inserted upstream of the gene of interest.
In one embodiment of the invention, the bacillus subtilis WB600 (Bacillus subtilis WB) is purchased from the China general microbiological culture collection center (CGMCC), address: the preservation number of the Chaoyang district of Beijing city in China is CGMCCNO:1.821.
in one embodiment of the present invention, the recombinant plasmid 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 invention also provides application of the recombinant bacillus subtilis WB600/pBHVY-G3-amyZ1 in improving the expression level of the raw starch alpha-amylase.
The invention also provides a production method of the raw starch alpha-amylase, which is prepared by fermenting the recombinant bacillus subtilis WB600/pBHVY-G3-amyZ1.
In one embodiment of the invention, the amino acid sequence of the raw starch alpha-amylase is shown in SEQ ID NO. 1.
In one embodiment of the invention, the method comprises the steps of inoculating the bacillus subtilis recombinant strain WB600/pBHVY-G3-amyZ1 into a seed culture medium, and culturing for 8-12 hours at 35-38 ℃ and 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.
In one embodiment of the invention, the recombinant bacillus subtilis is inoculated in a seed culture medium and cultured for 45-50 hours at the temperature of 30-37 ℃ and the rpm of 180-220 to obtain seed liquid.
In one embodiment of the invention, the seed medium comprises 8 to 12g/L peptone, 4 to 6g/L yeast powder, and 8 to 12g/L sodium chloride.
In one embodiment of the invention, the fermentation culture is shake flask fermentation culture, the seed liquid is inoculated into a shake flask fermentation culture medium according to the inoculum size of 1-3%, and the culture is carried out for 45-50 h at the temperature of 30-37 ℃ and the rpm of 180-220; the shake flask fermentation medium comprises: 8-12 g/L yeast powder, 14-18 g/L peptone, 4-6 g/L sodium chloride and CaCl 2 The initial pH of the fermentation medium is 6-8, the initial pH is 8-12 mM, and the initial pH is 20-40 mg/L of kanamycin.
In one embodiment of the present invention, the seed solution is inoculated into a shake flask fermentation medium and cultured at 180 to 220rpm at 30 to 37℃for 45 to 50 hours.
In one embodiment of the invention, the fermentation culture is upper tank fermentation culture, the seed liquid is inoculated into a seed culture medium according to the inoculation amount of 1-3%, the seed liquid is cultured for 8-12h at the temperature of 35-38 ℃ and the rpm of 180-220, the prepared secondary seed liquid is prepared, and the prepared secondary seed liquid is inoculated into an upper tank fermentation culture medium according to the inoculation amount of 8-10%, and the fermentation culture is carried out under the conditions of the pH of 6.5-7.5, the temperature of 30-37 ℃ and the dissolved oxygen of 20-40%; when the rising amplitude of dissolved oxygen exceeds 20%, the feeding culture medium is added, and the initial feeding flow rate is 14-18 mL/L.h.
In one embodiment of the invention, the secondary seed solution is inoculated into an upper tank fermentation medium, and 3-L tank fermentation is performed at pH 6.5-7.5, 30-37 ℃ and dissolved oxygen of 20-40%.
In one embodiment of the invention, the upper tank fermentation medium comprises: molasses 4-6 g/L, industrial yeast extract 23.5-27.5 g/L, soytone 6.5-10.5 g/L, sodium chloride 4-6 g/L, 2-4 mL/L ionic liquid and CaCl 2 2-4 mM, kanamycin of 20-40 mg/L, and initial pH of 6-8.
The ionic liquid of the upper tank culture medium is as follows: 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 medium comprises: 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, 20-40 mg/L kanamycin.
The ionic liquid of the feed medium is as follows: 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 invention also provides application of the recombinant bacillus subtilis WB600/pBHVY-G3-amyZ1 in preparing foods, washing, papermaking, spinning, alcohol and medicines containing raw starch alpha-amylase.
Advantageous effects
(1) The invention successfully realizes the efficient expression of the raw starch alpha-amylase AmyZ1 in the bacillus subtilis, adopts the technical scheme of the invention, takes the bacillus subtilis WB600 (Bacillus subtilis WB 600) as an expression host, takes the pBHVY as a carrier framework, optimizes the transcription, secretion and translation efficiency of the raw starch alpha-amylase AmyZ1 in the bacillus subtilis respectively by optimizing the promoter, the signal peptide and the RBS sequence, and constructs and obtains the bacillus subtilisBacillus recombinant Bacillus subtilis WB/pBHVY-RBS 1 -SP nucB -amyZ1(Bacillus subtilis WB600/pBHVY-G3-amyZ1)。
(2) The Bacillus subtilis WB/pBHVY-G3-amyZ 1 constructed by the invention can improve the enzyme activity of the raw starch alpha-amylase in shake flask fermentation supernatant to 4824.2U/mL, perform upper tank fermentation in a 3-L fermentation tank, and greatly improve the enzyme activity of the alpha-amylase by 49082U/mL, which are respectively 1.23 times and 1.96 times (Ref: he Li, et al enhanced extracellular raw starch-degradation alpha-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization. Biochemical Engineering Journal,2022,189) of the highest shake flask fermentation enzyme activity (3915.2U/mL) and 3-L tank fermentation enzyme activity (25070U/mL) reported in the prior literature, and has great application prospects in industrial production.
Preservation of biological materials
Bacillus subtilis recombinant strain WB600/pBHVY-G3-amyZ1, taxonomic name Bacillus subtilis WB/pBHVY-G3-amyZ 1, was deposited in China Center for Type Culture Collection (CCTCC) No: m2023021, the preservation address is Wuhan, university of Wuhan, china.
Drawings
FIG. 1 is a schematic diagram of the vector construction logic for promoter replacement according to the present invention.
FIG. 2 is a schematic diagram of the vector construction logic for signal peptide replacement according to the present invention.
FIG. 3 is a schematic diagram of the carrier construction logic of the RBS replacement of the present invention.
FIG. 4 is recombinant B.subilis WB600/pBHVY-RBS1-SP nucB amyZ1 shake flask fermentation SDS-PAGE electrophoresis.
FIG. 5 is a graph showing the fermentation enzyme activity of 3-L tank of different recombinant Bacillus subtilis.
FIG. 6 is an OD600 curve of 3-L tank fermentation of different recombinant Bacillus subtilis strains.
FIG. 7 is a graph showing the enzyme activity of recombinant B.subilis WB600/pBHVY-G3-amyZ1 fed-batch medium 3-L tank fermentation.
FIG. 8 is a graph showing OD600 of 3-L tank fermentation of recombinant bacterium B.subilis WB600/pBHVY-G3-amyZ1 feed medium with different carbon to nitrogen ratios.
Detailed Description
The methods of implementation in the following examples are conventional, unless otherwise specified.
The detection method involved in the following examples is as follows:
the method for detecting the enzyme activity of the alpha-amylase comprises the following steps:
0.3mL of 2% rice starch solution and 0.27mL of 50mM phosphate buffer solution with pH of 7.0 are fully and uniformly mixed, the mixture is preheated at 40 ℃ for 10min, 0.03mL of crude enzyme solution is added, the mixture is uniformly mixed by shaking, 0.3mL of DNS is added after the mixture reacts for 10min, the mixture is vibrated and boiled for 15min and then rapidly cooled, 12000g of the mixture is centrifuged for 1min, and the analytical degree (taking inactivated enzyme solution as a reference) is measured at 540 nm.
Under the above conditions, the amount of enzyme required to produce 1. Mu. Mol of maltose per unit time was defined as 1U.
The following examples relate to the following media:
seed culture medium: 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride.
Shake flask fermentation medium: yeast powder 10g/L, peptone 16g/L, sodium chloride 5g/L, caCl 2 The initial pH of the fermentation medium was 7.0 at 10mM, kanamycin, 30 mg/L.
LB solid medium: 10g/L peptone, 5g/L yeast extract powder, 10g/L NaCl and 0.2g/L agar powder.
LB liquid medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl.
Salt solution (T-base): 2g/L (NH) 4 ) 2 SO 4 K at 18.3g/L 2 HPO 4 ·3H 2 KH of O, 6g/L 2 PO 4 Sodium citrate 2H at 1g/L 2 O。
GMI medium: salt solution (Tbase) 20mL, 50% (w/v) glucose 0.2mL, 2% (w/v) MgSO 4 0.2mL, 10% (w/v) yeast extract 0.2mL, 1% (w/v) casein hydrolysate 0.4mL, 2mg/mL tryptophan solution 0.5mL.
GMII medium: 10mL of salt solution (Tbase), 0.1mL of 50% (w/v) glucose, 2% (w/v) MgSO 4 0.1mL, 10% (w/v) yeast extract 0.04mL, 1% (w/v) casein hydrolysate 0.02mL, 6% (w/v) CaCl 2 0.01mL、10％(w/v)MgCl 2 0.05mL, 2mg/mL tryptophan solution 0.25mL.
Note that: among the ingredients of the GMI and GMII media, the solutions were sterilized separately except for the saline solution, tryptophan was filtered, and all components were mixed prior to use.
The basic culture medium A of the upper tank culture medium is 10g/L glycerol, 24g/L peptone, 15g/L yeast extract powder and 7.5g/L, caCl of sodium chloride 2 The initial pH of the fermentation medium was 7.0 at 30mg/L with 3mM kanamycin.
The feed medium A of the upper tank medium is as follows: 400g/L glycerol, 60g/L peptone, 20g/L yeast extract, 7.5g/L, caCl sodium chloride 2 The initial pH of the fermentation medium was 7.0 at 30mg/L with 3mM kanamycin.
The basic culture medium B of the upper tank culture medium is as follows: molasses 5g/L, industrial yeast extract 25.5g/L, soybean peptone 8.5g/L, sodium chloride 5g/L, 3mL/L ionic liquid, caCl 2 3mM, kanamycin 30mg/L of the fermentation medium at an initial pH of 7.0; 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。
The feed medium B of the upper tank medium is as follows: 360g/L molasses, 90g/L industrial yeast extract, 30g/L soybean peptone, 5g/L sodium chloride, 30mL/L ionic liquid and CaCl 2 3mM, kanamycin 30mg/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。
Example 1: construction of bacillus subtilis recombinant bacteria containing different single promoters and shake flask fermentation thereof
The method comprises the following specific steps:
(1) Obtaining of the Carrier skeleton
The vector backbone pBH-amyZ1 fragment was obtained from pBHYCO6 using primers P1/P2 (the construction method of the pBHYCO6 vector is disclosed in HeLi, et al enhanced extracellular raw starch-degradation alpha-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization. Microb Cell face journal.2022.21 (1). 127); the nucleotide sequence of the alpha-amylase amyZ1 is shown in SEQ ID NO. 2; the primer sequences are shown in Table 1.
Table 1: primer sequences
| Primer(s) | Sequence (5 '-3') |
| P1 | ACTTCTCAAAGATCCCATGTGCT |
| P2 | GGTACCCCCATAGATGAATCCGAA |
The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 60 ℃,10s, extension at 72 ℃,4min,30 cycles; 72℃for 10min. The PCR product was recovered by 1% agarose electrophoresis.
The vector backbone pBH-amyZ1 fragment was prepared.
(2) Obtaining of promoters
The bacillus subtilis (Bacillus subtilis) 168 genome is used as a template, and P3/P4, P5/P6, P7/P8, P9/P10 and P11/P12 are respectively usedP13/P14, single promoter P for primer amplification veg 、P ylB 、P srfA 、P spoVG 、P hag And P hapll Primer sequences are shown in Table 2:
table 2: primer sequences
The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 55 ℃,10s, extension at 72 ℃,20s,30 cycles; 72℃for 10min. The PCR product was recovered by electrophoresis on 1.5% agarose.
The single promoter P is prepared respectively veg 、P ylB 、P srfA 、P spoVG 、P hag And P hapll Fragments.
(3) Acquisition of recombinant bacteria containing different single promoters
Connecting the vector skeleton pBH-amyZ1 fragment obtained in the step (1) with the single promoter fragment obtained in the step (2) by using a POE-PCR method, wherein the POE-PCR reaction is shown in Table 3:
table 3: POE-PCR reaction system
The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 55 ℃,10s, extension at 72 ℃,15min,30 cycles; 72℃for 20min.
Transforming the PCR product into bacillus subtilis WB600 competent cells; the recombinant bacillus subtilis is transformed, and the specific steps are as follows:
streaking bacillus subtilis WB600 on LB solid plate containing 1% starch, and culturing overnight at 37 ℃; one of the single clones was picked up with an inoculating loop and inoculated into 5mL of GMI solution, and cultured with shaking at 37℃and 200rpm for 10-12 hours. Transferring 2mL of fresh culture solution into 18mL of GMI solution in the next day, and culturing for 4.5h at 37 ℃ under shaking at 200 rpm; then 10mL of the culture solution is transferred into 90mL of GMII, and the competent cells are obtained after shaking culture for 1.5h at 37 ℃ and 200 rpm. Packaging competent cells into 500 mu L to 2mL sterilized centrifuge tubes for later use, wherein the competent cells are best used at present; at the time of transformation, an appropriate amount of DNA (. About.1. Mu.g) was added to 500. Mu.L of competent cells. Slowly shaking at 37 ℃ for 120 rpm) for 2.5 hours, then coating the mixture on a plate containing 30mg/L Kan and 1% soluble starch, and culturing at 37 ℃ overnight; the next day single colony with obvious transparent starch ring is picked, plasmid is extracted for sequencing, and the recombinant bacillus subtilis corresponding to the single promoter is obtained after the sequence analysis is correct.
Recombinant bacteria are prepared respectively: 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 bacillus subtilis containing different single promoters
Inoculating the bacillus subtilis recombinant bacteria obtained in the step (3) into a seed culture medium, culturing at 37 ℃ and 200rpm for 12 hours to obtain a seed solution, inoculating the seed solution into a shake flask fermentation culture medium according to an inoculum size of 2% (v/v), and culturing at 30 ℃ and 200rpm for 48 hours to obtain a fermentation broth.
And (3) centrifuging the fermentation liquor of the bacillus subtilis recombinant bacteria at the temperature of 4 ℃ and at the speed of 8000rpm for 10min, wherein the supernatant after centrifugation is crude enzyme liquor obtained by fermentation. The crude enzyme solutions obtained were each subjected to measurement of the activity of raw starch alpha-amylase, and the results are shown in Table 4.
Table 4: shake flask fermentation enzyme activity of different recombinant bacteria
| Strain | Enzyme activity (U/mL) |
| B.subtilis WB600/pBH-P veg -amyZ1 | 1655.9 |
| B.subtilis WB600/pBH-P ylB -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-P hag -amyZ1 | 1445.2 |
| B.subtilis WB600/pBH-P hapll -amyZ1 | 1390.8 |
The results showed that promoter P was used veg And P ylB The enzyme activity of the recombinant bacterium is preferably.
Example 2: construction of bacillus subtilis recombinant bacteria with tandem promoters and shake flask fermentation
The method comprises the following specific steps:
(1) Obtaining of tandem promoters
The two promoters P are obtained by using an overlay method veg And P ylB Ligation into double promoters P veg -P ylB 。P veg And P ylB The catalyst is added into a reaction system according to a molar ratio of 1:1, and the specific reaction system is shown in Table 5:
table 5: overlap reaction system
The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 55 ℃,10s, extension at 72 ℃,40s,30 cycles; 72℃for 10min. The PCR product was recovered by electrophoresis on 1.5% agarose.
(2) A vector backbone pBH-amyZ1 fragment was prepared as in example 1.
(3) Obtaining recombinant bacteria containing tandem promoters
The vector skeleton pBH-amyZ1 obtained in the step (2) and the double promoter P obtained in the step (1) are subjected to a POE-PCR method veg -P ylB And (3) connecting, then transforming the PCR product into bacillus subtilis WB600 competent cells, picking positive clones, extracting plasmids, and carrying out sequence analysis, wherein the correct expression vector is the double-promoter expression vector pBHVY-amyZ1 (shown in figure 1). Meanwhile, recombinant bacteria B.subtilis WB600/pBHVY-amyZ1 are prepared. POE-PCR reactions are shown in Table 6.
Table 6: POE-PCR reaction system
The PCR amplification procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 55 ℃,10s, extension at 72 ℃,15min,30 cycles; 72℃for 10min.
(4) Shake flask fermentation of correctly sequenced strains
The shake flask fermentation method is the same as that of the single-promoter shake flask fermentation in the step (4) of the above example 1, and crude enzyme liquid is prepared. The crude enzyme solution of the fermentation supernatant was tested for enzyme activity, and the results are shown in Table 7.
Table 7: shake flask fermentation enzyme activity of recombinant bacteria
| Strain | Shaking flask enzyme activity (U/mL) |
| B.subtilis WB600/pBHVY-amyZ1 | 3687.7 |
Example 3: construction and fermentation of bacillus subtilis recombinant bacteria containing different signal peptides
(1) Backbone plasmid and signal peptide acquisition
The backbone plasmid pBHVY-SP-amyZ1 was amplified using the plasmid pBHVY-amyZ1 constructed in example 2 as a template and P15/P16 as primers. Amplifying the signal peptide SP by using the Bacillus subtilis (Bacillus subtilis) 168 genome as a template and using P17/P18, P19/P20, P21/P22, P23/P24, P25/P26, P27/P28, P29/P30 and P31/P32 as primers nucB 、SP ypuA 、SP lipB 、SP pel 、SP ydbk 、SP yndA 、SP ywmC And SP yjcN 。
Primer sequences are shown in Table 8:
table 8: primer sequences
The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 55 ℃,10s, extension at 72 ℃,4min (backbone plasmid)/20 s (signal peptide SP) nucB ) 30 cycles; 72℃for 10min. The PCR products were recovered by 1% agarose electrophoresis (FIG. 2).
(2) Acquisition of recombinant bacteria containing different Signal peptides
Connecting the skeleton plasmid pBHVY-SP-amyZ1 with a signal peptide sequence by adopting a POE-PCR method, then transforming into competent cells of bacillus subtilis WB600, picking positive clones, extracting plasmids and carrying out sequence analysis to obtain correct plasmids, and simultaneously preparing recombinant bacteria B.subilis WB600/pBHVY-SP containing different signal peptides 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-SP yjcN amyZ1.POE-PCR reactions are shown in Table 9.
Table 9: POE-PCR reaction system
The PCR amplification procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 55 ℃,10s, extension at 72 ℃,15min,30 cycles; 72℃for 20min.
(3) Shake flask fermentation of correctly sequenced strains
The enzyme activities of the crude enzyme solutions of the fermentation supernatants were measured, and the results are shown in Table 10: the shake flask fermentation method is the same as in example 1, step (4).
Table 10: shake flask fermentation enzyme activity of different recombinant bacteria
| Strain | Shaking flask enzyme activity (U/mL) |
| B.subtilis WB600/pBHVY-SP nucB -amyZ1 | 4199.1 |
| B.subtilis WB600/pBHVY-SP ypuA -amyZ1 | 3687.7 |
| B.subtilis WB600/pBHVY-SP lipB -amyZ1 | 3160.5 |
| B.subtilis WB600/pBHVY-SP pel -amyZ1 | 3409.2 |
| B.subtilis WB600/pBHVY-SP ydbk -amyZ1 | 3442.6 |
| B.subtilis WB600/pBHVY-SP yndA -amyZ1 | 2922.1 |
| B.subtilis WB600/pBHVY-SP ywmC -amyZ1 | 3370.8 |
| B.subtilis WB600/pBHVY-SP yjcN -amyZ1 | 3009.2 |
As can be seen, bacillus subtilis WB600/pBHVY-SP nucB The shake flask fermentation of amyZ1 gives rise to the highest enzymatic activity of the starch alpha-amylase.
Example 4: construction of recombinant bacillus subtilis containing different RBS sequences and shake flask fermentation
(1) Acquisition of different RBS sequences
The tandem promoter P was introduced by RBS Calculator veg -P ylB Sequence to RBS region as constant upstream sequence, downstream sequence from signal peptide SP nucB To the TAA of AmyZ1, subspecies Bacillus subtilis were selected as host, the library range of RBS mutants was set to a maximum of 8000, a mutant library was constructed containing 8000 mutants, the sequences described above were sequenced according to translation initiation rate and gibbs free energy to obtain the best 10 RBS sequences as shown in table 11, and they were integrated into plasmids and transformation was completed.
Table 11: different RBS sequences
| Sequence ordering | 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 |
RBS was amplified with primers P36/P35, P37/P35, P38/P35, P39/P35, P40/P35, P41/P35, P42/P35, P43/P35, P44/P35 and P45/P35, respectively 1 、RBS 2 、RBS 3 、RBS 4 、RBS 5 、RBS 6 、RBS 7 、RBS 8 、RBS 9 、RBS 10 Sequence. Primer sequences are shown in Table 12.
TABLE 12 primer sequences
| Primer(s) | 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 procedure was as follows: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃,10s, annealing at 55 ℃,10s, extension at 72 ℃,2min (backbone plasmid)/2 min (RBS sequence), 30 cycles; 72℃for 10min. The PCR products were recovered by 1% agarose electrophoresis (FIG. 3).
(2) Acquisition of recombinant bacteria containing different RBS sequences
Plasmid pBHVY-SP constructed in example 3 above nucB Amplification of backbone plasmid pBHVY-SP using-amyZ 1 as template and P33 and P34 as primers nucB -amyZ1。
The backbone plasmid pBHVY-SP was subjected to POE-PCR nucB amyZ1 and RBS 1 ～RBS 10 The sequences were ligated and POE-PCR reactions are shown in Table 13:
table 13: POE-PCR reaction system
Then the connection product is transformed into bacillus subtilis WB600 competent cells, positive clones are selected, plasmids are extracted and sequence analysis is carried out, and the plasmids are correctly pBHVY-RBS 1～10 -SP nucB amyZ1. Meanwhile, recombinant bacteria containing different RBS sequences are prepared: subtilis WB600/pBHVY-RBS 1～10 -SP nucB -amyZ1。
(3) Shake flask fermentation of recombinant bacillus subtilis with different RBS sequences:
inoculating the obtained bacillus subtilis recombinant bacteria into a seed culture medium, culturing at 37 ℃ and 200rpm for 12 hours to obtain a seed solution, inoculating the seed solution into a shake flask fermentation culture medium according to an inoculum size of 2% (v/v), and culturing at 30 ℃ and 200rpm for 48 hours to obtain a fermentation broth.
And (3) centrifuging the fermentation liquor of the bacillus subtilis recombinant bacteria at the temperature of 4 ℃ and at the speed of 8000rpm for 10min, wherein the supernatant after centrifugation is crude enzyme liquor obtained by fermentation. Wherein recombinant bacterium B.subilis WB600/pBHVY-RBS 1 -SP nucB The activity of raw starch alpha-amylase was measured on the crude enzyme solutions obtained by the SDS-PAGE electrophoresis of amyZ1 shake flask fermentation as shown in FIG. 4, and the results are shown in Table 14.
Table 14: shake flask fermentation enzyme activity of different recombinant bacteria
| Strain | 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 |
As can be seen, bacillus subtilis WB600/pBHVY-RBS 1 -SP nucB The activity of the raw starch alpha-amylase in the fermentation supernatant of amyZ1 can reach 4824.2U/mL, and the raw starch alpha-amylase is recombinant bacillus subtilis WB600/pBHVY-SP nucB 1.14 times of amyZ1 (4199.1), recombinant Bacillus subtilis WB600/pBHVY-RBS 1 -SP nucB amyZ1 was designated Bacillus subtilis WB/pBHVY-G3-amyZ 1.
Example 5: 3-L tank fermentation of different bacillus subtilis recombinant bacteria
The method comprises the following specific steps:
(1) Activated recombinant bacillus subtilis WB600/pBH-P respectively ylB amyZ1 (example 1), B.subulis WB600/pBHVY-amyZ1 (example 2), B.subulis WB600/pBHVY-SP nucB amyZ1 (example 3) and B.subilis WB600/pBHVY-G3-amyZ1 (example 4) were inoculated into 5ml seed medium containing 30mg/L kanamycin, placed at 37℃and cultured at 200rpm for 10-12 hours, and primary seed solutions were prepared respectively;
(2) Inoculating the first seed solution into shake flasks containing 100mL of seed culture medium containing 30 μg/mL kan at 2% (v/v), shaking at 37deg.C, and culturing at 200rpm for 8-10 hr to obtain second seed solution;
(3) Inoculating the secondary seed solution with an inoculum size of 8-10% (v/v) to a seed solution containing 1100mL of kan containing 30 μg/mL and 3mM CaCl 2 In the 3-L fermentation tank of the basic culture medium A of the upper tank culture medium, 3-L fermentation is carried out at the pH of 7.5 and 30 ℃ and with dissolved oxygen of 20-40 percent. When the rise of dissolved oxygen exceeds 20%, the feed (feed medium A) was added at an initial feed flow rate of 16 mL/L.h. Samples were taken every 4 hours until the enzyme activity did not increase any more as the fermentation end point, samples were taken at different fermentation times and the biomass and fermentation enzyme activity of the samples were determined.
Centrifuging the fermentation broth of the bacillus subtilis recombinant bacteria obtained by sampling at 4 ℃ and 8000rpm for 10min, wherein the supernatant after centrifugation is crude enzyme obtained by fermentationAnd (3) liquid. The crude enzyme solutions obtained were subjected to raw starch alpha-amylase activity assay, respectively, wherein B.subtilis WB600/pBH-P ylB amyZ1 (example 1), B.subulis WB600/pBHVY-amyZ1 (example 2), B.subulis WB600/pBHVY-SP nucB Maximum enzyme activities were obtained at 60, 68, 64 and 68h for amyZ1 (example 3) and B.subtilis WB600/pBHVY-G3-amyZ1 (example 4), respectively (FIG. 5), the results are shown in Table 15, and the 3-L tank fermentation OD600 curves for the different Bacillus subtilis recombinants described above are shown in FIG. 6.
Table 15: 3-L tank enzyme activity of recombinant bacteria
| Strain | 3-L tank enzyme activity (U/mL) |
| B.subtilis WB600/pBH-P ylB -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 |
As can be seen, the activity of the raw starch alpha-amylase in the fermentation supernatant of the recombinant bacillus subtilis WB600/pBHVY-G3-amyZ1 can reach 41251.3U/mL, and the recombinant bacillus subtilis WB600/pBH-P ylB -2.55 times amyZ1.
Example 6: 3-L tank horizontal fermentation optimization of recombinant bacterium Bacillus subtilis WB/pBHVY-G3-amyZ 1
The carbon-nitrogen ratio in the feed medium B is regulated, the total amount of carbon and nitrogen sources is set to be 480g/L, molasses is used as the carbon source, industrial yeast extract and soybean peptone are used as composite nitrogen sources, and the ratio of the industrial yeast extract to the soybean peptone in the composite nitrogen sources is 3:1, adjusting the carbon-nitrogen ratio in the culture medium, namely adjusting the ratio of a carbon source to a composite nitrogen source, wherein the adjustment results are shown in table 16, and the specific steps are as follows:
(1) Recombinant Bacillus subtilis WB/pBHVY-G3-amyZ 1 glycerol as a glycerol stock of-80℃was streaked on LB solid medium containing kanamycin resistance (30 mg/L) and 1% soluble starch, the streaked LB solid medium was allowed to stand at 37℃for 12 hours, and monoclonals were picked from the LB solid medium into LB liquid medium and cultured at 200rpm at 37℃for 12 hours to obtain a seed solution.
(2) Inoculating the seed solution obtained in the step (1) to a seed solution containing 30 mug/mL kan and 3mM CaCl according to an inoculum size of 8-10% (v/v) 2 In a 3-L fermentation tank of the basic culture medium B of the upper tank culture medium, at 30 ℃ and pH 7.5; after the thalli are inoculated to the basic culture medium of the upper tank for culture for 6-8 hours, when the rising amplitude of dissolved oxygen exceeds 20%, the feeding culture medium B of the tank is added to the basic culture medium of the upper tank, and the initial feeding flow rate is 16 mL/L.h;
wherein, adjust the carbon-nitrogen ratio in the feed medium (set total amount of carbon-nitrogen source to 480g/L, wherein molasses is carbon source, industrial yeast extract and soybean peptone are compound nitrogen source, in compound nitrogen source, the ratio of industrial yeast extract and soybean peptone is 3:1, adjust the carbon-nitrogen ratio in the medium, namely adjust the ratio of carbon source and compound nitrogen source) respectively to be 7:1, 5: 1. 3:1, 5:1, 1:1 and 1:2 (shown in Table 16) and controlling the dissolved oxygen content in the fermentation broth in the fermentation process to be 20-40% by coupling stirring rotation speed (200-800 rpm) and pure oxygen content in ventilation (0-30%); in addition, the pH value of the fermentation liquid in the fermentation process is regulated and controlled to be maintained at about 7.0 by dilute hydrochloric acid (7-8% and sterile water) so as to be beneficial to the stable expression of the raw starch alpha-amylase.
Table 16: different carbon-nitrogen ratio feed medium B component
As a result, it was found (FIG. 7) that the extracellular starch alpha-amylase activity was highest at a carbon-nitrogen ratio of 3:1 and 49082U/mL, which was 1.96 times the highest level reported in the prior literature (see He Li, et al enhanced extracellular raw starch-degradation alpha-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization. Biochemical Engineering Journal,2022,189), when the recombinant Bacillus subtilis was fermented for 68 hours. Recombinant bacterium B.subilis WB600/pBHVY-G3-amyZ1 different carbon to nitrogen ratio feed medium 3-L tank fermentation OD600 curves are shown in FIG. 8.
Comparative example 1:
the specific embodiment is the same as in example 3, except that the recombinant strain is modified to be Bacillus subtilis WB600/pBHSS 142 -C1-amyZ1 (BZYACO 6): plasmid was adjusted and the adapted signal peptide SP was selected ypuA And adopts optimized Shine-Dalgarno (SD) sequence, constructs double 5' untranslated region (double UTR), optimizes UTR spacer region, then uses recombinant expression vector pBHSS 142 -SP ypuA amyZ1 was transformed into bacillus subtilis WB600 competent by chemical transformation, constructed to give: bacillus subtilis WB600/pBHSS 142 C1-amyZ1 (BZYACO 6) (the construction method of BZYACO6 is described in HeLi, et al enhanced extracellular raw starch-degradation alpha-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization. Biochemical Engineering Journal,2022,189).
The bacillus subtilis recombinant strain BZYACO6 is cultivated for 12 hours at 37 ℃ and 200rpm to obtain a seed solution, and the seed solution is inoculated into a shake flask fermentation medium according to the inoculum size of 2% (v/v) and cultivated for 48 hours at 30 ℃ and 200rpm to obtain a fermentation broth. After shake flask fermentation optimization, the enzyme activity of the bacillus subtilis recombinant BZYACO6 extracellular starch alpha-amylase is 3915.2U/mL.
Recombinant bacterium B.subilis WB600/pBHVY-RBS in example 4 1 -SP nucB The activity of the amyZ1 shake flask fermentation extracellular starch alpha-amylase is up to 4824.2U/mL, which is improved by 1.23 times compared with the activity of the recombinant BZYACO6 extracellular starch alpha-amylase.
Thus, it can be seen that the finally obtained recombinant Bacillus subtilis WB/pBHVY-G3-amyZ 1 is indeed more suitable for recombinant expression of raw starch alpha-amylase than BZYACO6 by optimizing the promoter, signal peptide and RBS sequences of the plasmid pBHE-amyZ1 and optimizing the feed process for the 3-L tank fermentation of the recombinant.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended 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.
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