CN102492645A - Recombinant bacillus subtilis with high aminopeptidase yield, construction method thereof, and application thereof - Google Patents

Abstract

The invention discloses a method for constructing a recombinant bacillus subtilis secreting and expressing aminopeptidase and a method for producing aminopeptidase by using the recombinant bacterium, belonging to the technical field of genetic engineering. The method is efficient and safe, and the obtained aminopeptidase is an extracellular enzyme, which is beneficial to the extraction and purification of large-scale production in the later stage, and the product meets the food requirements.

Description

A recombinant Bacillus subtilis with high aminopeptidase production and its construction method and application

Technical field:

A recombinant Bacillus subtilis with high aminopeptidase production and its construction method and application, in the technical field of bioengineering.

Background technique:

Aminopeptidases are a class of exoproteolytic enzymes that can hydrolyze the N-terminal amino acids of proteins and polypeptides, and are widely found in animals, plants and microorganisms. Aminopeptidase can be divided into leucine aminopeptidase, lysine aminopeptidase, phenylalanine aminopeptidase, etc. according to the most reactive substrate. Aminopeptidase plays an important role in the debittering of protein hydrolyzate and the deep hydrolysis of protein. It is mainly used in the food industry, including fish and meat processing industry, plant processing industry and processing industry with microorganisms as raw materials. It can increase The amount of free amino acids can improve the flavor and nutritional value, and can also be used as an additive for seasonings such as soy sauce.

Invention content:

The first object of the present invention is to provide a recombinant Bacillus subtilis with high aminopeptidase production, which is a genetically engineered bacterium obtained by introducing a mature aminopeptidase gene from Bacillus subtilis Zj016 into Bacillus subtilis.

For the Bacillus subtilis Zj016, see Tian Yaping and Xu Yingmin for details. Purification and enzymatic properties of a Bacillus subtilis aminopeptidase[J]. Food and Fermentation Industry, 2006, 32(3): 7-10.

The nucleotide sequence of the gene encoding mature aminopeptidase is shown in SEQ ID NO.1.

The Bacillus subtilis is Bacillus subtilis WB600, see xuchu Wu, wilson Lee, louise Tran, and suilam Wong for details. Engineering a Bacillus subtilis expression-secretion system with a strain deficient insix extracellular proteases[J].JOURNAL 91 LOF 491 BACTER -4958.

The large intestine-subtilis shuttle carrier is PMA5, see EVA ZYPRIAN and HANS MATZURA.Characterization of signal promoting gene expression on the Staphylococcus aureus plasmamid pUB110 and development of a Gram-positive expression system[J].DNA,1986,5:219-2

The second object of the present invention is to provide a method for constructing recombinant Bacillus subtilis that secretes and expresses aminopeptidase.

The construction method of the recombinant Bacillus subtilis is as follows:

1) Design primers according to the gene of Bacillus subtilis aminopeptidase, and use the genome of the original bacteria as a template to clone the target gene containing the coding signal peptide sequence.

2) Connect the cloned target gene SEQ ID NO.1 to the vector PUC19, transform E.coli JM109, coat a resistant LB plate, verify positive transformants by PCR and double enzyme digestion, and obtain the recombinant plasmid PUC19-SAP , the identified recombinant plasmids were sequenced and analyzed.

3) The recombinant plasmid PUC19-SAP and the large intestine-subtilis shuttle vector PMA5 were subjected to double enzyme digestion, the gel was cut to recover the target fragment, ligated and transformed into E.coli JM109, coated with a resistant LB plate, and the transformant was picked for PCR , double enzyme digestion verification, to obtain the recombinant plasmid PMA5-SAP.

4) Transform the recombinant plasmid PMA5-SAP into Bacillus subtilis WB600 to obtain the recombinant bacteria, which is to be verified for expression.

The third object of the present invention is to provide a method for producing aminopeptidase using the recombinant Bacillus subtilis, which is to inoculate the recombinant Bacillus subtilis in a fermentation medium after seed cultivation.

The specific method of using recombinant Bacillus subtilis to produce aminopeptidase is as follows:

1) Pick a single colony of recombinant bacteria from the preservation plate and inoculate it in 5mL LB medium (Kana 50μg/mL) for overnight culture at 200rpm at 37°C, then transfer to 50mL TB medium (Kana 50μg/mL) according to the inoculation amount of 1%. mL) at 37°C 200rpm for 36h.

2) Centrifuge the obtained fermentation broth at 8000 rpm for 5 minutes to remove bacteria, and the obtained fermentation supernatant is the crude enzyme liquid.

The invention provides a recombinant Bacillus subtilis with high aminopeptidase production and its construction method and application. The method is efficient and safe, and the enzyme activity reaches 32U/mL, which is 15 times that of the original strain. The obtained aminopeptidase It is an extracellular enzyme, which is beneficial to the extraction and purification of large-scale production in the later stage, and the product meets the food requirements.

Description of drawings:

Figure 1: PCR results of aminopeptidase target gene (1: target gene)

Figure 2: Results of double digestion of recombinant plasmid PUC19-SAP (1: PUC19; 2: target gene)

Figure 3: Schematic diagram of the recombinant plasmid PMA5-SAP

Figure 4: SDS-PAGE results of recombinant Bacillus subtilis expression (M: marker; 1: expression results of recombinant bacteria containing PMA5 empty plasmid; 2: expression results of recombinant bacteria containing PMA5-SAP plasmid; 3: aminopeptidase)

Detailed ways:

Materials and Testing Methods

LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0.

TB medium: 1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17Mm KH ₂ PO ₄ , 72mM K ₂ HPO ₄ .

Aminopeptidase activity assay method: use L-leucine-p-nitroaniline as substrate, add enzyme solution and substrate (1mM) diluted by a certain number of times in 50mM Tris-HCl buffer solution with pH 8.5, and put in water bath at 50℃ After reacting for 10 min, the absorbance was measured at 405 nm. Definition of enzyme activity unit: at 50°C, the amount of enzyme needed to decompose L-leucine-p-nitroaniline in 1 minute to produce 1 μM p-nitroaniline is one enzyme activity unit (1U).

Example 1

In order to realize the secretory expression of aminopeptidase in Bacillus subtilis, a pair of primers are designed from the signal peptide, i.e. upstream primers (comprising Nde I restriction site):

5′-GGAATTCCATATGAAAAAGCTTTTGACTGT-3′

Downstream primers (including BamH I restriction site):

5′-CGCGGATCCTTATTTGATATCTTCAAAAA-3′

Using the original bacterial genome as a template, clone the target gene containing the coding signal peptide sequence. The PCR system is: 1 μL each of 10 μM primers P1 and P2, 5 μL of 2 mM dNTPS, 5 μL of 10×KOD-Plus-NeoBuffer, 1 U/μL of KOD-Plus- NeoDNA polymerase 1 μL, template 0.5 μL, add double distilled water to make up 50 μL. PCR conditions: pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 1min and 30s, 30 cycles. The PCR product and carrier PUC19 were subjected to double enzyme digestion and then gel-cut to recover, ligated overnight at 16°C, transformed into E.coli JM109, coated with LB plates containing ampicillin (100 μg/mL) resistance, and incubated at 37°C for 10-12h, Pick 3 transformants, extract recombinant plasmids and perform PCR and double enzyme digestion verification, and then determine the DNA sequence of the verified recombinant plasmids, and the positive clones are PUC-SAP. The recombinant plasmid PUC19-SAP and the large intestine-subtilis shuttle vector PMA5 were subjected to double enzyme digestion and then gel-cut to recover, ligated at 16°C overnight, transformed into E.coli JM109, and coated with LB plates containing ampicillin (100 μg/mL) resistance, Cultivate at 37°C for 10-12 hours, pick 3 transformants, extract recombinant plasmids and verify by PCR and double enzyme digestion, the positive clone is PMA-SAP.

Transform the obtained recombinant plasmid PMA-SAP into Bacillus subtilis WB600. The transformation method is as follows: Pick a single colony of Bacillus subtilis WB600 from the preservation plate and put it into 2 mL of SPI medium (both use 50 mL centrifuge tubes), and cultivate it overnight at 37°C and 200 rpm. Transfer to 5mL SP I medium according to 10% inoculation amount, cultivate at 37°C 200rpm to the logarithmic growth stage (about 4-5h), then transfer to 2mL SPII medium according to 10% inoculum amount, After culturing at 37°C at 200rpm for 90min, add 20uL of 10mM EGTA, and then incubate at 37°C at 200rpm for 10min. Dispense into 0.5mL tubes, add plasmid DNA, incubate at 37°C 100rpm for 90min, take the bacterial solution and spread it on a screening plate containing kana (50μg/mL), incubate at 37°C for 10h-12h, pick 3 transformants for preservation , pending expression verification.

SP salt: 0.2% (NH ₄ ) ₂ SO ₄ , 1.4% K ₂ HPO ₄ , 0.6% KH ₂ PO ₄ , 0.02% MgSO ₄ ·7H ₂ O, 0.1% sodium citrate;

SP I medium: add 1% volume concentration of SP salt solution to 50% glucose solution, 1% volume 100×CAYE solution;

SP II medium: SP I medium was added with 1% volume of 50mM CaCl ₂ solution and 1% volume of 250mM MgCl ₂ solution.

Example 2

Pick a single colony of recombinant bacteria from the preserved plate and inoculate it in 5mL LB medium (Kana 50μg/mL) for overnight culture at 37°C 200rpm, then transfer to 50mL TB medium (Kana 50μg/mL) according to the inoculation amount of 1%. Incubate at 37°C and 200rpm for 36h. The obtained fermentation liquid was centrifuged at 8000 rpm for 5 minutes to remove bacteria, and the obtained fermentation supernatant was the crude enzyme liquid. The activity of the crude enzyme solution was determined to reach 32U/mL.

Claims (6)

1. producing recombined bacillus subtilis for one kind, is that the gene that derives from coding aminopeptidase among the subtilis Zj016 is imported the genetic engineering bacterium that subtilis obtains.

2. genetic engineering bacterium according to claim 1 is characterized in that said encoding mature aminopeptidase gene nucleotide series is shown in SEQ ID NO.1.

3. genetic engineering bacterium according to claim 1 is characterized in that said subtilis is Bacillus subtilis WB600.

4. the construction process of the said recombined bacillus subtilis of claim 1 is characterized in that, step is following:

1) the goal gene SEQ ID NO.1 that clones is connected on the carrier PUC19; Transformed E .coli JM109, coating has the LB flat board of resistance, PCR, double digestion checking positive transformant; Obtain recombinant plasmid PUC19-SAP, the recombinant plasmid after identifying is carried out sequencing analysis.

2) recombinant plasmid PUC19-SAP and large intestine-withered grass shuttle vectors PMA5 are carried out double digestion respectively; Cut glue and reclaim the purpose fragment, connect and Transformed E .coli JM109, coating has the LB flat board of resistance; The picking transformant carries out PCR, double digestion checking, obtains recombinant plasmid PMA5-SAP.

5. the method that application rights requires 1 described recombined bacillus subtilis to produce aminopeptidase is that recombined bacillus subtilis is cultivated through being inoculated in fermention medium after the seed culture, from fermented liquid, extracts aminopeptidase.

6. like the said method of claim 5, it is characterized in that concrete steps are following:

1) contains 37 ℃ of 200rpm incubated overnight the kantlex 50 μ g/mL LB substratum from the single colony inoculation of the dull and stereotyped picking reorganization of preservation bacterium in 5mL, be forwarded to 50mL by 1% inoculum size again and contain that 37 ℃ of 200rpm cultivate 36h in the kantlex 50 μ g/mL TB substratum;

2) the centrifugal 5min of fermented liquid 8000rpm that obtains is removed thalline, the fermented supernatant fluid of acquisition is crude enzyme liquid.

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