CN102703368A - Genetically engineered strain capable of producing keratinase and application thereof - Google Patents

Abstract

The invention discloses a genetically engineered strain capable of producing keratinase and application thereof and belongs to the technical field of genetic engineering. Gene of keratinase from Bacillus licheniformis BBE11-1 is cloned and connected to a bacillus subtilis expression vector pMA 0911 by DNA recombination technology and is converted into bacillus subtilis WB600. The recombinant bacillus subtilis strain WB600-pMA 0911-ker capable of producing a high amount of keratinase is obtained by screening and identifying. The recombinant bacillus subtilis strain WB600-pMA 0911-ker is preserved in China center for type culture collection under the number of CCTCC NO.M 2012066 on March 8 2012. Activity of the keratinase expressed by the genetically engineered strain is 522U/mL about two times of total enzyme activity of wild mushrooms. Therefore, solid foundation for large-scale production of the keratinase is laid.

Description

A kind of genetic engineering bacterium and application thereof of producing M-Zyme

Technical field

The present invention relates to a kind of genetic engineering bacterium and application thereof of producing M-Zyme, belong to gene engineering technology field.

Background technology

M-Zyme (Keratinase) is a kind of keratic enzyme of can specificity degrading, by multiple microorganisms such as bacterium, actinomycetes and fungies.Feather is as Poultry farming and to butcher the by product annual output of industry huge, and protein and aminoacids content enrich, and is potential fine protein resource.The reasonable utilization of feather can reduce the pollution of waste to environment on the one hand, can be used as a kind of raising that section's protein is applied to livestock and poultry of raising simultaneously.Utilize the main acid and alkali hydrolysis that adopts in the feather process in tradition.Methods such as thermal destruction, there is environmental pollution problem in the former, and the latter is bigger to energy consumption, and can destroy partial amino-acid, has reduced the nutritive value of product.Other common Keratin sulfate are the hair of animal, send out etc. like ox hair, wool and people, and outside this keratin like protein matter part was processed as commercial materials, all the other were many as waste treatment, have also caused the pollution of environment and the waste of protein resource.It is polypeptide and amino acid that M-Zyme can make keratin degrading, both can be used for the production of agricultural fertilizer, can be used as the livestock and poultry feed protein source again; M-Zyme is the important invasion and attack factor of dermatophytes, and its research aspect skin diseases treatment awaits further to excavate.In addition, M-Zyme can " digest " toxalbumin that causes mad cow disease and human Keyashi's syndrome, thereby can be applicable to the purification of medical treatment and laboratory apparatus; It also can be used for leather depilation tanning, cosmetics such as preparation skin cream, bath soap, shampoo and depilatory cream.

Research about M-Zyme at present mainly concentrates on the screening and the relevant enzyme Study on Properties of producing bacterial strain.Yet, produce the bacterial strain that has only in the bacterium seldom at these M-Zymes and have business development value.Expression also exists many problems to need to solve in heterologous host, occurs with the inclusion body form like expressing protein; The albumen of this heterogenous expression limits the growth of host bacterium sometimes to host bacterium toxigenicity, and in the process of expression amount accumulation, this Sumizyme MP also can cause the degraded of self; The recombinant plasmid that makes up is very unstable sometimes, in the expression process, loses easily, so just causes the reduction or the disappearance of expressing quantity.Making up suitable carriers is the key that this proteinoid is expressed with selecting correct host.

Summary of the invention

The invention provides a kind of genetic engineering bacterium that produces M-Zyme; Be that the M-Zyme gene is imported the genetic engineering bacterium that subtilis obtains; Be preserved in Chinese typical culture collection center on March 8th, 2012; The preservation address is Chinese Wuhan, Wuhan University, and deposit number is CCTCC NO:M 2012066, taxonomy called after subtilis (Bacillus subtilis) WB600-pMA 0911-ker.

The present invention also provides a kind of construction process that produces salt hydrolysis enzyme genetic engineering bacterium, comprises the steps:

1) adopt PCR method or chemical total synthesis method to obtain the ker gene;

2) the ker gene is connected to coli expression carrier pMA 0911, obtains recombinant vectors pMA 0911-ker;

3) recombinant vectors transforms Bacillus subtilis WB600.

The PCR method primer sequence is following:

ker1-F：5’-GGAATTC CATATGATGAGGAAAAAGAGTTTTTGG-3’

ker1-R：5’CGC GGATCCTTATTGAGCGGCAGCTTCG-3’

PCR reaction system: be sequentially added into following reagent in the 0.2mL PCR pipe: 5 * prime STAR PCR buffer II (Mg ²⁺Plus) 5 μ l; DNTP Mixture 4 μ l; Template DNA 1 μ l; Each 1 μ l of upstream and downstream primer; Taq enzyme 0.5 μ l; Adding distilled water to final volume is 50 μ l.

Pcr amplification condition: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 61 ℃ of annealing 15s, 72 ℃ are extended 1min 20s (30 circulations); 72 ℃ are extended 10min.

Glue reclaimed be connected to subtilis expression vector pMA0911 after PCR product behind the purifying is cut with Nde I and BamHI enzyme; Obtain recombinant vectors pMA 0911-ker; Transform Bacillussubtilis WB600, obtain reorganization bacterium Bacillus subtilis WB600-pMA 0911-ker through Screening and Identification.

M-Zyme enzyme activity determination method: with the centrifugal 10min (10000 * g of fermented liquid; 4 ℃) get fermented supernatant fluid; Draw the suitably enzyme liquid of dilution of 200uL; The substrate (Keratin sulfate) that adds 300uL 0.05mol/L gly-NaOH damping fluid (pH9.0) dissolving 1% is cultivated 20min for 50 ℃, adds 500uL 4M TCA solution with termination reaction.Centrifugal 10min draws the 200uL supernatant and is moved in the new test tube, after add 1mL forint phenol reagent and 200uL 0.5M Na according to this ₂CO ₃Back 50 degree colour developing 15min.Blank is when adding enzyme liquid, adds 500uL TCA solution, crosses cleaner liquid as blank through what identical process reacted.Detect light absorption value at the 660nm place, define according to the tyrosine typical curve and discharge 1ug tyrosine in every 20min and be defined as the enzyme unit that lives.

The present invention has made up a kind of engineering bacteria of stably express at gene; With this engineering bacteria serves as to produce bacterial strain to carry out fermentative prodn; The M-Zyme enzyme that this bacterial strain is expressed after fermentation ends is lived and is 522U/mL; Total enzyme work than wild bacterium has improved nearly 2 times, for the scale operation of M-Zyme is had laid a good foundation.

The biological material specimens preservation

One plant height produces the subtilis of M-Zyme, and called after Bacillus subtilis WB600-pMA 0911-ker has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M 2012066, and preservation date is on March 8th, 2011.

Description of drawings

Fig. 1: (a) amplification of goal gene.M:DL 2,000DNAMarker; The pcr amplification band of swimming lane 1 and swimming lane 2:ker gene; (b) bacterium colony PCR selects positive recombinant.M:DL 2,000DNAMarker; Swimming lane 1 and swimming lane 2: positive recombinant;

Fig. 2: (a) extraction of recombinant plasmid.M:DL 10,000DNA Marker; Swimming lane 1 and swimming lane 2: recombinant plasmid pMA0911-ker; (b) double digestion checking.M:DL 10,000DNAMarker; Swimming lane 1: recombinant plasmid pMA 0911-ker;

Swimming lane 2: the band that recombinant plasmid obtains after with BamH I and Nde I double digestion.

Fig. 3: protein electrophoresis (SDS-PAGE) experimental result.M: protein molecular weight standard (Protein Molecular Weight Marker); 1: contain plasmid pMA 0911-ker reorganization bacterium; 2: contain plasmid pMA 0911 reorganization bacterium.

Embodiment

Structure and the evaluation of embodiment 1 reorganization bacterium

1) said primer is according to NCBI website gene order design (GenBank:S78160.1), and primer sequence is following:

ker1-F：5’-GGAATTC CATATGATGAGGAAAAAGAGTTTTTGG-3’

ker1-R：5’CGC GGATCCTTATTGAGCGGCAGCTTCG-3’

Bacillus licheniformis Bacillus licheniformis BBE1 has been preserved in Chinese typical culture collection center, and deposit number is CCTCC M 2011319.

PCR reaction system: be sequentially added into following reagent in the 0.2mL PCR pipe: 5 * prime STAR PCR buffer II (Mg ²⁺Plus) 5 μ l; DNTP Mixture 4 μ l; Template DNA 1 μ l; Each 1 μ l of upstream and downstream primer; Taq enzyme 0.5 μ l; Adding distilled water to final volume is 50 μ l.Pcr amplification condition: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 61 ℃ of annealing 15s, 72 ℃ are extended 1min 20s (30 circulations); 72 ℃ are extended 10min.

2) reclaim pcr amplification product with 1% agarose gel electrophoresis checking and with 0.8% agarose gel electrophoresis, result's amplification obtains the ker gene fragment (Fig. 1 a-swimming lane 1, swimming lane 2) that conforms to the bibliographical information size.

3) with ker gene and carrier pMA 0911 behind restriction enzyme Nde I and the BamH I double digestion digestion purifying; With the T4DNA ligase enzyme in 16 ℃ of connections of spending the night; Connect product and transform host bacterium JM109 with chemical transformation; Transformed bacteria liquid is coated on the LB flat board that contains penbritin (100mg/mL), and 37 ℃ of incubated overnight are method evaluation positive colony (Fig. 1 b-swimming lane 1, swimming lane 2) that primer carries out bacterium colony PCR with ker1-F and ker1-R; The final recombinant expression plasmid pMA0911-ker (Fig. 2 a-swimming lane 1, swimming lane 2) that obtains to contain the ker gene verifies (Fig. 2 b-swimming lane 2) with double digestion.

The intestinal bacteria chemical conversion is: gets and connects product 5 μ l and join and contain in the 100 μ l JM109 competent cells, and fully behind the mixing, ice bath 30min; With the Eppendorf pipe that mixture is housed, 42 ℃ of water-bath heat-shocked 90s transfer to cooled on ice 2min with the Eppendorf pipe then immediately; Xiang Guanzhong adds 600 μ l LB liquid nutrient mediums, places on 37 ℃ of 200rpm constant temperature shaking tables and cultivates 1h, coats then on the flat board that contains kantlex, upwards places 1h for 37 ℃, and inversion is observed bacterium colony after cultivating 12-16h.

4) recombinant plasmid pMA 0911-ker is transformed Bacillus subtilis WB600 competent cell; Obtain can be on the LB flat board that contains kantlex (30mg/mL) genetic engineering bacterium of normal growth, and through identifying called after Bacillus subtilisWB600-pMA 0911-ker.

Enzyme activity determination and the protein electrophoresis of embodiment 2 reorganization bacterium

Substratum: seed and slant medium are LB substratum (1L): Tryptones 10g, yeast extract 5g, NaCl10g; Slant medium adds agar 15g; Basic fermention medium is for adding the LB substratum (1L) of glucose: Tryptones 10g, yeast extract 5g, NaCl 10g, glucose 10g;

Cultural method: change the seed of overnight cultures under 37 ℃, 200rpm over to basic fermention medium with 3% inoculum size, under 37 ℃, 200rpm condition, cultivate;

As contrast, obtain the protein band (see figure 3) that a molecular weight size is about 40kDa with empty carrier, measure the enzyme of this reorganization bacterium simultaneously and live through protein electrophoresis (SDS-PAGE), enzyme live for 522U/mL than total enzyme of wild bacterium live (320U/mL) improved nearly 2 times.

It is understandable that, concerning those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.

Claims (3)

1. a genetic engineering bacterium that produces M-Zyme is preserved in Chinese typical culture collection center on March 8th, 2012, and deposit number is CCTCC NO:M 2012066.

2. application rights requires the method for 1 said genetic engineering bacterium fermentative prodn M-Zyme; It is characterized in that to produce the M-Zyme genetic engineering bacterium serve as to produce bacterial strain; After slant activation prepared seed, the inoculum size with 3% changed basic fermention medium over to, under 37 ℃, 200rpm condition, cultivated 24 hours; Seed and slant medium are the LB substratum: Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L; Slant medium adds agar 15g; Basic fermention medium is for adding the LB substratum of glucose: Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L, glucose 10g/L; The construction process of said genetic engineering bacterium is: 1) adopt the complete synthesis or PCR method clone ker gene of chemistry;

2) the ker gene is connected to coli expression carrier pMA 0911, obtains recombinant vectors pMA 0911-ker;

3) recombinant vectors transforms Bacillus subtilis WB600 and obtains genetic engineering bacterium CCTCC NO:M 2012066.

3. the said genetic engineering bacterium of claim 1 is applied to fodder additives.

Images