CN108085327A - A kind of alkali protease heterogenous expression engineered strain in extreme environment source and its application - Google Patents
A kind of alkali protease heterogenous expression engineered strain in extreme environment source and its application Download PDFInfo
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- CN108085327A CN108085327A CN201711459372.0A CN201711459372A CN108085327A CN 108085327 A CN108085327 A CN 108085327A CN 201711459372 A CN201711459372 A CN 201711459372A CN 108085327 A CN108085327 A CN 108085327A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
- C12N9/6408—Serine endopeptidases (3.4.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21014—Microbial serine proteases (3.4.21.14)
Abstract
The invention discloses a kind of engineering bacterias of the alkali protease AP1 in heterogenous expression extreme environment source, strain was named E.coli BL21 (DE3)/pHSL AP1, it is to utilize E.coli BL21 (DE3) as starting strain, is obtained by electricity conversion in its intracellular importing by ap1 HSL10 genes of pET28a (+) plasmid carrying.The invention also discloses application of the engineering bacteria in alkali protease AP1 is expressed.Experiment confirms：Engineered strain provided by the invention is in expressing protein enzyme AP1; the glucose of addition 1% is particularly important for high efficient expression protease gene in SYN culture mediums; its application is expected to provide experiment and theoretical foundation for large-scale production alkali protease, has important value in commercial Application exploitation.
Description
Technical field
The present invention relates to the clone of alkali protease, engineered strain and its structure and applications, and in particular to a kind of extreme ring
The basic protein enzyme coding gene in border source simultaneously utilizes a kind of heterologous table of alkali protease in gene constructed extreme environment source
Expression engineered bacteria strain and its application, belong to biological technical field.
Background technology
In order to adapt to harsh extreme living environment, metabolic type, physiological structure can occur very Situation of Microorganism Under Extremity Environment
Big variation is evolved the result is that special metabolic pathway and metabolite can be generated, the enzyme including some specific functions, new
Biomaterial etc.【1-2】.Applicant was once separated to one plant of extreme microorganism in the salt alkali lake of Xinjiang, was accredited as oxidation microbacterium
Microbacterium oxydans HSL10【3】。
Alkali protease belongs to the serine protein hydrolase class in endopeptidase, and (pH9-10) being capable of water in alkaline conditions
Protein peptide bond is solved, generates polypeptide or amino acid.Alkali protease is initially found in pig pancreas, after in bacillus licheniformis
It is separated to [1].Studies on Microbial Alkaline Protease has obtained quick development since then.Alkali protease is mainly derived from bacterium, unwrapping wire
Bacterium, fungi.Alkali protease currently used for commercial use is mostly obtained from bacillus.In addition to alkaline environment, at some its
Alkali protease producing strains can also be screened in its extreme environment, such as polar region psychro-environment, ocean hypersaline environment, more even
There are the different separated reports of alkali protease producing strains in metallic pollution region.This seminar is micro- from the separated oxidation in salt alkali lake area
Bacillus Microbacterium oxydans HSL10 have the ability of production alkali protease【3】。
As important industrial enzymes, alkali protease is the class of enzymes being most widely used at present, enzyme is washed without phosphorus
Multiple industries such as the preparation of clothing powder, the processing of food/medicine/feed/leather, silver recovery, chemical industry and debirs processing
It is widely used.Wherein, serine protease (Serine protease) due to there is higher activity in alkaline conditions
And stability, obtain more extensive utilization in industrial circle.The active region of this fermentoid contains the serine residues there are one nucleophilic,
Catalytic triads are formed with aspartate and the required residues of histidine two.The optimal pH of this kind of protease is 7-11.Together
When, research shows most of with Substratspezifitaet in them【1-2】。
However, the alkali protease production bacterial strain with industrial development value is relatively fewer.At present, industry has been applied to
The producing strains of production or studied more alkali protease are mainly limited to two categories：Bacillus (such as Bacillus
alcalophilus ATCC 21522、B.alcalophilus、B.alcalophilus subsp、Halodurans
KP1239, B.amyloliquefaciens, B.circulans, B.coagulans etc.) and fungi (such as Aspergillus
Candidus, A.flavus, A.fumigatus, A.melleus, A.niger, A.oryzae etc.).Finding has basic protein
The novel strain of enzymatic activity is still a current heat subject.Retrieval at present finds to separate basic protein from extreme microorganism
Enzyme is rarely reported.Whether the alkali protease in extreme environment source has some sp acts, how to carry out high efficient expression, this is right
It is of great significance in the research of current alkali protease.
Bibliography：
[1]Gupta R,Beg QK,Lorenz P(2002)Bacterial alkaline proteases:
molecular approaches and industrial applications.Appl Microbiol Biotechnol
59:15–32
[2]Gupta R,Beg QK,KhanS andChauhanB(2002)An overview on fermentation,
downstream processing and properties of microbial alkaline proteases.Appl
Microbiol Biotechnol 60:381–395
[3]Jin Lü,Xiaodan Wu,Yali Jiang,Xiaofeng Cai,Luyao Huang,Yongbo Yang,
Huili Wang,Aibing Zeng and Aiying Li,An extremophile Microbacterium strain
and its protease production under alkaline conditions.J.Basic Microbiol.2014,
54,378–385
The content of the invention
In view of the deficiencies of the prior art, the problem to be solved in the present invention is that alkali protease production is screened from special habitats
Bacterial strain therefrom clones basic protein enzyme coding gene, builds a kind of alkali protease heterogenous expression engineering in extreme environment source
Bacterial strain and its application in alkali protease AP1 is expressed.
The basic protein enzyme coding gene in extreme environment source of the present invention, it is characterised in that：The basic protein
Enzyme gene is named as ap1-HSL10, and nucleotide sequence is as shown in table SEQ ID No.1；The gene order is micro- from aoxidizing
Bacillus (Microbacterium oxydans) HSL10, nucleotide sequence encode basic protein by 3210bp base compositions
Enzyme.
The alkali protease in extreme environment source of the present invention, it is characterised in that：The alkali protease is named as
AP1, amino acid sequence is as shown in SEQ ID No.2；The sequence is from oxidation microbacterium (Microbacterium
Oxydans) HSL10, amino acid sequence are made of 1070 amino acid, and the albumen of coding contains serine protease catalytic work
Property structural domain and catalytic activity triad residues.
Applicant was previously cloned into from basophilic oxidation microbacterium Microbacterium oxydans HSL10 genomes
One new alkaline protease gene is serine protease from zymologic property research deduction.It is produced for current alkali protease
The problem of life bacteria strain resource is relatively single, the wild strain of basic protein enzyme coding gene provided by the invention --- oxidation is micro-
Bacillus (Microbacterium oxydans) HSL10- is the actinomyces for the basophilic for being isolated from Xinjiang salt alkali lake, and the bacterial strain is not only
Activity with alkali protease, special saline-alkali environment cause such bacterial strain to have basophilic, salt tolerant, low temperature resistant isoreactivity.
The engineering bacteria of the alkali protease AP1 in heterogenous expression extreme environment source of the present invention, it is characterised in that：Institute
It states engineering bacteria and is named as E.coli BL21 (DE3)/pHSL-AP1, genotype is E.coli BL21 (DE3), kanamycin
resistance、ap1-HSL10、IPTG inducible PT7Promoter, it using E.coli BL21 (DE3) is bacterium germination to be
Strain is obtained by electricity conversion in its intracellular ap1-HSL10 gene carried by pET28a (+) plasmid that import.Wherein E.coli
BL21 (DE3) is common bacterium heterogenous expression host, and pHSL-AP1 is for heterogenous expression oxidation microbacterium HSL10 sources
The expression plasmid of serine protease AP1：3210bp protease genes ap1-HSL10 is inserted by digestion connection mode
On pET28a (+) expression vector, carry card and receive mycin resistant gene, size 8.58kb.
Specifically, the structure side of the engineering bacteria of the alkali protease AP1 in heterogenous expression extreme environment source of the present invention
Method, step are：
(1) aoxidize microbacterium HSL10 wild-type strains protease and carry out activity analysis.
(2) according to oxidation microbacterium HSL10 genome sequencings result to wherein alkaline protease gene ap1-HSL10's
Homology analysis.
(3) Yin WuF ＆R-AP1 of the design with restriction enzyme site (NdeI and HindIII), obtains aoxidizing micro- by PCR amplification
Bacillus HSL10 protease gene segments ap1-HSL10.
(4) NdeI and HindIII double digestions, the Insert Fragment obtained with step (3) are carried out to expression vector pET28a (+)
It is attached with T4 ligases, obtains expression plasmid pHSL-AP1.
(5) the expression plasmid pHSL-AP1 for obtaining step (4) directly electricity conversion E. coli BL21 (DE3),
Screening positive clone, and extract plasmid and carry out digestion verification, obtain the alkali protease AP1 in energy heterogenous expression extreme environment source
Engineered strain, be named as engineered strain E.coli BL21 (DE3)/pHSL-AP1.
The engineering bacteria of the alkali protease AP1 in heterogenous expression extreme environment source of the present invention is in expression alkali protease
Application in AP1.
Wherein：The condition of culture of the engineering bacteria of the alkali protease AP1 in the heterogenous expression extreme environment source is preferably：
1% glucose is added on the basis of SYN culture mediums and carries out induced expression, it is 0.4mmol/L to induce the IPTG concentration used.
The engineered strain E.coli BL21 of the alkali protease AP1 in heterogenous expression extreme environment source disclosed by the invention
(DE3)/pHSL-AP1 is the heterogenous expression realized for the first time to the alkali protease AP1 in extreme environment source.Experiment confirms：This hair
For the engineered strain of bright offer in expressing protein enzyme AP1, the glucose of addition 1% is for high efficient expression albumen in SYN culture mediums
Enzyme gene is particularly important, and application is expected to provide experiment and theoretical foundation for large-scale production alkali protease, in commercial Application
There is important value in exploitation.
Description of the drawings
Fig. 1：Influence of the metal ion to the enzyme activity of HSL10 protease As P1 is added in HSL10 cultural characters and culture medium
Wherein：(A) HSL10 can be grown at low temperature；(B) can be generated in using Casein as unique C sources culture medium
Protease hydrolytic circle；(C) the enzyme activity highest under SYN (for addition skimmed milk power and the culture medium of NaCl) condition of culture；(D)：Training
Metal ion adds the influence to enzyme activity during supporting：SYN+Ca2+：To add 10mmol/L Ca on the basis of SYN2+；SYN+
Mg2+：To add 10mmol/L Mg on the basis of SYN2+；SYN+K+：To add 10mmol/L K on the basis of SYN+。
Fig. 2：The influence of temperature and pH to the enzyme activity of HSL10 protease As P1
Wherein：(A) influence of the temperature to enzyme activity；(B) influences of the pH to enzyme activity.
Fig. 3：The Analysis of Heat Tolerance of HSL10 protease As P1.
Fig. 4：HSL10 protease A P1 structure domain analysis
Fig. 5：Aoxidize the gene magnification of microbacterium HSL10 protease As P1 and expression plasmid structure
Wherein：(A) band 1 is Dongsheng 1kb Marker；Band 2-3 carries out PCR amplification glue by primer of F-AP1＆R-AP1
Recycle band；B：Band 1 is Dongsheng 1kb Marker；NdeI, HindIII double digestion verification of band 2-3 positive colony plasmids.
Fig. 6：Glucose promotes the expression of protease A P1
Wherein：Scheme A：Containing kanamycins, the SYN culture mediums of 0.4mmol/L IPTG；Scheme B：Containing kanamycins, 0.4mmol/
L IPTG, the SYN culture mediums of 1% glucose.AP1-1 and two parallel bacterium colonies that AP1-2 is cell BL21/pHSL-AP1.
Specific embodiment
The present invention is described in detail below in conjunction with attached drawing and specific example, to more fully understand the present invention, but the content
It is not limited to the protection content of the present invention.
General explanation：Wild strain oxidation microbacterium (Microbacterium involved by following examples
Oxydans) HSL10 is isolated from the saline and alkaline lake region in Xinjiang【3】, genom sequence has been sequenced；The coding base of alkali protease AP1
Because being named as ap1-HSL10, oxidation microbacterium (Microbacterium oxydans) is obtained by Direct Cloning on HSL10 genomes
It arrives；E.coli BL21 (DE3) are common bacterium heterogenous expression hosts, carry out the plasmid vector that alkali protease AP1 expression uses
PET28a (+) is purchased from Novagen companies of the U.S..Gene sequencing in plasmid construction is completed by Huada gene company.It is other not carry
And plasmid is commercially available conventional plasmid.The reagent and consumptive material being related to are domestic.Experimental method and reagent in embodiment for example without
Specified otherwise is this field conventional method and commercial reagent.
Embodiment 1：Aoxidize microbacterium (Microbacterium oxydans) HSL10 wild-type strain basic protein enzyme activity
Property analysis.
(1) metal ion adds influence (Fig. 1) to HSL10 proteinase activities in HSL10 cultural characters and culture medium.
It concretely comprises the following steps：Culture oxidation microbacterium HSL10 under low temperature in the medium adds three kinds often with different culture media
See metallic ions Ca2+、Mg2+And K+, enzyme solution is obtained with dialysis, ammonium sulfate precipitation and over-molecular sieve protein isolate enzyme, and is detected
Activity.
It turns out that HSL10 have cold-resistant characteristic (Figure 1A), utilize using Casein as unique C sources culture medium in can generate
Protease hydrolytic circle (Figure 1B)【3】；It was found that (skimmed milk power 1.0g, 0.5% yeast extract and 1% in SYN fluid nutrient mediums
Sodium chloride.Adding deionized water, adjusting pH to HSL10, the protease of acquisition 7.0) is cultivated has high enzyme work (figure to 100ml
1C).Select three kinds of common metal ion Ca2+、Mg2+And K+It is added in the SYN culture mediums of optimization (Fig. 1 D), then compares enzyme
The activity of liquid；It was found that the addition of these three ions helps to improve enzyme activity.
Protease activity determination method：According to national relevant speciality standard (SB/T 10317-1999) it is suitably modified after
It is measured：For different pH requirements, can be realized by the way that Casein buffer pH can be adjusted.Do three groups of parallel examinations every time
It tests.The 1ml enzyme solutions of preheating and 1ml caseins are blended under the conditions of 40 DEG C and react 20min；Then 0.4M TCA (trichlorines are added in
Acetic acid) 2ml inactivates enzyme, continue heat preservation 20 minutes；The protein flocculent deposit after TCA is denatured is filtered out with filter paper；Take filter
Liquid 1ml adds in 5ml 0.4M Na in another test tube2CO3And 1mL forint phenol reagents, it shakes up, 40 DEG C of heat preservation color developments
20min.Then OD is carried out with ultra-violet and visible spectrophotometer660It measures.It is first to add TCA inactivations in blank control, then adds again
Add casein solution.
(2) influence of temperature and pH to HSL10 proteinase activities.
It concretely comprises the following steps：According to the growth characteristics of Microbacterium oxydans HSL10 wild-type strains, alkali is set
Property mmp reaction temperature gradient be 20 DEG C -80 DEG C.Protease activity determination method is the same as (1).
The optimal reactive temperature that experiment shows to aoxidize microbacterium HSL10 production alkali proteases is 55 DEG C；40 DEG C -65 DEG C compared with
Wide scope inner enzyme vigor all maintains higher level, and the result is shown in Fig. 2-A；The enzyme solution of acquisition is saturating in different pH gradient buffer solutions
Analysis, obtains condition of different pH enzyme solution.And by the use of the casein of corresponding different pH gradients as substrate, judge so as to more accurate
HSL10 generates the most suitable pH of protease.Protease produced by oxidation microbacterium HSL10 shows in the range of pH9.0-pH12.0
Most go out greater activity, wherein 9.0 enzymatic activity highests of pH (see Fig. 2-B).This, which also demonstrates again oxidation microbacterium, can generate alkali
Property protease.
(3) HSL10 protease Analysis of Heat Tolerance.
It concretely comprises the following steps：30min-180min is handled at 35 DEG C, 45 DEG C, 55 DEG C respectively by the enzyme solution to acquisition, is surveyed
Its fixed remaining enzyme activity.The result shows that proteinase activity is substantially reduced after the protease handles 60min at 55 DEG C.But at 35 DEG C
At a temperature of, enzyme activity is then stablized relatively, more than half enzyme activity, such as Fig. 3 still can be kept after 180min is handled.
(4) metal ion influences proteinase activity in external Enzyme activity assay.
It concretely comprises the following steps：Each metal ion species, final concentration of 10mM are added in HSL10 alkali protease reaction systems.
Enzyme activity assay shows Ca2+, Mg2+It can significantly improve protease activity, and K+, Mn2+Enzyme activity can also be improved to a certain extent；
Cu2+, Zn+, Fe2+, Co2+There is apparent inhibitory action to protease, such as table 1.
Table 1：Metal ion influences HSL10 proteinase activities
(5) other factors influence HSL10 proteinase activities in external Enzyme activity assay.
Further qualitative analysis is made to HSL10 protease, including addition detergent, organic solvent and metal ion-chelant
Agent and serpin etc..The result shows that HSL10 protease to 1.0% detergent SDS, Triton 80,
Triton X 100 are insensitive；The enzyme is equally insensitive for organic solvent DMSO, such as table 2.
Table 2：Other factors influence HSL10 proteinase activities
On the other hand, the PMSF of 50 μ g/ml has apparent inhibitory action to HSL10 protease, can tentatively illustrate the enzyme
For serine protease；And the EDTA of 50mM has no significant effect the enzyme, illustrates the enzyme for nonmetallic ion dependent form protease,
Such as table 2.
Embodiment 2：Aoxidize microbacterium (Microbacterium oxydans) HSL10 protease gene ap1-HSL10 tables
Structure and expression up to plasmid
(1) gene order-checking is carried out to Microbacterium oxydans HSL10, analyzes the possibility on its genome
Alkaline serine protease AP1 homology (Fig. 4).
The gene coded sequence (3210bp bases) of alkali protease AP1 is as shown in SEQ ID No.1, amino acid sequence
(1070 amino acid) as shown in SEQ ID No.2, the albumin A P1 of coding contains serine protease domain and catalysis is lived
Property triad residues.
(2) PCR amplification Microbacterium oxydans HSL10 protease genes ap1-HSL10 (Fig. 5 A)
It concretely comprises the following steps：Microbacterium oxydans HSL10 genomes are extracted, by designing Yin WuF ＆R-AP1
PCR amplification is carried out to genome and obtains Microbacterium oxydans HSL10 protease genes, two sections of bands of target gene
There are the restriction enzyme site of NdeI and HindIII, (glue recycling specific practice is with reference to Tiangeng kit explanation for gel extraction target fragment
Book).
F-AP1:cgccatatggcgCTACTCCGGCTTCGCCGTTT
R-AP1:(lowercase is restriction enzyme site and protection to cccaagcttgggATGGGTCGAACACCCCTCCG in primer
Base, capitalization are primer).
PCR amplification system：
Primer used is F-AP1 and R-AP1 in experimentation.Template is Microbacterium oxydans HSL10
Genomic DNA.
(3) Microbacterium oxydans HSL10 protease genes ap1-HSL10 expression plasmids pHSL-AP1
The structure (Fig. 5 B) of structure and expression engineered strain
It concretely comprises the following steps：Double digestion, and gel extraction are carried out to pET28a (+) with NdeI, HindIII, and carries out dephosphorization
It is acidified enzymatic treatment.By carrier：Insert Fragment is 1:1 to 1:3 ratio is attached with T4 ligases；Direct electricity is transferred to large intestine bar
In bacterium expressive host E.coli BL21, positive colony is screened with kanamycins.The solid medium of selection is LA.It is right
The positive colony chosen carries out liquid and is incubated overnight, and extracts plasmid, with NdeI, HindIII double digestion positive plasmid, agarose coagulates
Gel electrophoresis are verified.The obtained band of digestion is 5300bp and 3200bp or so (size of plasmid pET28a is 5369bp), is tested
The result is shown in Fig. 5 B for card.Plasmid names pHSL-AP1.
As a result confirm：The expression plasmid pHSL-AP1 electricity of structure is gone in E.coli BL21 (DE3), obtains heterologous table
Up to the engineered strain of alkali protease AP1, engineered strain E.coli BL21 (DE3)/pHSL-AP1 is named as.
Embodiment 3：Engineered strain E.coli BL21 (DE3)/pHSL-AP1 of the present invention is applied to alkali protease
The expression of AP1
In the culture of engineering bacteria, carried out using 0.0 to 1.2mmol/L difference gradient concentration IPTG in SYN culture mediums
Induced expression, 37 DEG C of cultures find that bacterium colony corresponds to culture basal part and has weakly hydrolyse circle for 24 hours, and (figure is enclosed in periphery without apparent hydrolysis
6).Then, applicant attempts to add 1% glucose on the basis of SYN culture mediums and carries out induced expression and 0.0 arrive again
1.2mmol/L difference gradient concentrations IPTG.The result shows that not only colony growth is good by heterogenous expression host, while can also generate
Sharp-edged transparent circle.Wherein, the inducing effect in IPTG concentration 0.4mmol/L is best (Fig. 6).
Based on above-mentioned experiment basis, the engineering of the alkali protease AP1 in heterogenous expression extreme environment source of the present invention
Bacterium is in the application in expressing alkali protease AP1：The engineering of the alkali protease AP1 in the heterogenous expression extreme environment source
The condition of culture of bacterium is preferably：1% glucose is added on the basis of SYN culture mediums and carries out induced expression, induces what is used
IPTG concentration is 0.4mmol/L.
Claims (5)
1. a kind of basic protein enzyme coding gene in extreme environment source, it is characterised in that：The alkaline protease gene name
For ap1-HSL10, nucleotide sequence is as shown in SEQ ID No.1；The gene order is from oxidation microbacterium
(Microbacterium oxydans) HSL10, nucleotide sequence encode alkali protease by 3210bp base compositions.
2. a kind of alkali protease in extreme environment source, it is characterised in that：The alkali protease is named as AP1, amino
Acid sequence is as shown in SEQ ID No.2；The sequence derives from oxidation microbacterium (Microbacterium oxydans) HSL10,
Its amino acid sequence is made of 1070 amino acid, and the albumen of coding contains serine protease catalytic active structure domain and catalysis
Active triad residues.
3. a kind of engineering bacteria of the alkali protease AP1 in heterogenous expression extreme environment source, it is characterised in that：The engineering bacteria life
Entitled E.coli BL21 (DE3)/pHSL-AP1, genotype are E.coli BL21 (DE3), kanamycin
resistance、ap1-HSL10、IPTG inducible PT7Promoter, it using E.coli BL21 (DE3) is bacterium germination to be
Strain is obtained by electricity conversion in its intracellular ap1-HSL10 gene carried by pET28a (+) plasmid that import.
4. the engineering bacteria of the alkali protease AP1 in heterogenous expression extreme environment source described in claim 3 is in expression basic protein
Application in enzyme AP1.
5. application as claimed in claim 4, it is characterised in that：The alkali protease in the heterogenous expression extreme environment source
The condition of culture of the engineering bacteria of AP1 is：1% glucose is added on the basis of SYN culture mediums and carries out induced expression, induction uses
IPTG concentration be 0.4mmol/L.
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| CN108085327B (en) | 2021-02-19 |
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