CN108085327B - Alkaline protease heterologous expression engineering strain from extreme environment and application thereof - Google Patents
Alkaline protease heterologous expression engineering strain from extreme environment and application thereof Download PDFInfo
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Abstract
本发明公开了一种异源表达极端环境来源的碱性蛋白酶AP1的工程菌，该菌株命名为E.coli BL21(DE3)/pHSL‑AP1，是利用E.coli BL21(DE3)为出发菌株，通过电转化在其细胞内导入由pET28a(+)质粒携带的ap1‑HSL10基因获得。本发明还公开了该工程菌在表达碱性蛋白酶AP1中的应用。实验证实：本发明提供的工程菌株在表达蛋白酶AP1时，SYN培养基中添加1％的葡萄糖对于高效表达蛋白酶基因尤为重要，其应用有望为规模化生产碱性蛋白酶提供试验和理论基础，在工业应用开发上具有重要价值。
The invention discloses an engineering bacterium that heterologously expresses alkaline protease AP1 derived from extreme environment. It was obtained by introducing the ap1-HSL10 gene carried by the pET28a(+) plasmid into its cells by electroporation. The invention also discloses the application of the engineering bacteria in expressing alkaline protease AP1. Experiments have confirmed that when the engineered strain provided by the present invention expresses the protease AP1, adding 1% glucose to the SYN medium is particularly important for the efficient expression of the protease gene, and its application is expected to provide an experimental and theoretical basis for the large-scale production of alkaline protease. It is of great value in application development.
Description
Technical Field
The invention relates to cloning of alkaline protease, an engineering strain, construction and application thereof, in particular to an alkaline protease coding gene from an extreme environment source, construction of an alkaline protease heterologous expression engineering strain from the extreme environment source by using the gene and application thereof, belonging to the technical field of biology.
Background
Extreme environment microorganism adaptationThe metabolic types and physiological structures of the extreme living environment which is harsh can be greatly changed, and the evolution result can generate special metabolic pathways and metabolic products, including special functional enzymes, novel biological materials and the like【1-2】. The applicant isolated an extreme microorganism in saline-alkali lake of Xinjiang and identified it as Microbacterium oxydans HSL10【3】。
Alkaline proteases belong to the group of serine proteolytic enzymes of endopeptidases, which are capable of hydrolyzing protein peptide bonds under alkaline conditions (pH9-10) to form polypeptides or amino acids. Alkaline protease is initially found in porcine pancreas and is subsequently isolated in Bacillus licheniformis [1 ]]. From this point on, the microbial alkaline proteases have been rapidly developed. The alkaline protease is mainly derived from bacteria, actinomycetes and fungi. Alkaline proteases currently used for commercial purposes are mostly obtained from bacillus. Besides alkaline environment, alkaline protease producing bacteria can be screened in other extreme environments, such as extremely cold environment, ocean high salt environment, and even some metal polluted areas, which have reports of different alkaline protease producing bacteria separation. Microbacterium oxydans HSL10 isolated from saline-alkali lake area in this subject group has alkaline protease producing ability【3】。
As an important industrial enzyme, alkaline protease is the most widely applied enzyme at present, and is widely applied to multiple industries such as preparation of phosphate-free enzyme-added washing powder, processing of food/medicine/feed/leather, silver recovery, chemical industry, organic waste treatment and the like. Among them, Serine protease (Serine protease) has high activity and stability under alkaline conditions, and thus is widely used in industrial fields. The active region of this enzyme contains a nucleophilic serine residue, which forms a catalytic triad with the two essential residues asparate and histidine. The optimum pH of the protease is 7-11. At the same time, studies have shown that most of them have substrate specificity【1-2】。
However, there are relatively few alkaline protease-producing strains having industrial development value. Currently, the alkaline protease producing bacteria which have been industrially used or much studied are mainly limited to two genera: bacillus (e.g., Bacillus alcalophilus ATCC 21522, B. alcalophilus subsp, Halodurans KP1239, B. amyloliquefaciens, B. circuita, B. coemulans, etc.) and fungi (e.g., Aspergillus candidus, A. flavus, A. fumigus, A.melleus, A.niger, A.oryzae, etc.). The search for novel strains with alkaline protease activity remains a hot topic today. The current search finds that the alkaline protease is separated from the extreme microorganisms and is only rarely reported. Whether the alkaline protease from extreme environment has some special activities or not and how to carry out high-efficiency expression are of great significance to the research of the alkaline protease at present.
Reference documents:
[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
disclosure of Invention
Aiming at the defects of the prior art, the invention aims to solve the problems of screening alkaline protease production strains from special habitats, cloning alkaline protease coding genes from the alkaline protease production strains, constructing an alkaline protease heterologous expression engineering strain with extreme environmental sources and application of the alkaline protease heterologous expression engineering strain in expressing the alkaline protease AP 1.
The alkaline protease coding gene of the extreme environment source is characterized in that: the alkaline protease gene is named as ap1-HSL10, and the nucleotide sequence of the alkaline protease gene is shown as SEQ ID No.1 in a table; the gene sequence is derived from Microbacterium oxydans (HSL 10), the nucleotide sequence of the gene sequence consists of 3210bp base, and alkaline protease is coded.
The alkaline protease derived from extreme environment according to the present invention is characterized in that: the alkaline protease is named as AP1, and the amino acid sequence of the alkaline protease is shown as SEQ ID No. 2; the sequence is derived from Microbacterium oxydans HSL10, the amino acid sequence of the sequence is composed of 1070 amino acids, and the coded protein contains serine protease catalytic activity structural domain and catalytic activity triplet residue.
The applicant previously cloned a new alkaline protease gene from the genome of Microbacterium oxydans HSL10, Microbacterium alkaligenes, and deduced serine protease from the study of enzymatic properties. Aiming at the problem that the resource of the existing alkaline protease producing strain is relatively single, the wild strain of the alkaline protease coding gene, namely Microbacterium oxydans (HSL 10), is an alkalophilic actinomycete separated from Xinjiang saline alkali lake, the strain not only has the activity of alkaline protease, but also has the activities of alkalophilic, salt-tolerant, low-temperature-resistant and the like under the special saline-alkaline environment.
The invention relates to an engineering bacterium for heterogeneously expressing alkaline protease AP1 from extreme environment, which is characterized in that: the engineering bacteria are named as E.coli BL21(DE3)/pHSL-AP1, and the genotypes of the engineering bacteria are E.coli BL21(DE3), kanamycin resistance, AP1-HSL10 and IPTG indicale PT7promoter was obtained by introducing the ap1-HSL10 gene carried by pET28a (+) plasmid into cells of e.coli BL21(DE3) as a starting strain by electroporation. Coli BL21(DE3) is a commonly used bacterial heterologous expression host, pHSL-AP1 is an expression plasmid for heterologous expression of serine protease AP1 derived from Microbacterium oxydans HSL 10: the 3210bp protease gene ap1-HSL10 was inserted into pET28a (+) expression vector by digestion ligation, carrying the kanamycin resistance gene, 8.58kb in size.
Specifically, the construction method of the engineering bacteria for heterologous expression of the alkaline protease AP1 from extreme environment comprises the following steps:
(1) the activity of protease of wild-type strain of Microbacterium oxydans HSL10 was analyzed.
(2) Homology analysis of alkaline protease gene ap1-HSL10 according to the sequencing result of the whole genome of Microbacterium oxydans HSL 10.
(3) Primers F & R-AP1 with enzyme cutting sites (NdeI and HindIII) are designed, and a microbacterium oxydans HSL10 protease gene fragment AP1-HSL10 is obtained by PCR amplification.
(4) The expression vector pET28a (+) was digested with NdeI and HindIII, and the insert obtained in step (3) was ligated with T4 ligase to obtain an expression plasmid pHSL-AP 1.
(5) And (3) directly electrically transforming the expression plasmid pHSL-AP1 obtained in the step (4) into escherichia coli E.coli BL21(DE3), screening positive clones, extracting plasmids, and performing enzyme digestion verification to obtain an engineering strain capable of heterologously expressing the alkaline protease AP1 from the extreme environment source, wherein the engineering strain is named as engineering strain E.coli BL21(DE3)/pHSL-AP 1.
The invention discloses application of engineering bacteria heterologously expressing alkaline protease AP1 from extreme environment sources in expression of alkaline protease AP 1.
Wherein: the culture conditions of the engineering bacteria for heterogeneously expressing the alkaline protease AP1 from the extreme environment are preferably as follows: 1% glucose was added to SYN medium to induce expression, and IPTG concentration used for induction was 0.4 mmol/L.
The invention discloses an engineering strain E.coli BL21(DE3)/pHSL-AP1 for heterologous expression of alkaline protease AP1 from an extreme environment source, which realizes the heterologous expression of the alkaline protease AP1 from the extreme environment source for the first time. The experiment proves that: when the engineering strain provided by the invention expresses the protease AP1, 1% of glucose added into the SYN culture medium is particularly important for efficiently expressing the protease gene, the application of the engineering strain is expected to provide experimental and theoretical basis for large-scale production of alkaline protease, and the engineering strain has important value in industrial application development.
Drawings
FIG. 1: the culture characteristics of HSL10 and the influence of metal ions added into the culture medium on the enzyme activity of HSL10 protease AP1
Wherein: (A) HSL10 can be grown at low temperature; (B) the protease hydrolysis loop can be generated in a culture medium taking Casein as the only C source; (C) the enzyme activity is highest under the culture condition of SYN (culture medium added with skimmed milk powder and NaCl); (D)): the influence of metal ion addition on enzyme activity in the culture process: SYN + Ca2+: adding 10mmol/L Ca on the basis of SYN2+；SYN+Mg2+: adding 10mmol/L Mg on the basis of SYN2+；SYN+K+: adding 10mmol/L K based on SYN+。
FIG. 2: effect of temperature and pH on the enzymatic Activity of the HSL10 protease AP1
Wherein: (A) the effect of temperature on enzyme activity; (B) influence of pH on enzyme activity.
FIG. 3: thermostability assay of HSL10 protease AP 1.
FIG. 4: HSL10 protease AP1 Domain analysis
FIG. 5: gene amplification and expression plasmid construction of Microbacterium oxydans HSL10 protease AP1
Wherein: (A) band 1 is Dongsheng 1kb Marker; the bands 2-3 are subjected to PCR amplification gel recovery by taking F-AP1& R-AP1 as primers; b: band 1 is Dongsheng 1kb Marker; NdeI and HindIII double enzyme digestion verification of the positive clone plasmid with the band 2-3.
FIG. 6: glucose-promoted expression of the protease AP1
Wherein: FIG. A: SYN medium containing kanamycin, 0.4mmol/L IPTG; and B: SYN medium containing kanamycin, 0.4mmol/L IPTG, 1% glucose. AP1-1 and AP1-2 are two parallel colonies of cells BL21/pHSL-AP 1.
Detailed Description
The invention will be described in detail below with reference to the attached drawings and specific examples in order to better understand the invention, but the invention is not limited to the protection content of the invention.
General description: the wild strain Microbacterium oxydans HSL10, referred to in the following examples, was isolated from saline lake region of Xinjiang【3】The complete sequence of the genome is sequenced; the coding gene of the alkaline protease AP1 is named as AP1-HSL10, and Microbacterium oxydans (Microbacterium oxydans) is directly cloned from HSL10 genome; coli BL21(DE3) is a commonly used bacterial heterologous expression host, and the plasmid vector pET28a (+) used for expression of the alkaline protease AP1 was purchased from Novagen, usa. Gene sequencing in plasmid constructionDageney Co Ltd. The plasmids are all commercially available conventional plasmids. The related reagents and consumables are all made in China. Unless otherwise specified, the experimental methods and reagents in the examples are those conventionally used in the art and those commercially available.
Example 1: alkaline protease activity assay of Microbacterium oxydans (Microbacterium oxydans) HSL10 wild-type strain.
(1) The culture characteristics of HSL10 and the effect of metal ion addition in the culture medium on the enzymatic activity of HSL10 protease (FIG. 1).
The method comprises the following specific steps: culturing Microbacterium oxydans HSL10 in culture medium at low temperature, adding three common metal ions Ca in different culture media2+、Mg2+And K+The enzyme solution is obtained by separating protease by dialysis, salting out with ammonium sulfate and sieving with molecular sieve, and the activity is measured.
As a result, it was found that HSL10 has cold-resistant property (FIG. 1A), and a proteolytic loop can be generated in a culture medium containing Casein as the only C source (FIG. 1B)【3】(ii) a It was found that by culturing HSL10 in SYN broth (skim milk powder 1.0g, yeast extract 0.5% and sodium chloride 1% in deionized water to 100ml, pH adjusted to 7.0), the obtained protease had higher enzyme activity (FIG. 1C). Three common metal ions Ca are selected2+、Mg2+And K+Addition to optimized SYN medium (fig. 1D), and then comparison of the activity of the enzyme solutions; it was found that the addition of these three ions contributes to the improvement of the enzyme activity.
Protease activity determination method: the assay was performed with appropriate modification according to the national relevant professional Standard (SB/T10317-1999): for different pH requirements, this can be achieved by adjusting the pH of the casein buffer. Three parallel runs were made each time. Mixing preheated 1ml enzyme solution and 1ml casein, and reacting at 40 deg.C for 20 min; then adding 2ml of 0.4M TCA (trichloroacetic acid) to inactivate the enzyme, and continuing to preserve heat for 20 minutes; filtering the protein flocculent precipitate after the denaturation of the TCA by using filter paper; 1ml of the filtrate was taken in another tube and 5ml of 0.4M Na was added2CO3And 1mL of a Folin phenol reagent, shaking up, and keeping the temperature at 40 ℃ for 20min for color development. Followed by OD with UV-visible spectrophotometer660And (4) measuring. Blank controlThe method comprises the steps of adding TCA for inactivation and then adding a casein solution.
(2) Temperature and pH effects on enzymatic activity of HSL10 protease.
The method comprises the following specific steps: setting the reaction temperature gradient of alkaline protease at 20-80 ℃ according to the growth characteristics of a wild strain of Microbacterium oxydans HSL 10. Protease activity was measured in the same manner as in (1).
Experiments show that the optimal reaction temperature for producing the alkaline protease by the microbacterium oxydans HSL10 is 55 ℃; the enzyme activity was maintained at a high level over a wide range of 40 ℃ to 65 ℃ and the results are shown in FIG. 2-A; dialyzing the obtained enzyme solution in buffer solutions with different pH gradients to obtain the enzyme solution with different pH conditions. And casein with corresponding different pH gradients was used as a substrate, thereby more precisely determining the most suitable pH for the protease produced by HSL 10. The protease produced by Microbacterium oxydans HSL10 showed the highest activity in the pH range of 9.0 to 12.0, with the highest activity at pH9.0 (see FIG. 2-B). This again demonstrates that Microbacterium oxydans is capable of producing alkaline proteases.
(3) HSL10 protease thermostability assay.
The method comprises the following specific steps: the obtained enzyme solution is treated at 35 deg.C, 45 deg.C and 55 deg.C for 30-180 min, and the residual enzyme activity is determined. The result shows that the protease activity is obviously reduced after the protease is treated for 60min at 55 ℃. However, at 35 ℃, the enzyme activity is relatively stable, and more than half of the enzyme activity can still be maintained after 180min of treatment, as shown in fig. 3.
(4) The influence of metal ions on the enzyme activity of the protease in-vitro enzyme activity detection.
The method comprises the following specific steps: various metal ions were added to the HSL10 alkaline protease reaction system to a final concentration of 10 mM. The enzyme activity detection shows that Ca2+，Mg2+Can obviously improve the protease activity, and K+，Mn2+The enzyme activity can also be improved to a certain extent; cu2+，Zn+，Fe2+，Co2+Has obvious inhibiting effect on protease, as shown in the table 1.
Table 1: effect of Metal ions on enzymatic Activity of HSL10 protease
(5) Other factors in the in vitro enzyme activity test have influence on the activity of the HSL10 protease.
Further qualitative analysis of the HSL10 protease was performed by adding detergents, organic solvents, and metal ion chelators and serine protease inhibitors. The results show that the HSL10 protease is not sensitive to 1.0% of detergents SDS, Triton 80 and Triton X100; the enzyme was also insensitive to the organic solvent DMSO, as shown in Table 2.
Table 2: effect of other factors on HSL10 protease Activity
On the other hand, PMSF of 50 mu g/ml has obvious inhibition effect on HSL10 protease, which can preliminarily indicate that the enzyme is serine protease; while EDTA at 50mM had no significant effect on the enzyme, indicating that the enzyme is a non-metal ion-dependent protease, as shown in Table 2.
Example 2: construction and expression of expression plasmid of Microbacterium oxydans (Microbacterium oxydans) HSL10 protease gene ap1-HSL10
(1) Microbacterium oxydans HSL10 was genomically sequenced and its genome was analyzed for homology to the potential alkaline serine protease AP1 (FIG. 4).
The gene coding sequence (3210bp base) of the alkaline protease AP1 is shown in SEQ ID No.1, the amino acid sequence (1070 amino acids) is shown in SEQ ID No.2, and the coded protein AP1 contains a serine protease structural domain and a catalytic activity triplet residue.
(2) PCR amplification of Microbacterium oxydans HSL10 protease Gene ap1-HSL10 (FIG. 5A)
The method comprises the following specific steps: extracting a Microbacterium oxydans HSL10 genome, carrying out PCR amplification on the genome by designing a primer F & R-AP1 to obtain a Microbacterium oxydans HSL10 protease gene, carrying out two sections of enzyme cutting sites of NdeI and HindIII on a target gene, cutting glue and recovering a target fragment (the concrete method of glue recovery refers to the specification of a Tiangen kit).
F-AP1:cgccatatggcgCTACTCCGGCTTCGCCGTTT
R-AP1: cccaagcttgggATGGGTCGAACACCCCTCCG (lower case letters in the primer are enzyme cutting site and protecting base, upper case letters are primer).
PCR amplification System:
the primers used in the experimental procedure were F-AP1 and R-AP 1. The template was Microbacterium oxydans HSL10 genomic DNA.
(3) Construction of Microbacterium oxydans HSL10 protease gene AP1-HSL10 expression plasmid pHSL-AP1 and construction of expression engineering strain (FIG. 5B)
The method comprises the following specific steps: pET28a (+) was digested with NdeI and HindIII, and the gel was recovered and dephosphorylated. And (3) mixing the carrier: the inserts were ligated at a ratio of 1:1 to 1:3 using T4 ligase; coli BL21, and positive clones were screened with kanamycin. The solid medium selected was LA. The selected positive clone is cultured overnight in liquid, plasmid is extracted, the positive plasmid is cut by NdeI and HindIII enzyme, and agarose gel electrophoresis is used for verification. The bands obtained by the enzyme digestion were about 5300bp and 3200bp (the size of the plasmid pET28a was 5369bp), and the results of the verification are shown in FIG. 5B. The plasmid was named pHSL-AP 1.
The results confirmed that: the constructed expression plasmid pHSL-AP1 is electrically transferred into E.coli BL21(DE3) to obtain an engineering strain for heterologous expression of alkaline protease AP1, and the engineering strain is named as engineering strain E.coli BL21(DE3)/pHSL-AP 1.
Example 3: the engineering strain E.coli BL21(DE3)/pHSL-AP1 is applied to expression of alkaline protease AP1
In the culture of engineering bacteria, induced expression is carried out in SYN culture medium by using IPTG with different gradient concentrations of 0.0 to 1.2mmol/L, and the bacterial colony has weak hydrolysis ring corresponding to the culture base part and no obvious hydrolysis ring at the periphery after being cultured for 24h at 37 ℃ (figure 6). Then, the applicants tried to add 1% glucose to the SYN medium for induction of expression, and different gradient concentrations of IPTG from 0.0 to 1.2 mmol/L. The results show that the heterologous expression host not only has good colony growth, but also can generate transparent circles with clear edges. Among them, the induction effect was the best at an IPTG concentration of 0.4mmol/L (FIG. 6).
Based on the experimental basis, the engineering bacteria heterologously expressing the alkaline protease AP1 from the extreme environment is applied to the expression of the alkaline protease AP1: the culture conditions of the engineering bacteria for heterogeneously expressing the alkaline protease AP1 from the extreme environment are preferably as follows: 1% glucose was added to SYN medium to induce expression, and IPTG concentration used for induction was 0.4 mmol/L.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1487080A (en) * | 2002-06-26 | 2004-04-07 | 花王株式会社 | alkaline protease |
| CN1505679A (en) * | 2001-05-02 | 2004-06-16 | �����ɷ� | Alkaline protease variants, and detergents and cleaning agents containing the same |
| CN103013960A (en) * | 2012-12-21 | 2013-04-03 | 青岛蔚蓝生物集团有限公司 | Alkaline protease and recombinant expression engineering bacterium thereof |
| JP2016111951A (en) * | 2014-12-12 | 2016-06-23 | 公立大学法人 富山県立大学 | Method for producing lumichrome |
| CN106676024A (en) * | 2015-11-05 | 2017-05-17 | 中国科学院天津工业生物技术研究所 | Bacillus amyloliquefaciens for high yield of alkali protease and application of bacillus amyloliquefaciens |
| CN107384897A (en) * | 2017-08-02 | 2017-11-24 | 北京科为博生物科技有限公司 | A kind of alkali protease and its gene and application |
-
2017
- 2017-12-28 CN CN201711459372.0A patent/CN108085327B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1505679A (en) * | 2001-05-02 | 2004-06-16 | �����ɷ� | Alkaline protease variants, and detergents and cleaning agents containing the same |
| CN1487080A (en) * | 2002-06-26 | 2004-04-07 | 花王株式会社 | alkaline protease |
| CN103013960A (en) * | 2012-12-21 | 2013-04-03 | 青岛蔚蓝生物集团有限公司 | Alkaline protease and recombinant expression engineering bacterium thereof |
| JP2016111951A (en) * | 2014-12-12 | 2016-06-23 | 公立大学法人 富山県立大学 | Method for producing lumichrome |
| CN106676024A (en) * | 2015-11-05 | 2017-05-17 | 中国科学院天津工业生物技术研究所 | Bacillus amyloliquefaciens for high yield of alkali protease and application of bacillus amyloliquefaciens |
| CN107384897A (en) * | 2017-08-02 | 2017-11-24 | 北京科为博生物科技有限公司 | A kind of alkali protease and its gene and application |
Non-Patent Citations (3)
| Title |
|---|
| An extremophile Microbacterium strain and its protease production under alkaline conditions;Jin Lü et al.;《Journal of basic microbiology》;20141231;第54卷;第378-385页 * |
| 一株极端环境微生物生理特性研究以及菌株鉴定;吴小丹;《中国优秀硕士论文全文数据库-基础科学辑》;20150815(第8期);A006-75页 * |
| 极端环境分离的氧化微杆菌的嗜碱性及其基因组文库的构建;吴小丹等;《浙江农业学报》;20110331;第23卷(第2期);第409-414页 * |
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