CN113801801B - Recombinant strain for efficiently producing alkaline pectinase and application thereof - Google Patents
Recombinant strain for efficiently producing alkaline pectinase and application thereof Download PDFInfo
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Abstract
The invention relates to a recombinant strain for efficiently producing alkaline pectinase and application thereof, wherein the recombinant strain is prepared by connecting a mutant pelA of a bacillus subtilis alkaline pectinase gene to a pichia pastoris expression vector pPIC9K, optimizing signal peptide types (MF 4I, HFBI, W1, albumin and Invertase 2) and chaperone factors (BMH 2, HAC1, PDI1 and KEX 2) and transforming the signal peptide types and the chaperone factors into the pichia pastoris for recombining and expressing the alkaline pectinase. The invention mainly adopts the strategies of signal peptide, co-expression molecular chaperone and the like to improve the secretion expression level of the alkaline pectinase in pichia pastoris.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a recombinant strain for efficiently producing alkaline pectinase and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Pectinase is a complex enzyme which can break down pectin polymers into unsaturated oligogalacturonans.
There are many strains producing pectinases, such as Aspergillus, penicillium, streptomyces, pseudomonas, bacillus, and Klebsiella. Pectinase has been widely used, and has been used industrially for approximately 50 years. Pectinase is divided into acid pectinase and Alkaline Pectinase (APs) according to the difference of optimal reaction pH, wherein the APs are mainly widely applied to the aspects of fruit juice clarification, tea fermentation, textile processing, degumming of plant bast fiber, pectin wastewater treatment, paper making and the like. The enzyme method is applied to the related reactions in the field, and the method has the advantages of reducing energy consumption, protecting environment, protecting fiber, improving refining efficiency and the like. The enzyme activity of the alkaline pectinase which is found is relatively low and cannot meet the requirements of industrial application. However, the molecular modification of APs is currently relatively rare and commercial APs are not sufficient.
At present, hosts for expressing alkaline pectinase mainly comprise escherichia coli, pichia pastoris and bacillus subtilis. The pichia pastoris (Pichiapastoris) has low nutritional requirement, high proliferation rate and high thallus density, and the pichia pastoris expression protein is easy to purify, has high yield and is suitable for large-scale production. However, the excessive expression of heterologous proteins can cause the unfolded protein effect (UPR) caused by growth stress pressure or the unfolded proteins can not be effectively modified, so that the yield of the alkaline pectinase can not be further improved, and the industrial production of the alkaline pectinase is limited. No reports are found about the research on the improvement of alkaline protease by the signal peptide and molecular chaperone co-expression strategy.
Disclosure of Invention
In order to overcome the problems, the invention provides a recombinant strain for efficiently expressing alkaline pectinase. The recombinant bacteria are obtained by connecting a mutant pelA of a bacillus subtilis alkaline pectinase gene to a pichia pastoris expression vector pPIC9K, optimizing signal peptide types (MF 4I, HFBI, W1, albumin and Invertase 2) and chaperone factors (BMH 2, HAC1, PDI1 and KEX 2), and transforming the recombinant bacteria into the pichia pastoris for recombinant expression of the alkaline pectinase.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention provides a construction method of a recombinant bacterium for efficiently producing alkaline pectinase, which comprises the following steps:
respectively connecting the mutant pelA gene of the alkaline pectinase gene and the signal peptide gene segment to an expression vector pPIC9K and optimizing to obtain a recombinant plasmid I;
respectively connecting the chaperone factor gene fragments to an expression vector pGAPZA and optimizing to obtain a recombinant plasmid II;
transforming the recombinant plasmid I into pichia pastoris for recombinant expression of alkaline pectinase to obtain a secretion-enhanced genetic engineering strain;
and transforming the recombinant plasmid II into the secretion-enhanced genetic engineering strain to obtain the alkaline pectinase secretion-enhanced genetic engineering strain.
The invention mainly adopts the strategies of signal peptide, co-expression molecular chaperone and the like to improve the secretory expression level of the alkaline pectinase in the pichia pastoris.
In a second aspect of the present invention, there is provided a recombinant bacterium constructed by the above method for efficiently producing alkaline pectinase.
In a third aspect of the present invention, there is provided a method for efficiently producing alkaline pectinase, comprising:
fermenting and culturing any recombinant bacterium.
The invention has the beneficial effects that:
(1) Compared with the original genetically engineered strain GS 115/alpha-factor-pelA, the genetically engineered strain GS115/KEX2-MF4I-pelA has the advantages that the enzyme activity is improved by 14.7 times during shake flask fermentation, the maximum enzyme activity of GS115/KEX2-MF4I-pelA reaches 2301.05U/mL during 5L fermentation tank culture, and the efficient expression of alkaline pectinase is realized. The alkaline pectinase of the invention can catalyze and crack alpha-1,4 glycosidic bond of polygalacturonic acid under alkaline condition, and can be widely applied to industries of food, textile, paper making and the like.
(2) The method is simple in operation method, high in expression efficiency and easy for large-scale production.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1: schematic representation of cloning expression plasmids.
FIG. 2: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis is carried out on fermentation supernatant in the fed-batch fermentation process; lanes 2-7 are induction 60h, 72h, 84h, 96h, 120h, 132h fermentation supernatant samples, respectively.
FIG. 3: fermentation performance of the recombinant strain in a 5L fermentor.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A recombinant strain for efficiently expressing alkaline pectinase.
The recombinant bacteria are obtained by connecting mutant pelA of bacillus subtilis alkaline pectinase gene to pichia pastoris expression vector pPIC9K, optimizing signal peptide types (MF 4I, HFBI, W1, albumin and Invertase 2) and chaperone factors (BMH 2, HAC1, PDI1 and KEX 2) and transforming the mutant pelA into pichia pastoris for recombinant expression of alkaline pectinase.
The nucleotide sequence of the pelA gene is shown in SEQ ID NO. 1.
The invention also provides a construction method of the recombinant bacterium, which comprises the following steps:
(1) Synthesizing to obtain a gene pelA, and synthesizing to obtain signal peptide gene fragments (MF 4I, HFBI, W1, albumin and Invertase 2) and chaperone factor gene fragments (BMH 2, HAC1, PDI1 and KEX 2).
(2) Respectively connecting pelA and signal peptide gene fragments (MF 4I, HFBI, W1, albumin and Invertase 2) obtained in the step (1) to an expression vector pPIC9K to obtain recombinant plasmids pPIC9K-MF4I-pelA, pPIC 9K-BI HFHFHFPElA, pPIC9K-W1-pelA, pPIC9K-Albumin-pelA and pPIC 9K-Invertase-pelA; and (2) respectively connecting the chaperone factor gene fragments (BMH 2, HAC1, PDI1 and KEX 2) obtained in the step (1) to an expression vector pGAPZA to obtain recombinant plasmids pGAPZA-BMH2, pGAPZA-HAC1, pGAPZA-PDI1 and pGAPZA-KEX2.
(3) And (3) transforming the recombinant plasmids pPIC9K-MF4I-pelA, pPIC9K-HFBI-pelA, pPIC9K-W1-pelA, pPIC9K-Albumin-pelA and pPIC9K-Invertase-pelA obtained in the step (2) into pichia pastoris for expressing alkaline pectinase in a recombinant mode to obtain the secretion enhancement type genetic engineering strain.
(4) And (3) transforming the recombinant plasmids pGAPZA-BMH2, pGAPZA-HAC1, pGAPZA-PDI1 and pGAPZA-KEX2 obtained in the step (2) into the secretion-enhanced genetic engineering strain in the step (3) to obtain a new alkali pectinase secretion-enhanced genetic engineering strain.
The invention also provides a method for producing the alkaline pectinase by using the recombinant bacterium.
In one embodiment of the invention, the recombinant strain is activated and inoculated into growth medium YPD for 24h at 30 ℃ and 220rpm, and then inoculated into induction medium BMGY at 1% inoculation rate, and subjected to shake culture at 30 ℃ and 220rpm until OD600 is about 20. Then, the cells were transferred into an induction medium BMMY, and 1% methanol was added every 24 hours at 23 ℃ and 220rpm to induce the expression of alkaline pectinase. The medium YPD (1L): 1% yeast powder, 2% peptone and 2% glucose; the medium BMGY (1L): 1% yeast extract, 2% peptone, 0.34% YNB,1% (NH) 4 ) 2 SO 4 100mmol/L potassium phosphate buffer (pH 6.0), 1% glycerol.
In one embodiment of the invention, the method is to inoculate the activated bacteria liquid into a fermentation medium with the liquid loading amount of 20-30%, the initial stirring speed is 400-550rpm, the ventilation amount is 1.5-2vvm, the pH is controlled to be 5.5-6.0, and the culture temperature in the growth phase is 28-30 ℃. (ii) a When the glycerol is exhausted and the dissolved oxygen rebounds, the glycerol is supplemented, and the DO is maintained at about 20-30% by adopting a dissolved oxygen and rotating speed coupling mode. When the glycerol is exhausted again and the dissolved oxygen rebounds, starvation culture is carried out for 0.5-2h, the induction culture medium is fed, the temperature is reduced to 24-26 ℃, the stirring speed is increased to 700-900rpm, and APs expression is induced.
In one embodiment of the present invention, the methanol induction stage employs a dissolved oxygen-related fed-batch approach: DO is maintained at about 0-10% and DO is maintained at about 10-20%.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Culture medium:
seed medium YPD:1% of yeast powder, 2% of peptone and 2% of glucose.
Growth medium BMGY (1L): 1% of yeast powder, 2% of peptone and 2% of glucose; the medium BMGY (1L): 1% yeast extract, 2% peptone, 0.34% YNB,1% (NH) 4 ) 2 SO 4 100mmol/L potassium phosphate buffer (pH 6.0), 1% glycerol.
Induction medium BMMY (1L): 1% yeast powder, 2% peptone and 2% glucose; the medium BMGY (1L): 1% yeast extract, 2% peptone, 0.34% YNB,1% (NH) 4 ) 2 SO 4 100mmol/L potassium phosphate buffer (pH 6.0), 1% methanol.
And (3) measuring the enzyme activity of alkaline pectinase:
the measurement is carried out by spectrophotometry. Taking 10 μ L of the diluted crude enzyme solution with a certain concentration, adding 190 μ L of the diluted crude enzyme solution containing 0.2% pectin and 0.1mM CaCl 2 The enzymatic reaction was started in the buffer (glycine-sodium hydroxide pH 9.5), the reaction was carried out at 55 ℃ for 15min, and then the reaction was terminated by adding 300. Mu.L of 0.03mol/L phosphoric acid, and the absorbance value was measured at 235 nm. The control group is prepared by mixing 300. Mu.L of 0.03mol/LPhosphoric acid and 10. Mu.L of the crude enzyme solution were mixed well, and then a substrate was added for reaction. One standard enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. Mu. Mol unsaturated polygalacturonic acid per minute.
Example 1: plasmid vector construction of recombinant alkaline pectinase and pichia pastoris electric conversion method
(1) Designing a base sequence of a pichia pastoris codon preference optimization coding alkaline pectinase gene pelA and artificially synthesizing, wherein the base sequence is shown as SEQ ID NO 1, inserting the base sequence into a pichia pastoris methanol inducible secretion type plasmid pPIC9K, realizing the insertion of the gene pelA into the pichia pastoris methanol inducible secretion type plasmid pPIC9K by using a double enzyme digestion technology, wherein enzymes used in the double enzyme digestion are EcoRI and NotI respectively, carrying out enzyme ligation after the double enzyme digestion is finished, and transforming the gene pelA into an E.coli DH5 alpha competent cell after purification. The plasmid vector pPIC9K-pelA is successfully constructed by coating the plasmid vector pPIC9K-pelA on an LB (peptone 10g/L, yeast extract 5g/L, naCl 10g/L,20g/L agar powder containing 100ug/mL Amp) plate, and selecting positive transformants for colony PCR identification and gene sequencing.
(2) The sequences of the signal peptides MF4I, HFBI, W1, albumin and Invertase2 are shown in the following table, synthesized by Tianlin biology company, and are subjected to double enzyme digestion with BamHI and EcoR I together with pPIC9K-pelA, recovered through agarose gel, and then connected by Ligation Mix ligase, positive transformants are picked for colony PCR identification and gene sequencing, and plasmid vectors pPIC9K-MF4I-pelA, pPIC9K-HFBI-pelA, pPIC9K-W1-pelA, pPIC9K-Albumin-pelA and pPIC9K-Invertase-pelA are successfully constructed.
(3) Construction of chaperone factor related plasmid: the PCR products of BMH2, HAC1, PDI1 and KEX2 are obtained by obtaining the gene sequences of molecular chaperones BMH2 (GenBank accession number: XM-002490942.1), HAC (GenBank accession number: XP-002490039.1), PDI (GenBank accession number: CAC 33588.1) and KEX2 (GenBank accession number: XM-002491154.1) from NCBI websites, designing primers according to the gene sequences, respectively amplifying by using the genome of P.pastoris as a template, obtaining the PCR products of BMH2, HAC1, PDI1 and KEX2, carrying out single enzyme digestion on the vector APZA by EcoR I, recovering the above gene fragments and the vector fragments by agarose gel, fusing by using an homologous recombination (InfusionZAkitts) kit, picking positive transformants for colony PCR identification and gene identification, and successfully constructing the vectors pGAPH-BMH 2, pGAPC-HAC 1, pGAPZA-PDI1 and KEX2.
(4) After the expression vector pPIC9K-pelA is linearized by Sal I, recovered and concentrated, the electric shock is transformed into Pichia pastoris GS115, and the electric transformation conditions are as follows: the voltage was 1500V, 200. Omega., 25. Mu.F, 5-10ms,2mm. Immediately after electric shock, 1mol/L sorbitol solution in cold was added, followed by shaking pre-incubation at 30 ℃ for 1.5h, and then plating onto MD (agar 20g/L,10mL of 1 XYNB, 40mL of 50% glucose, 2mL of 500 Xbiotin) plates. After 2-3 days, gradually picking transformants in the MD plate to YPD (yeast extract 10G/L, peptone 20G/L and glucose 20G/L) plates containing G418 geneticin different mass concentrations (0.5,1.0,2.0 and 3.0 mg/mL), screening a recombinant strain containing multiple copies of genes, and carrying out colony PCR (polymerase chain reaction) verification to construct a recombinant strain GS 115/alpha-factor-pelA. Similarly, the recombinant bacteria GS115/MF4I-pelA, GS115/HFBI-pelA, GS115/W1-pelA, GS115/Albumin-pelA and GS 115/Invertase-pelA.
(5) Chaperone factor recombinant plasmids pGAPZA-BMH2, pGAPZA-HAC1, pGAPZA-PDI1 and pGAPZA-KEX2 are subjected to single enzyme digestion by AvrII, linearized plasmids are introduced into competent cells of the recombinant bacteria GS115/MF4I-pelA by an electrical transformation method, 1mol/L of sorbitol solution which is exposed to cold is immediately added after electric shock, and then the cells are pre-cultured for 1.5h by placing the cells on a shaker at 30 ℃, and then the cells are coated on a Zeocin plate of 0.1mg/mL and cultured for 2 to 3 days at 28 ℃. Recombinant bacteria GS115/BMH2-MF4I-pelA, GS115/HAC1-MF4I-pelA, GS115/PDI1-MF4I-pelA and GS115/KEX2-MF4I-pelA for obtaining co-expression chaperone factors.
Example 2: enzyme activity determination and protein electrophoresis of coexpression gene engineering strain
The culture method comprises the following steps: the strain is inoculated to a basic fermentation culture medium YPD after the seed activation, is cultured for 24 hours under the conditions of 30 ℃ and 220rpm, is cultured to the OD600 of about 20-50 under the conditions of 30 ℃ and 220rpm of a transferred growth culture medium BMGY, is transferred to an induction culture medium BMMY at 23 ℃ and 220rpm, and is added with 1 percent of methanol every 24 hours to induce the expression of alkaline pectinase.
The enzyme activity determination conditions are as follows:
centrifuging the fermentation liquid at 15000rpm for 10min, collecting extracellular APs in the fermentation supernatant, and detecting. Taking 10 μ L of the diluted crude enzyme solution with a certain concentration, adding 190 μ L of the diluted crude enzyme solution containing 0.2% pectin and 0.1mM CaCl 2 The enzymatic reaction was started in the buffer (glycine-sodium hydroxide pH 9.5), the reaction was carried out at 55 ℃ for 15min, and then the reaction was terminated by adding 300. Mu.L of 0.03mol/L phosphoric acid, and the absorbance value was measured at 235 nm.
The Biyuntian SDS-PAGE gel electrophoresis kit is selected to prepare 10% separation gel and 5% concentrated gel, and the specific operation method is shown in the product specification. Mixing the crude enzyme sample with 5 xSDS sample buffer 4:1, heating in 100 deg.C metal bath for 10min, and centrifuging to obtain supernatant as alkaline pectinase sample. During electrophoresis, the electrophoresis is performed at 180V for 75min. The gel was stained with Coomassie Brilliant blue stain overnight and destained after staining (SDS-PAGE pattern shown in FIG. 2).
Example 3: high-density fermentation culture in 5L fermentation tank
Streaking the high-yield strain on a YPD plate, selecting a single colony, inoculating the single colony in a 5mL YPD test tube, and carrying out shake cultivation at 30 ℃ and 220r/min for 24h; the suspension was inoculated at 1% into 100mL YPD shake flasks and shake-cultured at 30 ℃ and 220r/min until the OD600 was about 20. The seed culture was inoculated into a 5L fermentor at 10% inoculum size. The fermentation conditions were as follows: initial liquid loading amount: 2L, stirring speed of 400r/min, and ventilation volume of 2vvm; the temperature in the growth stage is 30 ℃, ammonia water (30%) and phosphoric acid (30%) are adopted to adjust the pH value to 5.5, and 50%, ( V / V ) The glycerol (2) adopts a coupling mode of dissolved oxygen and rotating speed to maintain DO at about 20%. When the OD600 is more than or equal to about 250, stopping the glycerol feeding. After the strain is starved for 60min, beginning to add methanol for induction expression, reducing the culture temperature to 26 ℃, and increasing the stirring speed to 800rpm and also adopting a dissolved oxygen coupling mode for adding. Sampling is carried out every 12h, and the expression and the enzyme activity of the alkaline pectinase are determined.
When the strain is subjected to shake flask induction fermentation for 96 hours, the enzyme activity of the recombinant strain GS115/KEX2-MF4I-pelA is 732.60U/mL, and is improved by 14.7 times compared with the original strain GS 115/alpha-factor-pelA (49.73U/mL). When the fermentation culture is carried out in a 5L fermentation tank, the maximum enzyme activity of the recombinant bacteria GS115/KEX2-MF4I-pelA reaches 2301.05U/mL (the batch feeding fermentation performance of the recombinant bacteria is shown in figure 3), and the high-efficiency expression of the alkaline pectinase is realized.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Zaozhuang, jie Nuo Bio-enzyme Co Ltd, jiangnan university
<120> recombinant bacterium for efficiently producing alkaline pectinase and application thereof
<130> 2021.8.31
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1203
<212> DNA
<213> Artificial Synthesis
<400> 1
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Claims (3)
1. A method for efficiently producing alkaline pectinase, which comprises the following steps:
connecting the mutant pelA gene and the signal peptide gene of the alkaline pectinase gene to an expression vector pPIC9K to obtain a recombinant plasmid I;
connecting the chaperone factor gene to an expression vector pGAPZA to obtain a recombinant plasmid II;
transforming the recombinant plasmid I into pichia pastoris GS115 to obtain a secretion-enhanced genetic engineering strain;
the recombinant plasmid II is transformed into the secretion-enhanced genetic engineering strain to obtain a recombinant strain for efficiently producing the alkaline pectinase;
the signal peptide is MF4I;
the chaperone factor is KEX2;
the nucleotide sequence of the mutant pelA gene of the alkaline pectinase gene is shown as SEQID NO. 1;
fermenting and culturing the recombinant bacteria for efficiently producing the alkaline pectinase, comprising the following steps: activating the recombinant bacteria, inoculating the activated recombinant bacteria into a growth culture medium YPD, culturing for 24-28h under the conditions of 30-34 ℃ and 220-300rpm, then inoculating into an induction culture medium BMGY according to an inoculation rate of 1-1.5%, and performing shake culture at 30-34 ℃ and 220-300rpm until OD600 is 18-24; then transferring the cells into an induction culture medium BMMY, and adding 1-1.5% methanol every 24-28h at 23-25 ℃ and 220-240rpm to induce the expression of alkaline pectinase;
the culture medium YPD is counted by 1L: 1% yeast powder, 2% peptone and 2% glucose; the culture medium BMGY, calculated as 1L: 1% yeast extract, 2% peptone, 0.34% YNB,1% (NH) 4 ) 2 SO 4 100mmol/L potassium phosphate buffer pH 6.0,1% glycerol.
2. The method of claim 1, wherein the methanol induction stage employs a dissolved oxygen coupled fedbatch approach: DO is maintained at 10-20%.
3. The method for producing alkaline pectinase by using the recombinant bacteria for efficiently producing alkaline pectinase as defined in claim 1, wherein the activated bacteria liquid is inoculated into a fermentation medium with a liquid loading amount of 20-30%, the initial stirring speed is 400-550rpm, the ventilation amount is 1.5-2vvm, the pH is controlled to be 5.5-6.0, and the culture temperature in the growth phase is 28-30 ℃; supplementing glycerol when the dissolved oxygen of the glycerol is exhausted and the glycerol rebounds, and maintaining the DO at 20-30% by adopting a dissolved oxygen and rotating speed coupling mode; when the glycerol is used up again and the dissolved oxygen rebounds, starvation culture is carried out for 0.5-2h, the induction culture medium is fed, the temperature is reduced to 24-26 ℃, the stirring speed is increased to 700-900rpm, and the alkaline pectinase expression is induced.
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Non-Patent Citations (3)
| Title |
|---|
| Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide;WANG Ye等;《J. Cent. South Univ.》;20151231;第22卷;2088-2095 * |
| 信号肽序列对毕赤酵母表达外源蛋白质的影响;熊爱生等;《生物化学与生物物理学报》;20031231;第35卷(第2期);154-160 * |
| 枯草芽孢杆菌来源的碱性果胶酶在毕赤酵母中的高效表达;陈洲等;《食品科技》;20221231;第47卷(第02期);1-9 * |
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