CN105802867B - A kind of alkaline pectase secretes enhanced bacterial strain and its application - Google Patents
A kind of alkaline pectase secretes enhanced bacterial strain and its application Download PDFInfo
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- CN105802867B CN105802867B CN201610343046.2A CN201610343046A CN105802867B CN 105802867 B CN105802867 B CN 105802867B CN 201610343046 A CN201610343046 A CN 201610343046A CN 105802867 B CN105802867 B CN 105802867B
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Abstract
The invention discloses a kind of alkaline pectases to secrete enhanced bacterial strain and its application, belongs to gene engineering technology field.The UPR Cloning of Genes Related of Pichia pastoris is connected to yeast expression vector pPGAZA using gene recombination technology by the present invention, and it converts into Pichia pastoris GS115-pPIC9K-PGL bacterial strain, obtain the bacterial strain GS115-PGL/pGAPZA-HAC1 of several plants of more original bacterial strain enhancing secreting, expressing alkaline pectases, GS115-PGL/pGAPZA-ERO1, GS115-PGL/pGAPZA-UBC1 etc., compared to using the bacterial strain enzyme activity before this method to be respectively increased 24.1%, 35.5%, 22.3%, it is significantly improved, it haves laid a good foundation for the large-scale production of alkaline pectase.Alkaline pectase of the invention can be catalyzed the α by trans-elimination polygalacturonic acid-Isosorbide-5-Nitrae glycosidic bond cracking under alkaline condition, be widely used in the industries such as food, textile and paper.
Description
Technical field
The present invention relates to a kind of alkaline pectases to secrete enhanced bacterial strain and its application, belongs to gene engineering technology field.
Background technique
Pectase is a kind of complex enzyme, and Pectin polymers can be resolved into unsaturated oligogalacturonans.The enzyme point
Cloth is extensive, is found in section parasitic nematode, plant and microorganism.Pectase is widely used, and has industry in more than 40 years
Using history.Pectase is divided into acid pectase and alkaline pectase PGL according to the difference of optimal reaction pH.Wherein acid pectin
Enzyme is mainly used in clear juice fruit wine, extracts juice, fruit decortication etc..PGL application is mainly used in weaving, food
Product, paper industry and environmental area.Acting on above-mentioned field correlated response using enzyme process has environmental protection, saves raw material consumptive material and reaction
The advantages that mild condition.However it is less to PGL progress molecular modification research at present, the PGL being commercialized is also seldom.
Studying more deep bacterial strain to alkaline pectase at present is mainly Pichia pastoris, bacillus subtilis and large intestine bar
Bacterium.The different hosts that can express alkaline pectase are comprehensively compared, Pichia anomala expression albumen is easy to purify, and yield is high, but heterologous
Albumen overexpression will lead to growth stress pressure and cause unfolded protein effect (UPR), lead to the yield of alkaline pectase not
It can be further improved, limit the industrialized production of alkaline pectase.
Summary of the invention
The invention solves first technical problem be to provide a kind of alkaline pectase and secrete enhanced bacterial strain.
The alkaline pectase secrete enhanced bacterial strain by by Pichia pastoris HAC1, ERO1, BMH2, GCN4, UBC1,
HRD1, SSO2, SEC53, PDI or GSH2 gene are connected to yeast expression vector pGAPZA, and convert to recombinant expression alkali
In the Pichia pastoris of property pectase.
The Pichia pastoris GS115-pPIC9K-PGL of the recombinant expression alkaline pectase is by core
Nucleotide sequence alkaline pectinase gene as shown in SEQ ID NO.1, is connected on Expression vector pPIC9K, is then transformed into complete
Obtained in red yeast GS115.
The present invention also provides the construction methods that above-mentioned alkaline pectase secretes enhanced bacterial strain, and steps are as follows:
(1) using Pichia pastoris GS115 reverse transcription cDNA as template, target gene is obtained by PCR method；
(2) target gene that step (1) obtains is connected on yeast expression vector pGAPZA, obtains recombination matter
Grain；
(3) Pichia pastoris for the recombinant plasmid transformed recombinant expression alkaline pectase that step (2) obtain secretion is obtained to increase
Strong type engineering strain.
The present invention also provides the methods of the application recombinant bacterium fermentation production of alkaline pectic enzyme.
In one embodiment of the invention, the method is that growth medium BMGY is seeded to after activating recombinant bacterium
In cultivated under the conditions of 30 DEG C, 220rpm for 24 hours, it is every under 23 DEG C, 220rpm in the induced medium BMMY that then transfers again
The expression of the methanol induction alkaline pectase of addition 1.5% for 24 hours.The growth medium BMGY (1L): peptone 20g, yeast
Powder 10g, glycerol 40g, YNB13.4g, with the phosphate buffer tune pH to pH6.0 of the 0.1M of pH6.0.The induced medium
BMMY (1L): peptone 20g, yeast powder 10g, methanol 9%, YNB 13.4g, with the phosphate buffer tune of the 0.1M of pH6.0
PH to pH6.0.
The utility model has the advantages that for existing engineering strain Pichiapastoris GS115-pPIC9K-PGL,
Engineering strain Pichiapastoris GS115-pPIC9K-PGL/pGAPZA-HAC1 enzyme activity of the invention improves
24.1%, Pichia pastoris GS115-pPIC9K-PGL/pGAPZA-ERO1 enzyme activity improve 35.5%, Pichia
Pastoris GS115-pPIC9K-PGL/pGAPZA-UBC1 enzyme activity improves 22.3%.Alkaline pectase of the invention can be
Catalysis is widely used in food, is spun by α-Isosorbide-5-Nitrae glycosidic bond cracking of trans-elimination polygalacturonic acid under alkaline condition
It knits and the industries such as papermaking.
Detailed description of the invention
Fig. 1: clonal expression plasmid schematic diagram.
Fig. 2: alkaline pectase SDS-PAGE analysis；1-5 swimming lane respectively represents GS115-pPIC9K sky, GS115-
pPIC9K-PGL/pGAPZA-HAC1、GS115-pPIC9K-PGL、GS115-pPIC9K-PGL/pGAPZA-UBC1、GS115-
pPIC9K-PGL/pGAPZA-ERO1。
Fig. 3: the performance comparison of coexpression recombinant bacterial strain fermentation production of alkaline pectic enzyme.
Specific embodiment:
Culture medium:
Seed culture medium YPD: tryptone 20g/L, yeast powder 10g/L, glucose 20g/L.
Growth medium BMGY (1L): peptone 20g, yeast powder 10g, glycerol 40g, YNB13.4g, with the 0.1M of pH6.0
Phosphate buffer tune pH to pH6.0.
Induced medium BMMY (1L): peptone 20g, yeast powder 10g, methanol 9%, YNB13.4g, with the 0.1M of pH6.0
Phosphate buffer tune pH to pH6.0.
Alkaline pectase enzyme activity determination:
It is measured using spectrophotometry.The definition of unit enzyme activity: the unit time cracks polygalacturonic acid and generates 1 μm of ol not
It is saturated enzyme amount used in polygalacturonic acid.Enzyme activity determination condition are as follows: enzyme activity detection: fermentation liquid 8000rpm is centrifuged 10min,
Extracellular PGL is contained among fermented supernatant fluid, takes a certain amount of detect.PGL reaction system: contain 0.2% polygalacturonic acid
Glycine-NaOH buffer (the 0.2molL of (substrate)-1, 0.44mmolL-1CaCl2, pH9.4) and 2mL, sample to be tested 20
μ L, inactive enzyme solution are blank control.PGL reaction condition are as follows: reaction system is placed in water-bath 15min at 45 DEG C, with 3mL phosphorus
Acid solution (0.03molL-1) reaction is terminated, absorbance value is measured at 235nm.
Embodiment 1: the building and identification of recombinant bacterium
Pichia pastoris GS115 RNA, reverse transcription cDNA are extracted, using cDNA as template, design primer passes through the method for PCR
It obtains UPR related gene (HAC1, ERO1, SLY1, BMH2, GCN4, UBC1, HRD1, SSO2, SEC53, BIP, PDI, GSH2),
It is cloned on expression vector pGAPZA, obtaining recombinant plasmid pGAPZA-X, (clonal expression plasmid schematic diagram is shown in attached drawing 1, X generation
Table HAC1, ERO1, SLY1, BMH2, GCN4, UBC1, HRD1, SSO2, SEC53, BIP, PDI or GSH2), recombinant vector is converted
Pichiapastoris GS115-pPIC9K-PGL obtains coexpression recombinant bacterial strain Pichiapastoris through screening and identification
GS115-pPIC9K-PGL/pGAPZA-X。
The Pichia pastoris GS115-pPIC9K-PGL of the recombinant expression alkaline pectase is by core
Nucleotide sequence alkaline pectinase gene as shown in SEQ ID NO.1, is connected on Expression vector pPIC9K, is then transformed into complete
Obtained in red yeast host bacterial strain GS115.
Primer is as follows:
The conversion of Pichia pastoris uses electrotransformation.
Specific step is as follows: the single colonie of picking yeast recipient bacterium is inoculated in 25mLYPD fluid nutrient medium, 30 DEG C of shaking tables
Overnight；With 5% inoculum concentration switching 50mLYPD fluid nutrient medium, 30 DEG C of shaking table cultures to OD=1.3-1.5；4 DEG C of centrifugations,
5000rpm, 5min abandon supernatant；Thallus is resuspended with 50mL ice pre-cooling sterile water；4 DEG C of centrifugations 5000rpm, 5min abandon supernatant；
Thallus is resuspended with 25mL ice pre-cooling sterile water；4 DEG C of centrifugations 5000rpm, 5min abandon supernatant；It is pre- with the ice of 5mL 1mol/L again
Cold sorbitol washes 1 time are resuspended, 4 DEG C, and 5000rpm is centrifuged 5min, abandon supernatant；The ice pre-cooling of appropriate volume 1mol/L is added
Sorbierite, be resuspended；Packing is into sterile EP tube, every 80 μ l of pipe, in case conversion.The coexpression vector pGAPZA-X of extraction is used
Enzyme AvrII linearisation is added the plasmid 1-5 μ g linearized with suitable restriction enzyme site in 80 μ l competent yeast cells and puts on ice
It sets 15 minutes, is rapidly added in 0.2cm electric shock cup (ice pre-cooling), 1500v electric shock is rapidly added the sorbierite of 1ml ice pre-cooling, applies
YPDS plate containing 200 μ g/ml Zeocin, picking monoclonal after culture 3-4 days.
Embodiment 2: the enzyme activity determination and protein electrophoresis of co-expression gene engineered strain
Cultural method: bacterial strain is inoculated into basic fermentation medium YPD after seed activation, under the conditions of 30 DEG C, 220rpm
Cultivate 14h, the growth medium BMGY culture after being forwarded to optimization based on 30 DEG C, cultivate for 24 hours under the conditions of 220rpm, then by bacterial strain
23 DEG C, methanol of the 220rpm per addition 1.5% for 24 hours, the expression of inducible alkaline pectase are transferred in induced medium BMMY.
Enzyme activity determination condition are as follows: fermentation liquid 8000rpm is centrifuged 10min, and extracellular PGL is contained among fermented supernatant fluid,
Take a certain amount of detect.PGL reaction system: contain the glycine-NaOH buffer of 0.2% polygalacturonic acid (substrate)
(0.2mol·L-1, 0.44mmolL-1CaCl2, pH9.4) and 2mL, 20 μ L of sample to be tested, inactive enzyme solution is blank pair
According to.PGL reaction condition are as follows: reaction system is placed in water-bath 15min at 45 DEG C, with 3mL phosphoric acid solution (0.03molL-1) terminate
Reaction measures absorbance value at 235nm.
Green skies PAGE gel electrophoresis kit is selected to prepare 12% separation gel and 5% concentration glue, concrete operations side
Method is shown in product description.Sample is mixed with 5 × sample-loading buffer with volume ratio 4:1, boiling water bath 10min, loading after cooling.Electrophoresis
When, 80V constant-voltage, after to be instructed dose enters separation gel, voltage is adjusted to 150V, to be instructed dose to glue bottom when terminate electrophoresis.With
Coomassie brilliant blue staining liquid dyes gel, dyes 1h rear decoloring (SDS-PAGE map is shown in attached drawing 2).
Embodiment 3: the purifying of alkaline pectase
Will recombination fermented liquid 8000r/min be centrifuged 20min, take supernatant, add ammonium sulfate carry out gradient saltout, low temperature from
The heart collects 30~50% ammonium sulfate precipitation parts, and the enzyme of salt precipitation is dissolved in Glycine-NaOH buffer solution
(pH7.5) in, for 24 hours with 20mmol/L Glycine-NaOH buffer solution dialysis treatment.Centrifugation gained supernatant is through cation
Displacement chromatography is further isolated and purified.
Compared to than the starting strain Pichia pastoris GS115-pPIC9K-PGL before coexpression UPR related gene
Shaking flask induction fermentation 96h enzyme activity is 301.32U/ml, secretes enhanced recombinant bacterium Pichia pastoris in the present invention
GS115-PGL/pGAPZA-HAC1 enzyme activity is that 373.94U/ml improves 24.1%, Pichia pastoris GS115-PGL/
PGAPZA-ERO1 enzyme activity 408.27U/ml improves 35.5%, Pichia pastoris GS115-PGL/pGAPZA-UBC1 enzyme
368.54U/ml living improves 22.3%, and other some coexpression recombinant bacterial strains also improve or reducing effect is (total
The performance comparison of expression recombinant bacterial strain fermentation production of alkaline pectic enzyme is shown in attached drawing 3).
Claims (6)
1. a kind of alkaline pectase secretes enhanced bacterial strain, which is characterized in that complete by the way that Pichia pastoris ERO1 gene to be connected to
Red Yeast expression carrier pGAPZA, and convert into the Pichia pastoris of recombinant expression alkaline pectase and obtain；The recombinant expression
The Pichia pastoris of alkaline pectase is that nucleotide sequence alkaline pectinase gene as shown in SEQ ID NO.1 is connected to table
Up on carrier pPIC9K, then it is transformed into obtained in Pichia pastoris GS115Pichia pastoris GS115-pPIC9K-
PGL。
2. the method that alkaline pectase described in a kind of building claim 1 secretes enhanced bacterial strain, which is characterized in that step is such as
Under:
(1) using Pichia pastoris GS115 reverse transcription cDNA as template, ERO1 gene is obtained by PCR method；
(2) the ERO1 gene that step (1) obtains is connected on yeast expression vector pGAPZA, obtains recombinant plasmid；
(3) Pichia pastoris for the recombinant plasmid transformed recombinant expression alkaline pectase that step (2) obtain is obtained secreting enhanced
Engineering strain.
3. a kind of method for secreting enhanced bacterial strain production alkaline pectase using alkaline pectase described in claim 1.
4. according to the method described in claim 3, it is characterized in that, being seeded to after recombinant bacterium is activated in growth medium BMGY
24 h are cultivated under the conditions of 30oC, 220 rpm, it is every under 23oC, 220 rpm in the induced medium BMMY that then transfers again
The expression of the methanol induction alkaline pectase of 24 h addition 1.5%.
5. according to the method described in claim 4, it is characterized in that, the growth medium BMGY contains based on every liter: albumen
20 g of peptone, 10 g of yeast powder, glycerol 40 g, YNB13.4 g, with the phosphate buffer tune pH to pH6.0 of the 0.1M of pH6.0；
The induced medium BMMY contains based on every liter: 20 g of peptone, 10 g of yeast powder, methanol 9%, 13.4 g of YNB, with
The phosphate buffer tune pH to pH6.0 of the 0.1M of pH6.0.
6. alkaline pectase described in claim 1 secretes application of the enhanced bacterial strain in food, weaving, environmental protection or papermaking.
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| CN106520587B (en) * | 2016-10-18 | 2019-03-15 | 江南大学 | It is a kind of produce alkaline pectase recombinant bacterium and its application |
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