CN105754884A - Strain capable of efficiently expressing alkaline pectinase and application of strain - Google Patents
Strain capable of efficiently expressing alkaline pectinase and application of strain Download PDFInfo
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- CN105754884A CN105754884A CN201610170070.0A CN201610170070A CN105754884A CN 105754884 A CN105754884 A CN 105754884A CN 201610170070 A CN201610170070 A CN 201610170070A CN 105754884 A CN105754884 A CN 105754884A
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- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02002—Pectate lyase (4.2.2.2)
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Abstract
The invention discloses a strain capable of efficiently expressing alkaline pectinase and an application of the strain and belongs to the technical field of gene engineering. Alkaline pectinase genes are expressed in Pichia pastoris GS115 with a gene recombination technology, and the strain GS115-PIC9K-PGL is obtained and has a remarkably improved yield by comparison with the yield before sequence optimization. According to the strain, the enzyme activity of the alkaline pectinase fermented for 72 h in a shaking flask is 301.9 U/ml and is improved by about 25%, and a good basis is laid for large-scale production of the alkaline pectinase. The alkaline pectinase produced with the strain can catalyze alpha-1,4 glycosidic bonds of polygalacturonic acid to be pyrolysed through trans-elimination under the alkaline condition and is widely applied to food, textile, papermaking and other industries.
Description
Technical field
The present invention relates to bacterial strain and the application thereof of a kind of high efficient expression alkaline pectase, belong to gene engineering technology field.
Background technology
Pectase is a kind of compound enzyme, it is possible to Pectin polymers resolves into unsaturated oligogalacturonans.This enzyme is widely distributed,
It is found in section parasitic nematicide, plant and microorganism.Pectase is widely used, the commercial Application history of existing more than 40 year.
Pectase is divided into acid pectase and alkaline pectase PGL by the difference according to optimal reaction pH.Wherein acid pectase is main
It is applied to the aspects such as clear juice fruit wine, extraction fruit and vegetable juice, fruit decortication.PGL application is mainly used in weaving, food, makes
Paper industry and environmental area.Enzyme process effect above-mentioned field correlated response has environmental protection, economize in raw materials consumptive material and reaction condition temperature in application
With etc. advantage.But it is less that PGL carries out molecular modification research at present, the PGL carrying out commercialization is the most little.
The host being presently expressed by alkaline pectase is mainly Pichia sp., bacillus subtilis and escherichia coli.Although by fermentation
The means such as optimization can be effectively improved the yield of alkaline pectase, but when the yield reaching certain limit alkaline pectase can not enter one
Step improves, and limits the industrialized production of alkaline pectase, therefore need to solve to limit the factor of alkaline pectin expression of enzymes from source.
Although Pichia sp. host has expressing protein is prone to the advantages such as purification, but current alkaline pectase is in Pichia sp.
During expression, have that expression is the highest or exogenous gene original nucleotide sequences is not very suitable for asking of Pichia sp. host
Topic, thus limit the alkaline pectase high efficient expression in Pichia sp..
Therefore, it is necessary to improve alkaline pectase further to produce the production capacity of bacterial strain more to adapt to industrialized needs.
Summary of the invention
In order to solve the problems referred to above, the invention provides the alkaline pectase of a kind of optimization, and express finishing of this alkaline pectase
Red yeast gene engineering bacteria.
Described alkaline pectinase gene nucleotide sequence sequence as shown in SEQ ID NO.1.
Described pichia yeast genetic engineering bacteria, is nucleotide sequence alkaline pectinase gene as shown in SEQ ID NO.1 to be connected
On Expression vector pPIC9K, then it is transformed into and Pichia sp. host strain obtains.
In one embodiment of the invention, described expression vector is pPIC9K.
In one embodiment of the invention, described Pichia sp. host is Pichiapastoris GS115.
The present invention also provides for the construction method of a kind of described pichia yeast genetic engineering bacteria, and step is as follows:
(1) use chemistry complete synthesis codon optimized after alkaline pectinase gene PGL;
(2) alkaline pectinase gene that step (1) obtains is connected on yeast expression vector pPIC9K, is recombinated
Plasmid pPIC9K-PGL;
(3) it is excellent that recombiant plasmid pPIC9K-PGL conversion Pichia pastoris GS115 step (2) obtained obtains codon
The engineering strain expressing alkaline pectase changed.
The present invention also provides for a kind of method utilizing described engineering bacteria fermentation to produce alkaline pectase, is to be lived by genetic engineering bacterium
Be seeded to after change in growth medium BMGY in 30 DEG C, cultivate 24h under the conditions of 220rpm, transfer the most again into inducing culture
In base BMMY, in 23 DEG C, shake flask fermentation, the expression of inducible alkaline pectase under the conditions of 220rpm.
In one embodiment of the invention, described activation is to be inoculated in seed culture medium YPD cultivation 14h;Again with 5%
Inoculum concentration proceed to growth medium BMGY.
In one embodiment of the invention, during described induction, every 24h adds the methanol of 1.5% (v/v).
The present invention is also claimed the application in terms of food, weaving or papermaking of the described genetic engineering bacterium.
Beneficial effects of the present invention:
For the original engineering strain before gene optimization, the engineering strain Pichia pastoris of the present invention
GS115-pPIC9K-PGL enzyme is lived and is improve 25%, and the enzyme in shaking flask level is lived and be can reach 301.9U/ml (72h), is existing
Top level in the Pichia sp. having been reported that, higher enzyme work can be realized by tank fermentation and medium optimization on fermentation tank,
Large-scale production for alkaline pectase is had laid a good foundation.The alkaline pectase that the present invention obtains can be urged in the basic conditions
Change and cracked by the α of trans-elimination polygalacturonic acid-Isosorbide-5-Nitrae glycosidic bond, be widely used in the industry such as food, textile and paper.
Figure of description
Fig. 1: clonal expression plasmid map;
Fig. 2: alkaline pectase SDS-PAGE analyzes;1-3 swimming lane represents GS115-pPIC9K, GS115-pPIC9K-PGL respectively
After optimization, before GS115-pPIC9K-PGL optimization;
The performance comparison of the alkaline pectase that strain fermentation produces before and after the optimization of Fig. 3: alkaline pectinase gene.
Detailed description of the invention:
Culture medium:
Seed culture medium YPD (1L): tryptone 20g, yeast powder 10g, glucose 20g.
Growth medium BMGY (1L): peptone 20g, yeast powder 10g, glycerol 40g, YNB 13.4g, pH6.0's
The phosphate buffer of 0.1M.
Inducing culture BMMY (1L): peptone 20g, yeast powder 10g, methanol 9%, YNB 13.4g, pH6.0's
The phosphate buffer of 0.1M.
Alkaline pectase enzyme activity determination:
Use spectrophotometry.Unit enzyme is lived and is defined: unit interval cracking polygalacturonic acid produces the unsaturation of 1 μm ol
Enzyme amount used by polygalacturonic acid.Enzyme activity determination condition is: enzyme activity detects: fermentation liquid 8000rpm is centrifuged 10min, born of the same parents
Outer PGL is i.e. contained among fermented supernatant fluid, takes a certain amount of detection.PGL reaction system: containing 0.2% polygalacturonic acid
Glycine-NaOH buffer (the 0.2mol L of (substrate)-1, 0.44mmol L-1CaCl2, pH9.4) 2mL, to be measured
Sample 20 μ L, inactive enzyme liquid is blank.PGL reaction condition is: reaction system is placed in water-bath 15min at 45 DEG C,
With 3mL phosphoric acid solution (0.03mol L-1) terminate reaction, at 235nm, measure absorbance.
Embodiment 1: the structure of recombinant bacterium and qualification
Alkaline pectinase gene PGL of chemistry complete synthesis sequence such as SEQ ID NO.1, is redesigned primer, is obtained by the method for PCR
Alkaline pectinase gene PGL that must optimize, is cloned on Expression vector pPIC9K, it is thus achieved that recombiant plasmid pPIC9K-PGL
(clonal expression plasmid map is shown in accompanying drawing 1), converts Pichiapastoris GS115 by recombinant vector, obtains restructuring through Screening and Identification
Bacterial strain Pichiapastoris GS115-pPIC9K-PGL.
Primer is following (sequence is respectively as shown in SEQ ID NO.2 and SEQ ID NO.3):
PGL upstream: GCTGAAGCTTACGTAGAATTCGCTGATTTGGGTCATCAAACACTTG
PGL downstream: AAGGCGAATTAATTCGCGGCCGCTTAGTTCAATTTTCCAGCACCTGCT
The conversion of Pichia sp. uses electrotransformation.
Specifically comprise the following steps that single colony inoculation of picking yeast recipient bacterium in 25mLYPD fluid medium, 30 DEG C of shaking table mistakes
Night;With 5% inoculum concentration switching 50mL YPD fluid medium, 30 DEG C of shaking tables are cultivated to OD600=1.3-1.5;4 DEG C centrifugal 5000
Rpm, 5min, abandon supernatant;With 50mL ice pre-cooling sterilized water, thalline is resuspended;4 DEG C of centrifugal 5000rpm, 5min, abandon supernatant;
With 25mL ice pre-cooling sterilized water, thalline is resuspended;4 DEG C of centrifugal 5000rpm, 5min, abandon supernatant;Again with 5mL 1mol L-1's
The sorbitol washes of ice pre-cooling 1 time, resuspended, 4 DEG C, 5000rpm is centrifuged 5min, abandons supernatant;Add appropriate volume 1mol L-1
The sorbitol of ice pre-cooling, resuspended;In subpackage extremely aseptic EP pipe, often pipe 80ul, in case converting.The coexpression vector that will extract
PPIC9K-PGL enzyme Sal I linearisation, adds linearizing plasmid 1-5 μ g in 80 μ l competent yeast cells and places on ice
15 minutes, being rapidly added in 0.2cm electric shock cup (ice pre-cooling), 1500v shocks by electricity, is rapidly added the sorbitol of 1mL ice pre-cooling,
Being coated with MD flat board, after cultivating 3-4 days, picking monoclonal carries out next step screening.
Embodiment 2: the alkaline pectase enzyme activity determination of engineering strain and protein electrophoresis
Cultural method: bacterial strain is inoculated into basic fermentation medium YPD after seed activation, in 30 DEG C, cultivate under the conditions of 220rpm
14h, be forwarded to the growth medium BMGY after optimizing cultivate based on 30 DEG C, cultivate 24h under the conditions of 220rpm, then by bacterial strain
Proceeding to 23 DEG C, 220rpm in inducing culture BMMY, every 24h adds the methanol of 1.5%, the expression of inducible alkaline pectase.
Selecting green skies PAGE gel electrophoresis kit to prepare 12% separation gel and 5% and concentrate glue, concrete operation method is shown in product
Product description.Sample and 5 × sample-loading buffer mix with volume ratio 4:1, boiling water bath 10min, loading after cooling.During electrophoresis, 80V
Constant-voltage, after to be instructed dose enters separation gel, voltage is adjusted to 150V, and to be instructed dose to terminating electrophoresis time at the bottom of glue.Use coomassie
Gel is dyeed by light blue dyeing liquor, dyeing 1h rear decoloring (accompanying drawing 2 is shown in by SDS-PAGE collection of illustrative plates).
Determine the alkaline pectase enzyme in the fermentation liquid that above-mentioned shake-flask culture method obtains to live simultaneously.Result shows, expresses SEQ
The recombinant bacterium Pichiapastoris GS115-pPIC9K-PGL of the alkaline pectinase gene of ID NO.1, the enzyme work in shaking flask is
301.9U/ml (72h), than the recombinant bacterium enzyme of the alkaline pectinase gene (sequence is as shown in SEQ ID NO.4) expressed before optimizing
Live 242.6U/ml (72h) improve about 25% and Biomass be basically unchanged (Fig. 3).The present invention is the big of alkaline pectase
Large-scale production is had laid a good foundation.
Additionally, inventor has also tested the alkaline pectinase gene after other multiple sequence optimisation, with Pichiapastoris GS115,
PPIC9K is vector construction recombinant bacterium, it was found that expression effect is not as the present invention, such as expresses the base of SEQ ID NO.5
The recombinant yeast pichia pastoris of cause, alkaline pectase enzyme under the same conditions is lived and is only 253.2U/ml (72h), is far smaller than this
Bright.
Embodiment 3: the purification of alkaline pectase
Recombinant bacterium fermentation liquid 8000r/min is centrifuged 20min, takes supernatant, add ammonium sulfate and carry out gradient and saltout, low-temperature centrifugation
Collection 30~50% ammonium sulfate precipitation part, be dissolved in the enzyme of salt precipitation in Glycine-NaOH buffer solution (pH7.5),
By 20mmol/L Glycine-NaOH buffer solution dialysis treatment 24h.Centrifugal gained supernatant enters through cation-exchange chromatography
Row is the most isolated and purified.
Claims (8)
1. a pichia yeast genetic engineering bacteria, it is characterised in that described genetic engineering bacterium expresses nucleotide sequence such as SEQ ID
Alkaline pectinase gene shown in NO.1.
Genetic engineering bacterium the most according to claim 1, it is characterised in that described genetic engineering bacterium is with Pichia pastoris
GS115 is that host builds and obtains.
Genetic engineering bacterium the most according to claim 1, it is characterised in that described genetic engineering bacterium is with pPIC9K as table
Reach carrier, Pichia pastoris GS115 is host expresses alkaline pectinase gene.
4. the method for a fermentation production of alkaline pectic enzyme, it is characterised in that be to utilize the genetic engineering bacterium described in claim 1.
Method the most according to claim 4, it is characterised in that described method is raw by being seeded to after genetic engineering bacterium activation
In long culture medium BMGY in 30 DEG C, cultivate 24h under the conditions of 220rpm, transfer in inducing culture BMMY the most again,
In 23 DEG C, the expression of inducible alkaline pectase under 220rpm.
Method the most according to claim 5, it is characterised in that in Induction Process, every 24h adds the methanol of 1.5%.
7. the genetic engineering bacterium of claim 1 produces the alkaline pectase obtained.
8. the alkaline pectase of claim 7 application in terms of food, weaving, environment or papermaking.
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Cited By (3)
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CN106399337A (en) * | 2016-10-18 | 2017-02-15 | 江南大学 | Alkaline pectinase producing recombinant bacteria and application thereof |
CN106399286A (en) * | 2016-10-18 | 2017-02-15 | 江南大学 | Method for promoting Pichia pastoris to produce alkaline pectinase |
CN106520587A (en) * | 2016-10-18 | 2017-03-22 | 江南大学 | Recombinant strain producing alkaline polygalacturonate lyase and application thereof |
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CN103966195A (en) * | 2014-05-07 | 2014-08-06 | 江南大学 | Alkaline pectinase mutant with improved specific and enzyme activity |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106399337A (en) * | 2016-10-18 | 2017-02-15 | 江南大学 | Alkaline pectinase producing recombinant bacteria and application thereof |
CN106399286A (en) * | 2016-10-18 | 2017-02-15 | 江南大学 | Method for promoting Pichia pastoris to produce alkaline pectinase |
CN106520587A (en) * | 2016-10-18 | 2017-03-22 | 江南大学 | Recombinant strain producing alkaline polygalacturonate lyase and application thereof |
CN106399286B (en) * | 2016-10-18 | 2019-12-24 | 江南大学 | Method for promoting pichia pastoris to produce alkaline pectinase |
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