CN103966194A - PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance - Google Patents

PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance Download PDF

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CN103966194A
CN103966194A CN201410190780.0A CN201410190780A CN103966194A CN 103966194 A CN103966194 A CN 103966194A CN 201410190780 A CN201410190780 A CN 201410190780A CN 103966194 A CN103966194 A CN 103966194A
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pgl
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alkaline
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CN103966194B (en
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陈坚
堵国成
刘松
汪明星
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Jiangnan University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • 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 PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance and belongs to the technical field of bioengineering. A 58-th isoleucine I point of PGL deriving from Bacillus sp.WSHB04-02 is mutated into valine V. The extracellular enzyme activity of the mutated PGL is improved to 337.58 U/mL, and is 2.60 times that of the PGL which is not mutated, and the expression quantity of extracellular secretion is obviously increased. In addition, the optimum reaction temperature of the mutant is improved by 5 DEG C, and the optimum enzyme activity can be better shown under the condition of high temperature; Km is obviously improved, the capacity of the mutant in combination with a substrate is improved to be 4.5 times than that of the mutant which is not mutated, and kcat, t1/2,kcat/Km and the like are improved. The enzymatic property of the PGL provided by the invention is greatly improved, and requirements for industrial production and social production are met.

Description

The alkaline pectin enzyme mutant that a kind of secernment property improves
Technical field
The present invention relates to the alkaline pectin enzyme mutant that a kind of secernment property improves, belong to technical field of bioengineering.
Background technology
Alkaline pectase (E.C.4.2.2.2 is called for short PGL), full name polygalacturonic acid lyase, this enzyme is extensively present in bacterium, yeast, fungi, plant and and some parasitic nematode.Alkaline pectase is widely used in the every field such as food, weaving, papermaking, environment, biotechnology, extracts, plays a great role containing pectin Industrial Wastewater Treatment etc. at tea and coffee fermentation, weaving and vegetable fibre processing, oil.Alkaline pectase has high reactivity under alkaline condition, can utilize trans-elimination to cut off the α-Isosorbide-5-Nitrae-glycosidic link of poly lactose aldehydic acid, and pectin substance is decomposed into undersaturated oligogalacturonans.
Contain a lot of accompanying impurities in the most surface of cotton fibre primary cell wall, as: the non-cellulose class materials such as pectin substance, wax, nitrogenous substances and protein, inclusion becomes complicated huge hydrophobic reticulated structure mutually.In textile industry, comprise refinement step, its object is exactly the impurity of removing on cotton fibre, improves the moisture absorbing of cotton fabric, then carries out following process.Traditional chemistry weaving method of refining instant heating alkaline process, it need to consume a large amount of chemical and water resources, and environmental pollution is serious, and according to statistics, in dyeing process, approximately 70% waste water produces in this step.In addition, hot alkali treatment will cause major injury to fiber, and physical strength will decline to a great extent.
In recent ten years, along with the swift and violent fast development of molecular biology and industrial microorganism research, investigator starts to adopt genetic engineering means to build recombinant bacterium, to realizing the excessive recombinant expressed of alkaline pectase.At present, in succession there is utilizing the report as host expresses different sources polygalacturonase gene such as Escherichia coli, Bacillus subtilis and Pichia pastoris.Now the research of alkaline pectase is mainly concentrated on to synthetic alkaline pectase engineering bacteria and fermenting process control.
Nearly ten years, the research direction of external alkaline pectase focuses on the aspects such as separation and purification, enzymatic property and the research of protein structure feature thereof of enzyme, China mainly concentrates on the improvement aspect of strain improvement, zymotechnique and application art to the research of alkaline pectase, still lack the commodity alkaline pectase of tiring very high, for further realizing industrialization place mat, the research of this respect can not be ignored.
Summary of the invention
First technical problem that the present invention will solve is to provide the alkaline pectin enzyme mutant that a kind of secernment property improves.Described mutant be come from Bacillus sp.WSHB04-02 the alkaline pectase (wild-type) of aminoacid sequence as shown in SEQ ID NO.2 for basis, be α-amino-isovaleric acid V by the Isoleucine I point mutation of the 58th.
Second technical problem that the present invention will solve is to provide the method that builds described alkaline pectin enzyme mutant, is to be undertaken expressing after rite-directed mutagenesis by the gene of design primer pair coding alkaline pectase.
Described construction process, preferably suddenly change taking plasmid pET-20b (+)-pgl as template design primer and the plasmid conversion after sudden change is entered to E.coli JM109 and increase, then express the recombinant plasmid containing mutator gene with E.coli BL21 (DE3), obtain the alkaline pectin enzyme mutant that secernment property improves.
The construction process of described pET-20b (+)-pgl is referring to document: Overproduction of alkaline polygalacturonatelyase in recombinant Escherichia coli by a two-stage glycerol feeding approach.2011, ShuyingFang et al.Volume102, Issue22, p10671 – 10678.
The described primer for rite-directed mutagenesis is:
pglI58V primer1：5'-AATCATTTATGTTAAGGGAACGATT-3'
pglI58V primer2：5'-TTTGGCGTTGTGTTCGTTTC-3'
The 3rd technical problem that the present invention will solve is to provide the method for alkaline pectin enzyme mutant described in using gene engineering bacterium fermentative production, is that (after pET-20b (+)-pglI58V activation culture, access contains 100 μ g mL by the recombinant bacterium E.coli BL21 (DE3) of the gene that contains encoding mutant alkaline pectase -1in the fermention medium of penbritin, liquid amount is 50mL/500mL, in 37 DEG C, and 200r min -1cultivate.Thalli growth is to certain phase (OD 600=0.6), add final concentration 0.4mM IPTG to induce, temperature is adjusted into 30 DEG C simultaneously, induction fermentation 48h.
Described activation culture is from glycerine pipe, recombinant bacterium E.coli BL21 (DE3) (pET-20b (+)-pglI58V) to be inoculated in and to contain 100 μ g mL -1in the seed culture medium of penbritin, liquid amount is 20mL/250mL.37 DEG C of culture temperature, shaking culture 10h on 200rpm shaking table.
Described seed culture medium composition (g/L): yeast powder 5, Tryptones 10, NaCl10, glucose 20, pH7.0.
Described fermention medium composition: yeast powder 24g/L, Tryptones 12g/L, glycerine 5g/L, K 2hPO 472mmol L -1, KH 2pO 417mmol L -1.
The present invention, by Fixedpoint mutation modified alkaline pectase gene, lives coded PGL enzyme and improves, and thermotolerance strengthens.After sudden change, the work of alkaline pectase extracellular enzyme rises to 337.58U/mL, is 2.60 times before sudden change, and exocytosis expression amount obviously improves.In addition, the optimal reactive temperature of mutant has improved 5 DEG C, can under hot conditions, show better optimum enzyme and live; Its K mimprove obviously, strengthen as 4.5 times before sudden change k with Binding Capacity ability cat, t 1/2and k cat/ K mdeng being all improved.The zymologic property of PGL provided by the invention is improved largely, and is more applicable to suitability for industrialized production demand, meets the requirement of social production.
Brief description of the drawings
The secreting, expressing amount (SDS-PAGE protein electrophoresis) of Fig. 1 mutant strain and original strain, WT: the enzyme before sudden change; I58V: mutant.
The thermotolerance of Fig. 2 mutant strain and original strain, WT: the enzyme before sudden change; I58V: mutant.
Embodiment
Alkaline pectase enzyme activity detects: get a certain amount of fermented liquid, and the centrifugal 10min of 8000rpm, the outer alkaline pectase of born of the same parents is contained among fermented supernatant fluid.Alkaline pectase reaction system is: enzyme diluent 20 μ L, and containing glycine-NaOH damping fluid (0.2mol L of 0.2%PGA -1, 0.44mmol L -1caCl 2, pH9.4) and 2mL, the enzyme liquid of non-activity is blank.Alkaline pectase reaction conditions is: 45 DEG C of water-bath 15min, use 3mL phosphoric acid solution (0.03mol L -1) termination reaction, 235nm place assaying reaction thing absorbance.
Alkaline pectase enzyme live unit definition by: within 1 minute, cracking polygalacturonic acid produces the corresponding enzyme amount of 1 μ mol unsaturated polyester galacturonic acid.
Alkaline pectase enzyme method of calculation alive:
(OD 235× 10 6× system volume × enzyme liquid extension rate)/(10 3time of enzymatic reacting × enzyme liquid in × cuvette thickness × 4600 × enzyme reaction linearity range is long-pending)
Alkaline pectase purification condition: A liquid: glycine-NaOH, pH7.5; B liquid: glycine-NaOH, 2M ammonium sulfate, pH7.5; 5mL drainage column [HiTrap Phenvl (high sub) FF], 3mL/min, 30%B liquid wash-out obtains target protein; 30kDa millipore ultra-filtration centrifuge tube is concentrated, and to obtain SDS-PAGE electrophoresis pure.
Alkaline pectase heat stability test: measure enzyme activity according to standard method under differing temps, be decided to be 100%. to process preferment work, enzyme liquid is incubated to 24h at 30 DEG C respectively, 40 DEG C of insulation 2h, 50 DEG C of insulation 2h, are incubated 1h at 55 DEG C, after utilizing ice bath cooling rapidly, survey as stated above residual enzyme activity, investigate its thermostability.
Alkaline pectase K m, V maxdetect: under the concentration of substrate not waiting at 0.05-2g/L, measure, obtained by GraphPad Prism5 prediction.
Embodiment 1 structure of mutation expression plasmid and the acquisition of recombined bacillus subtilis
1, build mutation expression carrier taking pET-20b (+)-pgl plasmid as template
The nucleotide sequence of the gene of encoding wild type alkaline pectase and the signal peptide that is made up of 21 amino acid is as shown in SEQ IDNO.1, and the aminoacid sequence of the ripe alkaline pectase of wild-type is as shown in SEQ ID NO.2.By analyzing the three-dimensional structure of alkaline pectase, infer that the Isoleucine I of 58 has considerable influence to the secreting, expressing of alkaline pectase, design mutating experiment, becomes α-amino-isovaleric acid V by the isoleucine mutation of 58.
Taking pET-20b (+)-pgl plasmid as template, utilize the plasmid of PCR (polymerase chain reaction) amplification in vitro containing mutator gene, the isoleucine mutation of 58 is become to α-amino-isovaleric acid.
Primer for rite-directed mutagenesis:
pglI58V primer1：5'-AATCATTTATGTTAAGGGAACGATT-3'
pglI58V primer2：5'-TTTGGCGTTGTGTTCGTTTC-3'
PCR reaction system is as follows: (primer concentration is 20 μ mol/L)
Add water and mend to 50 μ l.
PCR reaction conditions: 94 DEG C of denaturation 10min, 98 DEG C of sex change 10s, 55 DEG C of annealing 15s, 72 DEG C are extended 5min, 30 rear 72 DEG C of extension 10min of circulation, 4 DEG C of preservations.By first the plasmid pET-20b (+) obtaining by PCR-pglI58V phosphorylation, connect after night (it is precious biological that test kit is purchased from TAKARA), reaction system and condition are as table 1:
Table 1: plasmid phosphorylation and ligation system and reaction conditions
The construction process of pET-20b (+)-pgl is referring to document: Overproduction of alkaline polygalacturonate lyase inrecombinant Escherichia coli by a two-stage glycerol feeding approach.2011, Shuying Fang et al.Volume102, Issue22, p10671 – 10678.
2, the conversion of mutation expression carrier
(1) preparation of competent cell
By E.coli BL21 (DE3) picking list bacterium colony on streak plate substratum, be inoculated in LB substratum.37 DEG C of vibrations (200rpm) are cultivated.Measure OD 600value, works as OD 600value reaches at 0.35～0.5 o'clock, and (approximately cultivating 5 hours) placed and stopped cultivating (if OD in ice 600value exceeds this scope and can not ensure the transformation efficiency of competent cell).Get above-mentioned bacterial culture fluid 1ml (amount is determined Microtube quantity as required) in 1.5ml Microtube.Centrifugal 5 minutes of 4 DEG C of 1,500 × g (the about 4000rpm of general microcentrifuge), abandon supernatant (noting as far as possible eliminating supernatant).In each Microtube, add the Solution A of precooling in 100l ice, springing Microtube suspends precipitation gently, forbid 4 DEG C of thermal agitation 1,500 × g (the about 4000rpm of general microcentrifuge) centrifugal 5 minutes, abandon supernatant (noting as far as possible eliminating supernatant).In each Microtube, add the Solution B of precooling in 100 μ L ice, springing Microtube suspends precipitation gently, forbids thermal agitation.
(2) checking of mutation expression carrier amplification
Competent cell completes.This competent cell can be directly used in the transformation experiment of DNA, also can in-80 DEG C, preserve, in order to use later.In the time preserving for-80 DEG C, can effectively preserve more than 1 year, but can not multigelation, once after melting, can not carry out again-80 DEG C of preservations.The DNA conversion of competent cell is placed in ice by the competent cell of-80 DEG C of preservations and melts 10 minutes.The competent cell of getting 100 μ L moves in new conversion tube.To the conversion DNA that adds 0.1ng～10ng (3 μ L～10 μ L) in competent cell, mix gently in rear ice and place 30 minutes.In 42 DEG C of water-baths, place after 90 seconds, in ice, place immediately 1～2 minute.Add the LB substratum of 890 μ L37 DEG C of pre-temperature.37 DEG C of shaking culture 1 hour.Get after appropriate painting flat board, flat-plate inverted is placed in to 37 DEG C of incubators and cultivates a night.Confirm to cultivate bacterium colony, carry out lower step experiment.
The suddenly change expression of PGL of embodiment 2
Seed culture medium composition (g/L): yeast powder 5, Tryptones 10, NaCl10, glucose 20, pH7.0.
Fermention medium composition: yeast powder 24g/L, Tryptones 12g/L, glycerine 5g/L, K 2hPO 472mmol L -1, KH 2pO 417mmol L -1.
From glycerine pipe, the recombinant bacterium E.coli BL21 (DE3) (pET-20b (+)-pglI58V) that contains mutation expression carrier pET-20b (+)-pglI58V is inoculated in and contains 100 μ g mL -1in the seed culture medium of penbritin, liquid amount is 20mL/250mL.37 DEG C of culture temperature, 200rpm min -1shaking culture 10h on shaking table.
The seed liquor of cultivating 10h is contained to 100 μ g mL with the inoculum size access of 3% (V/V) -1in the fermention medium of penbritin, liquid amount is 50mL/500mL, in 37 DEG C, and 200r min -1cultivate.Thalli growth is to certain phase (OD 600=0.6), add final concentration 0.4mM IPTG to induce, temperature is adjusted into 30 DEG C simultaneously, induction fermentation 48h.
Before and after embodiment 3 sudden changes, PGL extracellular enzyme is lived
Ferment with E.coli BL21 (DE3) and the mutant strain E.coli BL21 (DE3) (pET-20b (+)-pgl I58V) of expression vector pET-20b (+)-pgl containing without sudden change respectively according to the method described in embodiment 2.
After Fig. 1 SDS-PAGE protein electrophoresis shows sudden change, alkaline pectase I58V exocytosis expression amount obviously improves, before sudden change, alkaline pectase extracellular enzyme is lived as 129.65U/mL, after sudden change, the work of alkaline pectase extracellular enzyme rises to 337.58U/mL, is 2.60 times before sudden change.Associative list 1, after sudden change, alkaline pectase is brought up to 295.63U/mg than enzyme work from 179.14U/mg, is 1.65 times before sudden change.The secretion capacity that the rear alkaline pectase I58V of sudden change is described strengthens, and enzymic activity strengthens.
The zymologic property of PGL before and after embodiment 4 sudden changes
Respectively E.coli BL21 (DE3) (pET-20b (+)-pgl) and mutant strain E.coli BL21 (DE3) (pET-20b (+)-pglI58V) are fermented according to the method described in embodiment 2, after purifying, carry out the zymologic property analyses such as thermotolerance.As can be seen from Table 2, after sudden change, optimal reactive temperature has improved 5 DEG C, and the rear alkaline pectase of sudden change can better show optimum enzyme and live under hot conditions; K mimprove obviously, strengthen as 4.5 times before sudden change k with Binding Capacity ability cat, t 1/2and k cat/ K mdeng being all improved.The overall zymologic property that the rear alkaline pectase of sudden change is described all improves.
Table 2 mutant strain and original strain zymetology parameter
Result shows, at critical sites mutating acid, can make this enzyme secretion performance greatly improve by sequence alignment and structure combination, enzyme-to-substrate binding ability greatly strengthens, zymologic property obviously promotes, and this strategy can be widely used in the improvement of zymologic property.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims were defined.

Claims (10)

1. the alkaline pectin enzyme mutant that secernment property improves, is characterized in that, is that the Isoleucine I point mutation of the 58th of the alkaline pectase as shown in SEQ IDNO.2 by aminoacid sequence is α-amino-isovaleric acid V.
2. the gene of mutant described in coding claim 1.
3. carry carrier or the cell of gene described in claim 2.
4. obtaining a method for alkaline pectin enzyme mutant described in claim 1, is to be undertaken expressing after rite-directed mutagenesis by the gene of design primer pair coding alkaline pectase.
5. method according to claim 4, it is characterized in that, suddenly change taking plasmid pET-20b (+)-pgl as template design primer and the plasmid conversion after sudden change is entered to E.coli JM109 and increase, then express the recombinant plasmid containing mutator gene with E.coli BL21 (DE3), obtain the alkaline pectin enzyme mutant that secernment property strengthens.
6. method according to claim 5, is characterized in that, for the primer of rite-directed mutagenesis is:
pglI58V primer1：5'-AATCATTTATGTTAAGGGAACGATT-3'
pglI58V primer2：5'-TTTGGCGTTGTGTTCGTTTC-3'。
7. the method for alkaline pectin enzyme mutant described in using gene engineering bacterium fermentative production claim 1, is characterized in that, is access after the recombinant bacterium activation culture of the gene that contains encoding mutant alkaline pectase is contained to 100 μ g mL -1in the fermention medium of penbritin, in 37 DEG C, 200r min -1cultivate; Thalli growth is to OD 600=0.6, add IPTG to induce, temperature is adjusted into 30 DEG C, induction fermentation 48h simultaneously.
8. method according to claim 7, is characterized in that, described activation culture is that recombinant bacterium E.coli BL21 (DE3) (pET-20b (+)-pglI58V) is inoculated in and contains 100 μ g mL -1in the seed culture medium of penbritin, 37 DEG C of culture temperature, shaking culture 10h on 200rpm shaking table.
9. method according to claim 8, is characterized in that, described seed culture medium composition: yeast powder 5g/L, Tryptones 10g/L, NaCl10g/L, glucose 20g/L, pH7.0.
10. method according to claim 7, is characterized in that, described fermention medium composition: yeast powder 24g/L, Tryptones 12g/L, glycerine 5g/L, K 2hPO 472mmol L -1, KH 2pO 417mmol L -1.
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN105754981A (en) * 2014-12-16 2016-07-13 中国科学院微生物研究所 Alkaline pectinase and encoding genes thereof and application of alkaline pectinase and encoding genes of alkaline pectinase
CN106636049A (en) * 2016-12-27 2017-05-10 江南大学 Alkaline pectinase mutant with improved secretion property

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CN102899299A (en) * 2012-09-06 2013-01-30 青岛蔚蓝生物集团有限公司 Alkaline pectinase mutant and recombinant expression engineering bacteria thereof

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CN102899299A (en) * 2012-09-06 2013-01-30 青岛蔚蓝生物集团有限公司 Alkaline pectinase mutant and recombinant expression engineering bacteria thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105754981A (en) * 2014-12-16 2016-07-13 中国科学院微生物研究所 Alkaline pectinase and encoding genes thereof and application of alkaline pectinase and encoding genes of alkaline pectinase
CN105754981B (en) * 2014-12-16 2019-07-12 中国科学院微生物研究所 A kind of application of alkaline pectase and its encoding gene and they
CN106636049A (en) * 2016-12-27 2017-05-10 江南大学 Alkaline pectinase mutant with improved secretion property
CN106636049B (en) * 2016-12-27 2019-11-08 江南大学 A kind of alkaline pectin enzyme mutant that secernment property improves

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