CN103966194B - The alkaline pectin enzyme mutant that a kind of secernment property improves - Google Patents

The alkaline pectin enzyme mutant that a kind of secernment property improves Download PDF

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CN103966194B
CN103966194B CN201410190780.0A CN201410190780A CN103966194B CN 103966194 B CN103966194 B CN 103966194B CN 201410190780 A CN201410190780 A CN 201410190780A CN 103966194 B CN103966194 B CN 103966194B
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alkaline
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pectin enzyme
sudden change
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CN103966194A (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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    • 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 the alkaline pectin enzyme mutant that a kind of secernment property improves, belong to technical field of bioengineering.Will the present invention come from Bacillus? the Isoleucine I point mutation of the 58th of the alkaline pectase of sp.WSHB04-02 is α-amino-isovaleric acid V.After sudden change, alkaline pectase enzymatic activities rises to 337.58U/mL, and be 2.60 times before sudden change, exocytosis expression amount significantly improves.In addition, the optimal reactive temperature of mutant improves 5 DEG C, can show optimum enzyme better under the high temperature conditions and live; Its K mimprove obviously, strengthen as 4.5 times before suddenling change with substrate binding ability, k 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 industrial production demand, meets the requirement of social production.

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 galacturonan 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 at tea and coffee fermentation, weaving and vegetable fibre processing, oil, plays a great role containing pectin Industrial Wastewater Treatment etc.Alkaline pectase has high reactivity in the basic conditions, trans-elimination can be utilized to cut off the α-Isosorbide-5-Nitrae-glycosidic link of poly lactose aldehydic acid, pectin substance is decomposed into undersaturated oligogalacturonans.
A lot of accompanying impurities is contained 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, mutual inclusion becomes complicated huge hydrophobic reticulated structure.Comprise refinement step in textile industry, its object is exactly remove the impurity 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 needs to consume a large amount of chemical and water resources, and environmental pollution is serious, and according to statistics, in dyeing process, the waste water of about 70% 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, the swift and violent fast development studied along with molecular biology and industrial microorganism, 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 the report utilizing Escherichiacoli, Bacillussubtilis and Pichiapastoris etc. as host expresses different sources polygalacturonase gene.Now synthesis alkaline pectase engineering bacteria and fermentation processes are mainly concentrated on to the research of alkaline pectase.
Nearly ten years, the research direction of external alkaline pectase focuses on separation and purification, the aspect such as enzymatic property and protein structure properties study 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 realizing industrialization place mat further, 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 is based on the alkaline pectase of the aminoacid sequence coming from Bacillussp.WSHB04-02 as shown in SEQIDNO.2 (wild-type), is 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 building described alkaline pectin enzyme mutant, is to express after carrying out rite-directed mutagenesis by the gene of design primer pair encodes alkaline polygalacturonase.
Described construction process, be preferably that template design primer is carried out suddenling change and the Plastid transformation after sudden change is entered E.coliJM109 and increases with plasmid pET-20b (+)-pgl, then express the recombinant plasmid containing mutator gene with E.coliBL21 (DE3), obtain the alkaline pectin enzyme mutant that secernment property improves.
The construction process of described pET-20b (+)-pgl is see document: Overproductionofalkalinepolygalacturonatelyaseinrecombin antEscherichiacolibyatwo-stageglycerolfeedingapproach.20 11, ShuyingFangetal.Volume102, Issue22, p10671 – 10678.
The described primer for rite-directed mutagenesis is:
pglI58Vprimer1：5'-AATCATTTATGTTAAGGGAACGATT-3'
pglI58Vprimer2：5'-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 is containing 100 μ gmL by the recombinant bacterium E.coliBL21 (DE3) of the gene containing encoding mutant alkaline pectase -1in the fermention medium of penbritin, liquid amount is 50mL/500mL, in 37 DEG C, and 200rmin -1cultivate.Thalli growth is to certain phase (OD 600=0.6), add final concentration 0.4mMIPTG and induce, temperature is adjusted to 30 DEG C simultaneously, induction fermentation 48h.
Described activation culture is inoculated in recombinant bacterium E.coliBL21 (DE3) (pET-20b (+)-pglI58V) containing 100 μ gmL from glycerine pipe -1in the seed culture medium of penbritin, liquid amount is 20mL/250mL.Culture temperature 37 DEG C, 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 472mmolL -1, KH 2pO 417mmolL -1.
The present invention is by Fixedpoint mutation modified alkaline pectinase gene, and coded PGL enzyme is lived and improves, thermotolerance strengthens.After sudden change, alkaline pectase enzymatic activities rises to 337.58U/mL, and be 2.60 times before sudden change, exocytosis expression amount significantly improves.In addition, the optimal reactive temperature of mutant improves 5 DEG C, can show optimum enzyme better under the high temperature conditions and live; Its K mimprove obviously, strengthen as 4.5 times before suddenling change with substrate binding ability, k 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 industrial production demand, meets the requirement of social production.
Accompanying drawing explanation
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, the centrifugal 10min of 8000rpm, and namely 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, containing the glycine-NaOH buffer (0.2molL of 0.2%PGA -1, 0.44mmolL -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, uses 3mL phosphoric acid solution (0.03molL -1) termination reaction, 235nm place assaying reaction thing absorbance.
Alkaline pectase enzyme live unit definition for: within 1 minute, cracking polygalacturonic acid produces enzyme amount corresponding to 1 μm of ol 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 in × cuvette thickness × 4600 × enzyme reaction linearity range × enzyme liquid amasss)
Alkaline pectase purification condition: A liquid: glycine-NaOH, pH7.5; B liquid: glycine-NaOH, 2M ammonium sulfate, pH7.5; 5mL drainage column [HiTrapPhenvl (highsub) FF], 3mL/min, 30%B liquid wash-out obtains target protein; 30kDamillipore 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 at different temperatures, to process, preferment is alive is decided to be 100%., by enzyme liquid respectively at 30 DEG C of insulation 24h, 40 DEG C of insulation 2h, 50 DEG C of insulation 2h, are incubated 1h at 55 DEG C, after utilizing ice bath to cool rapidly, survey residual enzyme activity as stated above, investigate its thermostability.
Alkaline pectase K m, V maxdetect: measure under the concentration of substrate that 0.05-2g/L does not wait, predicted obtaining by GraphPadPrism5.
The structure of embodiment 1 mutation expression plasmid and the acquisition of recombined bacillus subtilis
1, with pET-20b (+)-pgl plasmid be template build mutant expression vector
Encoding wild type alkaline pectase and the nucleotide sequence of the gene of signal peptide that is made up of 21 amino acid are as shown in SEQIDNO.1, and the aminoacid sequence of wild-type mature alkaline pectase is as shown in SEQIDNO.2.By analyzing the three-dimensional structure of alkaline pectase, infer that the secreting, expressing of Isoleucine I to alkaline pectase of 58 has considerable influence, design mutating experiment, becomes α-amino-isovaleric acid V by the isoleucine mutation of 58.
With pET-20b (+)-pgl plasmid for template, utilize PCR (polymerase chain reaction) amplification in vitro containing the plasmid of mutator gene, the isoleucine mutation of 58 is become α-amino-isovaleric acid.
Primer for rite-directed mutagenesis:
pglI58Vprimer1：5'-AATCATTTATGTTAAGGGAACGATT-3'
pglI58Vprimer2：5'-TTTGGCGTTGTGTTCGTTTC-3'
PCR reaction system is as follows: (primer concentration is 20 μm of ol/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 extend 5min, 30 rear 72 DEG C of extension 10min of circulation, 4 DEG C of preservations.By the first phosphorylation of plasmid pET-20b (+)-pglI58V obtained by PCR, connect (it is precious biological that test kit is purchased from TAKARA) after night, 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 see document: Overproductionofalkalinepolygalacturonatelyaseinrecombin antEscherichiacolibyatwo-stageglycerolfeedingapproach.20 11, ShuyingFangetal.Volume102, Issue22, p10671 – 10678.
2, the conversion of mutant expression vector
(1) preparation of competent cell
By E.coliBL21 (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 600(about cultivate 5 hours) when value reaches 0.35 ~ 0.5 to place in ice and stop cultivating (if OD 600value exceeds the transformation efficiency that this scope can not ensure competent cell).Get above-mentioned bacterial culture fluid 1ml (amount determines Microtube quantity as required) in 1.5mlMicrotube.1,500 × g (general microcentrifuge is about 4000rpm) 4 DEG C is centrifugal 5 minutes, abandons supernatant (noting as far as possible eliminating supernatant).The SolutionA of precooling in 100l ice is added in each Microtube, springing Microtube makes precipitation suspend gently, forbid thermal agitation 1,500 × g (general microcentrifuge is about 4000rpm) 4 DEG C is centrifugal 5 minutes, abandons supernatant (noting as far as possible eliminating supernatant).In each Microtube, add the SolutionB of precooling in 100 μ L ice, springing Microtube makes precipitation suspend gently, forbids thermal agitation.
(2) the checking amplification of mutant expression vector
Competent cell completes.This competent cell can be directly used in the transformation experiment of DNA, also can preserve in-80 DEG C, in order to using later.When preserving for-80 DEG C, can effectively preserve more than 1 year, but can not multigelation, once after melting ,-80 DEG C of preservations can not be carried out again.The DNA of competent cell transforms and the competent cell that-80 DEG C are preserved is placed in ice thawing 10 minutes.The competent cell getting 100 μ L moves in new conversion tube.In competent cell, add the conversion DNA of 0.1ng ~ 10ng (3 μ L ~ 10 μ L), place 30 minutes in ice after mixing gently.After placing for 90 seconds in 42 DEG C of water-baths, place 1 ~ 2 minute in ice immediately.Add the LB substratum of 890 μ L37 DEG C of pre-temperature.37 DEG C of shaking culture 1 hour.After getting appropriate painting flat board, flat-plate inverted is placed in 37 DEG C of incubators and cultivates a night.Confirm to cultivate bacterium colony, carry out lower step experiment.
Embodiment 2 is suddenlyd change the expression of PGL
Seed culture medium composition (g/L): yeast powder 5, Tryptones 10, NaCl10, glucose 20, pH7.0.
Fermention medium forms: yeast powder 24g/L, Tryptones 12g/L, glycerine 5g/L, K 2hPO 472mmolL -1, KH 2pO 417mmolL -1.
From glycerine pipe, the recombinant bacterium E.coliBL21 (DE3) (pET-20b (+)-pglI58V) containing mutant expression vector pET-20b (+)-pglI58V is inoculated in containing 100 μ gmL -1in the seed culture medium of penbritin, liquid amount is 20mL/250mL.Culture temperature 37 DEG C, 200rpmmin -1shaking culture 10h on shaking table.
The seed liquor of cultivating 10h is contained 100 μ gmL with the access of the inoculum size of 3% (V/V) -1in the fermention medium of penbritin, liquid amount is 50mL/500mL, in 37 DEG C, and 200rmin -1cultivate.Thalli growth is to certain phase (OD 600=0.6), add final concentration 0.4mMIPTG and induce, temperature is adjusted to 30 DEG C simultaneously, induction fermentation 48h.
PGL enzymatic activities before and after embodiment 3 sudden change
According to the method described in embodiment 2 respectively to ferment containing without the E.coliBL21 (DE3) of expression vector pET-20b (+)-pgl of sudden change and mutant strain E.coliBL21 (DE3) (pET-20b (+)-pglI58V).
After Fig. 1 SDS-PAGE protein electrophoresis display sudden change, alkaline pectase I58V exocytosis expression amount significantly improves, before sudden change, alkaline pectase enzymatic activities is 129.65U/mL, after sudden change, alkaline pectase enzymatic activities rises to 337.58U/mL, is 2.60 times before sudden change.Associative list 1, after sudden change, alkaline pectase brings up to 295.63U/mg than enzyme work from 179.14U/mg, is 1.65 times before sudden change.After sudden change is described, the secretion capacity of alkaline pectase I58V strengthens, and enzymic activity strengthens.
The zymologic property of PGL before and after embodiment 4 sudden change
Respectively E.coliBL21 (DE3) (pET-20b (+)-pgl) and mutant strain E.coliBL21 (DE3) (pET-20b (+)-pglI58V) is fermented according to the method described in embodiment 2, after purifying, carry out the characterization analysis such as thermotolerance.As can be seen from Table 2, after sudden change, optimal reactive temperature improves 5 DEG C, and the rear alkaline pectase of sudden change better can show optimum enzyme under the high temperature conditions and live; K mimprove obviously, strengthen as 4.5 times before suddenling change with substrate binding ability, k cat, t 1/2and k cat/ K mdeng being all improved.After sudden change is described, the overall zymologic property of alkaline pectase all improves.
Table 2 mutant strain and original strain zymetology parameter
Result shows, by sequence alignment and structure combination at critical sites mutating acid, this enzyme secretion performance can be made greatly to improve, and 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 also not used 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, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (5)

1. the alkaline pectin enzyme mutant that improves of secernment property, it is characterized in that, be α-amino-isovaleric acid V by the Isoleucine I point mutation of the 58th of the alkaline pectase of aminoacid sequence as shown in SEQIDNO.2 the.
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 express after carrying out rite-directed mutagenesis by the gene of design primer pair encodes alkaline polygalacturonase; Described method is that template design primer is carried out suddenling change and the Plastid transformation after sudden change is entered E.coliJM109 and increases with plasmid pET-20b (+)-pgl, then express the recombinant plasmid containing mutator gene with E.coliBL21 (DE3), obtain the alkaline pectin enzyme mutant that secernment property strengthens; The described primer for rite-directed mutagenesis is:
pglI58Vprimer1：5'-AATCATTTATGTTAAGGGAACGATT-3'
pglI58Vprimer2：5'-TTTGGCGTTGTGTTCGTTTC-3'。
5. the method for alkaline pectin enzyme mutant described in using gene engineering bacterium fermentative production claim 1, is characterized in that, is that after the recombinant bacterium activation culture by the gene containing encoding mutant alkaline pectase, access contains 100 μ gmL -1in the fermention medium of penbritin, in 37 DEG C, 200rmin -1cultivate; Thalli growth, to OD600=0.6, adds IPTG and induces, and temperature is adjusted to 30 DEG C simultaneously, induction fermentation 48h; Described activation culture is inoculated in by recombinant bacterium containing 100 μ gmL -1in the seed culture medium of penbritin, culture temperature 37 DEG C, shaking culture 10h on 200rpm shaking table; Described seed culture medium composition: yeast powder 5g/L, Tryptones 10g/L, NaCl10g/L, glucose 20g/L, pH7.0; Described fermention medium composition: yeast powder 24g/L, Tryptones 12g/L, glycerine 5g/L, K 2hPO 472mmolL -1, KH 2pO 417mmolL -1; Described recombinant bacterium be with E.coliBL21 (DE3) be host, recombinant bacterium containing plasmid pET-20b (+)-pglI58V.
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