CN105316310A - Alkaline pectinase mutant with improved specific enzyme activity and heat stability - Google Patents

Alkaline pectinase mutant with improved specific enzyme activity and heat stability Download PDF

Info

Publication number
CN105316310A
CN105316310A CN201510828863.2A CN201510828863A CN105316310A CN 105316310 A CN105316310 A CN 105316310A CN 201510828863 A CN201510828863 A CN 201510828863A CN 105316310 A CN105316310 A CN 105316310A
Authority
CN
China
Prior art keywords
mutant
enzyme
alkaline
genetic engineering
pgl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201510828863.2A
Other languages
Chinese (zh)
Other versions
CN105316310B (en
Inventor
刘松
陈坚
堵国成
赵伟欣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN201510828863.2A priority Critical patent/CN105316310B/en
Publication of CN105316310A publication Critical patent/CN105316310A/en
Application granted granted Critical
Publication of CN105316310B publication Critical patent/CN105316310B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention discloses an alkaline pectinase mutant with improved specific enzyme activity and heat stability, and belongs to the field of enzyme engineering. Compared with an existing mutant PGL-S1, the specific enzyme activity of the mutant PGL-(GS)3-S1 is improved by 6 times and the half-life period at the temperature of 60 DEG C is prolonged by 1.3 times. Alkaline pectinase can be catalyzed under the alkaline condition, the alpha-1,4 glucosidic bond of polygalacturonic acid is split through the reverse eliminating effect, and the alkaline pectinase mutant can be widely applied to industries such as the food industry, the textile industry and the papermaking industry.

Description

A kind of than the alkaline pectin enzyme mutant that enzyme is lived and thermostability improves
Technical field
The present invention relates to a kind of than the alkaline pectin enzyme mutant that enzyme is lived and thermostability improves, belong to enzyme engineering field.
Background technology
Polygalacturonase is a kind of prozyme, Pectin polymers can be resolved into unsaturated oligogalacturonans.This enzyme is widely distributed, is all found in section parasitic nematode, plant and microorganism.Polygalacturonase is widely used, the industrial application history of existing more than 40 year.Polygalacturonase is divided into acid pectase and alkaline pectase PGL by the difference according to optimal reaction pH.Wherein acid pectase is mainly used in clear juice fruit wine, extracts garden spgarden stuff, the aspects such as fruit decortication.PGL application is mainly used in weaving, food, paper industry and environmental area.Application enzyme process effect above-mentioned field correlated response has environmental protection, consumptive material and the reaction conditions gentleness etc. of economizing in raw materials advantage.But it is less at present to carry out molecular modification research to PGL, it is also little to carry out commercial PGL.
At present to bacterial strain mainly pichia spp, subtilis and the intestinal bacteria that alkaline pectase research is more deep.Integrated comparative can express the different hosts of alkaline pectase, although pichia spp expressing protein is easy to purifying, output is high, and fermentation period is long, process is complicated, low temperature induction power consumption is high; Subtilis is not easily expressed or expresses enzyme and to live the shortcoming such as low.
Summary of the invention
In order to solve the problem, the present invention with early-stage Study at PGL albumen n end with PT-Linker (PTPPTTPTPPTTPTPT, i.e. SEQIDNO.3) connect parents' small peptide (AEAEAKAKAEAEAKAK that one section of positive and negative charge replaces (hydrophilic and hydrophobic is alternately), i.e. SEQIDNO.4), be called for short PGL-S1, for sequence of setting out, further molecular modification is carried out to it, PT-linker stronger for wherein rigidity is replaced with flexible stronger (GS) 3(GGGGSGGGGSGGGGS, i.e. SEQIDNO.5) linkerPGL, raising six times more alive than enzyme, and zymologic property and thermostability are also improved.
The invention provides a kind of alkaline pectin enzyme mutant improved than enzyme work and thermostability, its aminoacid sequence is the sequence shown in SEQIDNO.1, and mutant is called for short PGL-(GS) 3-S1.
In one embodiment of the invention, the nucleotide sequence of described mutant is the sequence shown in SEQIDNO.2.
In one embodiment of the invention, described mutant is that original fusion enzyme linker merging the alkaline pectase of parents' small peptide replaces.
In one embodiment of the invention, described mutant is flexible linker linker between original fusion rotein being replaced with (GS) n series.
The present invention also provides a kind of genetic engineering bacterium of expressing described mutant.
In one embodiment of the invention, described is intestinal bacteria based on engineering bacteria.
In one embodiment of the invention, described genetic engineering bacterium take intestinal bacteria as host, pET-22b (+) is carrier, expresses alkaline pectin enzyme mutant.
The nucleotide sequence of the present invention's also described mutant of claimed coding, and described mutant and the application of genetic engineering bacterium in food, weaving or papermaking.
Beneficial effect: for existing mutant PGL-S1, mutant PGL-(GS) of the present invention 3the ratio enzyme of-S1 is lived and is improve 6 times, improves 1.3 times the transformation period of 60 DEG C.Alkaline pectase of the present invention catalysis by the α-Isosorbide-5-Nitrae glycosidic link cracking of trans-elimination polygalacturonic acid, can be widely used in the industry such as food, textile and paper in the basic conditions.
Figure of description
Fig. 1: alkaline pectase SDS-PAGE analyzes; Wherein swimming lane 3 is mutant PGL-(GS) of the present invention 3-S1.
Embodiment:
Substratum:
Seed culture medium: Tryptones 10g/L, yeast powder 5g/L, NaCl10g/L, glucose 2g/L.
Fermention medium: peptone 12g/L, yeast powder 24g/L, glycerine 10g/L, KH 2pO 42.32g/L, K 2hPO 416.43g/L.
Alkaline pectase enzyme activity determination:
Adopt spectrophotometry.Unit enzyme is lived definition: unit time cracking polygalacturonic acid produces the unsaturated polyester galacturonic acid of 1 μm of ol enzyme amount used.Enzyme activity determination condition is: enzyme activity detects: the centrifugal 10min of fermented liquid 8000rpm, and namely the outer PGL of born of the same parents is contained among fermented supernatant fluid, gets a certain amount of detection.PGL reaction system: containing glycine-NaOH buffer (0.2molL-1, the 0.44mmolL of 0.2% polygalacturonic acid (substrate) -1caCl2, pH9.4) 2mL, testing sample 20 μ L, the enzyme liquid of non-activity is blank.PGL reaction conditions is: water-bath 15min at reaction system being placed in 45 DEG C, with 3mL phosphoric acid solution (0.03molL -1) termination reaction, measure absorbance at 235nm place.
Thermal stability determination:
The enzyme liquid packing of having diluted is placed in 60 DEG C of metal baths, carries out enzyme activity determination every 3min sampling, calculate the transformation period.
Embodiment 1: the acquisition of mutant strain
Adopt the method for pcr amplification or chemosynthesis, obtain the alkaline pectinase gene of aminoacid sequence as shown in SEQIDNO.1, then gene is connected to pET-22b (+), be transformed in E. coli BL21 (DE3) again, screening, correct transformant called after recombinant bacterium E.coliBL21 (DE3) (pET-22b (+)/PGL-(GS) 3-S1).
Embodiment 2: the checking of mutant strain
Seed culture: by recombinant bacterium E.coliBL21 (DE3) (pET-22b (+)/PGL-(GS) 3-S1) get from glycerine pipe and be inoculated in (100 μ gmL in LB substratum in right amount -1penbritin, 2% glucose), liquid amount is 20mL/250mL.37 DEG C, 200rmin -1shaking culture 10h on shaking table.
Shake flask fermentation: will the seed liquor of 10h be cultivated with the inoculum size of 3% (V/V) access fermention medium TB (100 μ gmL -1penbritin) in, liquid amount is 20mL/250mL, 37 DEG C, 200rmin -1be cultured to cell concentration OD600=0.6, add final concentration 0.04mMIPTG and induce, at 30 DEG C, induce 48h.
Embodiment 3: the purifying of alkaline pectase
By the centrifugal 20min of recombinant bacterium fermented liquid 8000r/min, get supernatant liquor, add ammonium sulfate to carry out gradient and saltout, low-temperature centrifugation collects 30 ~ 50% ammonium sulfate precipitation parts, the enzyme of salt precipitation is dissolved in Glycine-NaOH buffered soln (pH7.5), with 20mmol/L Glycine-NaOH buffered soln dialysis treatment 24h.Centrifugal gained supernatant liquor carries out further separation and purification through cation-exchange chromatography.
Embodiment 4: alkaline pectase pure enzyme liquor ratio enzyme is lived and the transformation period measures
By the alkaline pectase dilution after purifying, measure enzyme according to the method described above and live, with BSA determination of protein concentration kit measurement protein concentration, the PGL-S1 that sets out is more alive than enzyme is 458.52 ± 22.9U/mg, and mutant PGL-(GS) 3-S1 is than enzyme work up to 2793.17 ± 90.23U/mg, and 60 DEG C of half-life bring up to nearly 20min by original 15min.And compare with other mutant, the expression amount of mutant of the present invention increases, and is illustrated in fig. 1 shown below.
In addition, the present invention also compares mutant linker between original fusion rotein being replaced with other linker and obtain, and such as uses (EK) nseries linker (EAAAK) n (n=1,2,3,4,5) and (GS) nother linker of (n=1,2,3) series replace (GS) of the present invention 3, obtain a series of PGL-(Linker)-S1 mutant, result shows, compared with PGL-S1, (EK) nseries is more not obvious than enzyme increase alive, and 60 DEG C of transformation period decrease; (GS) nserial enzymes work is at n=1, and when 2, enzyme is lived and remained unchanged, and only the transformation period improves a little.

Claims (10)

1., than the alkaline pectin enzyme mutant that enzyme is lived and thermostability improves, it is characterized in that, the aminoacid sequence of described mutant is the sequence shown in SEQIDNO.1.
2. mutant according to claim 1, is characterized in that, the nucleotide sequence of described mutant is the sequence shown in SEQIDNO.2.
3. mutant according to claim 1, is characterized in that, described mutant is flexible linker linker between original fusion rotein being replaced with (GS) n series.
4. mutant according to claim 1, is characterized in that, described in be that the alkaline pectase fusion rotein linker wherein of fusion parents small peptide that early stage builds is replaced.
5. the nucleotide sequence of mutant described in coding claim 1.
6. the application of mutant described in claim 1 in food, weaving or papermaking.
7. express the genetic engineering bacterium of mutant described in claim 1.
8. genetic engineering bacterium according to claim 6, is characterized in that, described genetic engineering bacterium take intestinal bacteria as host, pET-22b (+) is carrier, expresses alkaline pectin enzyme mutant.
9. the application of genetic engineering bacterium described in claim 7 or 8 in fermentation production of alkaline pectic enzyme.
10. the application of genetic engineering bacterium described in claim 7 or 8 in food, weaving or papermaking.
CN201510828863.2A 2015-11-25 2015-11-25 A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising Active CN105316310B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510828863.2A CN105316310B (en) 2015-11-25 2015-11-25 A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510828863.2A CN105316310B (en) 2015-11-25 2015-11-25 A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising

Publications (2)

Publication Number Publication Date
CN105316310A true CN105316310A (en) 2016-02-10
CN105316310B CN105316310B (en) 2019-03-01

Family

ID=55244628

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510828863.2A Active CN105316310B (en) 2015-11-25 2015-11-25 A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising

Country Status (1)

Country Link
CN (1) CN105316310B (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106701724A (en) * 2016-12-27 2017-05-24 江南大学 Preparation method and application of alkaline pectinase active inclusion body
CN106754848A (en) * 2016-12-27 2017-05-31 江南大学 The alkaline pectin enzyme mutant that a kind of heat endurance is improved
CN109750014A (en) * 2019-03-27 2019-05-14 云南师范大学 The pattern of fusion rhizopus chinensis lipase and its application that a kind of thermostability improves
CN109852602A (en) * 2019-01-11 2019-06-07 江南大学 A method of improving enzyme stability
CN114149987A (en) * 2021-12-07 2022-03-08 安徽大学 Artificially-modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103540574A (en) * 2013-07-25 2014-01-29 江南大学 Method for improving specific activity and activation efficiency of transglutaminase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103540574A (en) * 2013-07-25 2014-01-29 江南大学 Method for improving specific activity and activation efficiency of transglutaminase

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
XIAOYING CHEN ET AL.: "Fusion Protein Linkers: Property, Design and Functionality", 《ADV DRUG DELIV REV.》 *
刘松等: "融合自组装双亲短肽提高碱性果胶酶热稳定性", 《食品与发酵工业》 *
孙明雪: "代谢工程改造酿酒酵母生产（S）-芳樟醇", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106701724A (en) * 2016-12-27 2017-05-24 江南大学 Preparation method and application of alkaline pectinase active inclusion body
CN106754848A (en) * 2016-12-27 2017-05-31 江南大学 The alkaline pectin enzyme mutant that a kind of heat endurance is improved
CN106754848B (en) * 2016-12-27 2020-11-03 江南大学 Alkaline pectinase mutant with improved thermal stability
CN109852602A (en) * 2019-01-11 2019-06-07 江南大学 A method of improving enzyme stability
CN109750014A (en) * 2019-03-27 2019-05-14 云南师范大学 The pattern of fusion rhizopus chinensis lipase and its application that a kind of thermostability improves
CN114149987A (en) * 2021-12-07 2022-03-08 安徽大学 Artificially-modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis
CN114149987B (en) * 2021-12-07 2024-02-13 安徽大学 Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis

Also Published As

Publication number Publication date
CN105316310B (en) 2019-03-01

Similar Documents

Publication Publication Date Title
US10472657B2 (en) Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes
JP6929499B2 (en) Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzyme
CN105316310A (en) Alkaline pectinase mutant with improved specific enzyme activity and heat stability
CN105316311A (en) Alkaline pectinase mutant with improved specific enzyme activity
CN103981167B (en) The alkaline pectin enzyme mutant that a kind of heat stability improves
Purama et al. Identification, effective purification and functional characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640
CN102260694B (en) Acidproof medium-temperature alpha-amylase and preparation method thereof
CN105886484A (en) Thermophilic cellulase, encoding gene thereof and application of thermophilic cellulase
CN103614303B (en) A kind of Li's Trichoderma strains of expressing saccharifying enzyme
CN101503681B (en) Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method
CN105112433A (en) Novel coding gene of Type-I pullulanase, and recombinant expression and application thereof
CN105154417B (en) The acidic cellulase and its gene of a kind of originated from fungus and application
CN103966195B (en) A kind of than the enzyme alkaline pectin enzyme mutant improving alive
CN106636049B (en) A kind of alkaline pectin enzyme mutant that secernment property improves
CN103224920A (en) Method for separation purification of high temperature-resistant beta-amylase from bacillus subtilis
DK2912049T3 (en) PROCEDURE FOR PREPARING SOPHOROSIS FROM SOPHOROLIPIDS
US8420355B2 (en) 4S-iota-carrageenan sulfatase and use thereof to obtain alpha-carrageenan
US8921078B2 (en) Method for transforming iota-carrageenan into alpha-carrageenan by means of a new class of 4S-iota-carrageenan sulfatase
CN103966194A (en) PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance
CN102994529A (en) Method for improving thermal stability of GH10 xylanase through N-terminal replacement
CN107043759A (en) A kind of high catalytic efficiency cellulase variants and its encoding gene and application
Housseiny Production of Purified Free and Immobilized Exo-Inulinase from Aspergillus terreus AUMC 11628 by Solid State Fermentation for Degradation of Dahlia Tubers and Chicory Roots Inulin Mixture and Ethanol Production
CN104531638A (en) Method for producing low temperature-resistance beta-mannase by using enterobacter
CN103981166A (en) Alkaline pectase mutant with improved heat resistance
BR112016019820B1 (en) GLUCANE SYNTHESIS METHOD AND HYDROLYSIS OF LEUCROSE BY-PRODUCT

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant