CN105316310B - A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising - Google Patents
A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising Download PDFInfo
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- CN105316310B CN105316310B CN201510828863.2A CN201510828863A CN105316310B CN 105316310 B CN105316310 B CN 105316310B CN 201510828863 A CN201510828863 A CN 201510828863A CN 105316310 B CN105316310 B CN 105316310B
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- 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 mutants that a kind of specific enzyme activity and thermal stability improve, and belong to enzyme engineering field.For existing mutant PGL-S1, mutant PGL- (GS) of the invention3The specific enzyme activity of-S1 improves 6 times, improves 1.3 times in 60 DEG C of half-life period.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 the alkaline pectin enzyme mutants that a kind of specific enzyme activity and thermal stability improve, and belong to enzyme engineering 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, although Pichia pastoris expression albumen is easy to purify, yield is high,
Fermentation period is long, process is complicated, low temperature induction energy consumption is high；Bacillus subtilis is not easy to express or express the disadvantages such as enzyme activity is low.
Summary of the invention
To solve the above-mentioned problems, the present invention with early-stage study in PGL albumen n end with PT-Linker
(PTPPTTPTPPTTPTPT, i.e. SEQ ID NO.3) connects parents' small peptide of one section of positive and negative charge alternating (hydrophilic and hydrophobic alternating)
(AEAEAKAKAEAEAKAK, i.e. SEQ ID NO.4), abbreviation PGL-S1 carry out further molecule to it and change for sequence of setting out
It makes, wherein will replace with flexible stronger (GS) by the stronger PT-linker of rigidity3(GGGGSGGGGSGGGGS, i.e. SEQ ID
NO.5) linker PGL, specific enzyme activity improves six times, and zymologic property and thermal stability are also improved.
The present invention provides the alkaline pectin enzyme mutant that a kind of specific enzyme activity and thermal stability improve, amino acid sequence is
Sequence shown in SEQ ID NO.1, mutant abbreviation PGL- (GS)3-S1。
In one embodiment of the invention, the nucleotide sequence of the mutant is sequence shown in SEQ ID NO.2
Column.
In one embodiment of the invention, the mutant is melting for the alkaline pectase of original fusion parents' small peptide
Synthase linker is replaced.
In one embodiment of the invention, the mutant is to replace with linker between original fusion protein
(GS) the flexible linker of n series.
The present invention also provides a kind of genetic engineering bacteriums for expressing the mutant.
In one embodiment of the invention, the engineering bacteria that is based on is Escherichia coli.
In one embodiment of the invention, the genetic engineering bacterium with Escherichia coli is host, pET-22b (+) is
Carrier expresses alkaline pectin enzyme mutant.
The nucleotide sequence for encoding the mutant and the mutant and genetic engineering bacterium is also claimed in the present invention
Application in terms of food, weaving or papermaking.
The utility model has the advantages that for existing mutant PGL-S1, mutant PGL- (GS) of the invention3- S1 ratio
Enzyme activity improves 6 times, improves 1.3 times in 60 DEG C of half-life period.Alkaline pectase of the invention can be catalyzed under alkaline condition
By α-Isosorbide-5-Nitrae glycosidic bond cracking of trans-elimination polygalacturonic acid, it is widely used in the row such as food, textile and paper
Industry.
Figure of description
Fig. 1: alkaline pectase SDS-PAGE analysis；Wherein swimming lane 3 is mutant PGL- (GS) of the invention3-S1。
Specific embodiment:
Culture medium:
Seed culture medium: tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, glucose 2g/L.
Fermentation medium: peptone 12g/L, yeast powder 24g/L, glycerol 10g/L, KH2PO42.32g/L、
K2HPO416.43g/L。
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 (0.2molL-1, the 0.44mmolL of (substrate)-1CaCl2, pH9.4) 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, uses 3mL
Phosphoric acid solution (0.03molL-1) reaction is terminated, absorbance value is measured at 235nm.
Thermal stability determination:
The enzyme solution diluted packing is placed in 60 DEG C of metal baths, is sampled every 3min and carries out enzyme activity determination, calculate half
It declines the phase.
Embodiment 1: the acquisition of mutant strain
Using PCR amplification or chemically synthesized method, amino acid sequence alkalinity fruit as shown in SEQ ID NO.1 is obtained
Then gene is connected to pET-22b (+) by glue enzyme gene, then be transformed into E. coli BL21 (DE3), screening,
Correct transformant is named as recombinant bacterium E.coli BL21 (DE3) (pET-22b (+)/PGL- (GS)3-S1)。
Embodiment 2: the verifying of mutant strain
Seed culture: by recombinant bacterium E.coli BL21 (DE3) (pET-22b (+)/PGL- (GS)3- S1) from glycerol tube
It takes and is inoculated in LB culture medium (100 μ gmL in right amount-1Ampicillin, 2% glucose), liquid amount 20mL/250mL.37
DEG C, 200rmin-1Shaken cultivation 10h on shaking table.
Shake flask fermentation: the seed liquor for cultivating 10h is accessed into fermentation medium TB (100 μ g with the inoculum concentration of 3% (V/V)
mL-1Ampicillin) in, liquid amount 20mL/250mL, 37 DEG C, 200rmin-1It cultivates to cell concentration OD600=0.6,
Final concentration 0.04mM IPTG is added to be induced, induces 48h at 30 DEG C.
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.
Embodiment 4: the pure enzyme solution specific enzyme activity of alkaline pectase and half-life period measurement
By alkaline pectase dilution after purification, enzyme activity is measured according to the method described above, with BSA determination of protein concentration kit
Protein concentration is measured, PGL-S1 specific enzyme activity of setting out is 458.52 ± 22.9U/mg, and mutant PGL- (GS)3- S1 specific enzyme activity is high
Up to 2793.17 ± 90.23U/mg, 60 DEG C of half-lifes are increased to nearly 20min by original 15min.And with other mutant phases
Than the expression quantity of mutant of the present invention increases, and is illustrated in fig. 1 shown below.
In addition, the present invention, which is also compared, replaces with the mutation that other linker are obtained for linker between original fusion protein
Body, for example use (EK)nSerial linker (EAAAK) n (n=1,2,3,4,5) and (GS)n(n=1,2,3) other serial
Linker replaces (GS) of the invention3, a series of PGL- (Linker)-S1 mutant is obtained, the results show that with PGL-S1 phase
Than (EK)nSerial specific enzyme activity increase is unobvious, and 60 DEG C of half-life period decrease；(GS)nSerial enzyme activity enzyme activity in n=1,2 is protected
Hold constant, only half-life period slightly improves.
Claims (10)
1. the alkaline pectin enzyme mutant that a kind of specific enzyme activity and thermal stability improve, which is characterized in that the amino of the mutant
Acid sequence is sequence shown in SEQ ID NO.1.
2. mutant according to claim 1, which is characterized in that the nucleotide sequence for encoding the mutant is SEQ ID
Sequence shown in NO.2.
3. encoding the nucleic acid of mutant described in claim 1.
4. application of the mutant described in claim 1 in terms of food, weaving or papermaking.
5. expressing the genetic engineering bacterium of mutant described in claim 1.
6. genetic engineering bacterium according to claim 5, which is characterized in that the genetic engineering bacterium is using Escherichia coli as place
Main, pET-22b (+) is carrier, expresses alkaline pectin enzyme mutant.
7. application of the genetic engineering bacterium described in claim 5 in terms of fermentation production of alkaline pectic enzyme.
8. application of the genetic engineering bacterium described in claim 6 in terms of fermentation production of alkaline pectic enzyme.
9. application of the genetic engineering bacterium described in claim 5 in terms of food, weaving or papermaking.
10. application of the genetic engineering bacterium described in claim 6 in terms of food, weaving or papermaking.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106701724B (en) * | 2016-12-27 | 2019-10-08 | 江南大学 | A kind of preparation and its application of alkaline pectase inactive inclusion body |
| CN106754848B (en) * | 2016-12-27 | 2020-11-03 | 江南大学 | Alkaline pectinase mutant with improved thermal stability |
| CN109852602B (en) * | 2019-01-11 | 2021-08-24 | 江南大学 | Method for improving enzyme stability |
| CN109750014B (en) * | 2019-03-27 | 2023-01-13 | 云南师范大学 | Fusion type rhizopus chinensis lipase with improved heat stability and application thereof |
| CN114149987B (en) * | 2021-12-07 | 2024-02-13 | 安徽大学 | Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103540574A (en) * | 2013-07-25 | 2014-01-29 | 江南大学 | Method for improving specific activity and activation efficiency of transglutaminase |
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| 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)
| Title |
|---|
| Fusion Protein Linkers: Property, Design and Functionality;Xiaoying Chen et al.;《Adv Drug Deliv Rev.》;20131015;第65卷(第10期);第1357-1369页 |
| 代谢工程改造酿酒酵母生产（S）-芳樟醇;孙明雪;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20140215(第02期);摘要 |
| 融合自组装双亲短肽提高碱性果胶酶热稳定性;刘松等;《食品与发酵工业》;20150910;第41卷(第11期);第1页左栏第1段，第2页第1.2.1-1.2.2节，第3页右栏第1段，第5页右栏第1段 |
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