CN110358786A - A kind of enzyme mutant - Google Patents

A kind of enzyme mutant Download PDF

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CN110358786A
CN110358786A CN201910698577.7A CN201910698577A CN110358786A CN 110358786 A CN110358786 A CN 110358786A CN 201910698577 A CN201910698577 A CN 201910698577A CN 110358786 A CN110358786 A CN 110358786A
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enzyme
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CN110358786B (en
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刘松
陈坚
堵国成
赵伟欣
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Jiangnan University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C12Y113/11012—Linoleate 13S-lipoxygenase (1.13.11.12)
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    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)
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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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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Abstract

The invention discloses a kind of enzyme mutants, belong to gene engineering technology field.For the method based on the functional polypeptide library constructed based on SAPs, the entire process for obtaining enzyme mutant is quick, efficient, convenient；Meanwhile the enzyme mutant that the present invention obtains, compared to wild type alkaline pectase, the enzymatic activities highest of the fusion enzyme mutant of alkaline pectase improves 15.32 times, and stability highest improves 3.86 times, and specific enzyme activity highest improves 2.55 times；Lipoxygenase fusion enzyme mutant enzyme activity highest intracellular improves 2.49 times, and stability highest improves 3.13 times, and specific enzyme activity highest improves 0.9 times；Asparaginase fusion enzyme mutant enzymatic activities highest improves 2.25 times, and stability highest improves 3.56 times, and specific enzyme activity highest improves 1.34 times.

Description

A kind of enzyme mutant
Technical field
The present invention relates to a kind of enzyme mutants, belong to genetic engineering and technical field of enzyme engineering.
Background technique
Based on expression quantity and thermal stability to the great influence of enzyme application performance, high expression quantity and high thermal stability are obtained Enzyme is always the research hotspot in enzyme engineering field.
, there is certain relationship of mutually promoting in expression quantity, expression quantity aspect is existing between thermal stability and enzymatic vigor Technology usually expresses label by the optimization of Expression element or terminal fusion to improve the expression quantity of protease, but Expression element Optimization there is certain uncertainty, and macromoleculars such as most common expression label MBP and GST, the later period application to enzyme All tool has a certain impact, and generally requires red tape and removes it；In terms of stability, as structure biology and biology are believed The development learned is ceased, researcher can be compared by the analysis (such as the B- factor, RMSF value) of some structural parameters or homologous sequence, The accurately amino acid residue or peptide fragment of positioning effects enzyme molecule thermal stability, and then carry out rite-directed mutagenesis to it and improve enzyme Thermal stability.Although having become the general categories of enzyme heat stability transformation, the technology that above-mentioned molecular modification technology still has its intrinsic Defect.The premise of rite-directed mutagenesis is to obtain accurate enzyme molecular structure information, and lactam enzyme by directional anagenesis in vitro then faces a large amount of mutant Screening, causes to be difficult to obtain the mutant that thermal stability is obviously improved in the short time.
Therefore, establishing efficient, the convenient and fast enzyme stabilization strategy of one kind becomes domestic and international researcher focus of attention.
It is worth noting that, fusion small peptide is in no enzymatic structure information and big to the raising of the expression quantity and thermal stability of enzyme It is completed under conditions of amount mutant screening, the efficiency compared with conventional molecular renovation technique significantly improves.It is by Japanese big earliest (the Nat that Urabe team, slope university finds when studying the thermal stability of bacillus stearothermophilus catalase Biotechnol,1999,17(1):58-61)；Researcher merges different length and amino acid sequence at the end catalase C- at random The small peptide of column obtains a series of mutant of high thermal stabilities；Hereafter, the fusion of the Osaka University Kanaya team discovery end C- is thermophilic Archaeal ribalgilase C- end heptapeptide (IGCIILT), the heat that can improve the ribalgilase of separate sources to some extent are steady Qualitative (PLoS ONE, 2011,6 (1): e16226).
SAPs is that a kind of hydrophobe amino acid is alternately distributed, and can be spontaneously assemble into the small peptide of nanostructure, distinctive two Close property makes it that can form hydrogel in water, to immobilize to destination protein or other small molecules.Based on this, In this research department's early-stage study (Appl Microbiol Biot, 2013,97 (21): 9419-9427), a kind of SAPs is merged Fusion enzyme heterogenous expression is carried out in enzyme N-terminal, discovery SAPs has the function of improving expression of enzymes amount and stability, wherein with special The S1 (AEAEAKAKAEAEAKAK) of distribution of charges has certain universality effect；(the Faraday such as Tsinghua University Lin Discuss, 2013,166:233) it is last in enzyme using similar SAP (LELELKLKLELELKLK) fusion is formed with S1 amino acid End, can promote generation inactive inclusion body, it is important to illustrate that the secreting, expressing of the pairs of SAPs fusion protein of amino acid group and stability have It influences.On the other hand, the group of the link peptide between SAPs and enzyme merges the expression of enzyme in pairs and stability also functions to important function (Enzyme Microb Tech,2016,82:105-109)。
Present invention trial constructs a kind of polypeptide libraries based on SAPs, with it is quick, efficient, easily obtain that there is high table It reaches, the protease mutant of high vigor and high stability.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of enzyme mutant for obtaining high expression, high vigor and high stability. The method is based on the functional polypeptide library constructed based on SAPs, and the entire process for obtaining enzyme mutant is quickly, efficiently, just It is prompt；Meanwhile using alkaline pectin enzyme mutant obtained by this functional polypeptide library, compared to wild type alkaline pectase, born of the same parents Outer enzyme activity highest improves 15.32 times, and stability highest improves 3.86 times, and specific enzyme activity highest improves 2.55 times；Fatty oxygen Synthase fusion enzyme mutant enzyme activity highest intracellular improves 2.49 times, and stability highest improves 3.82 times, and specific enzyme activity highest mentions It is 0.49 times high；Asparaginase fusion enzyme mutant enzymatic activities highest improves 2.33 times, and stability highest improves 1 Times, specific enzyme activity highest improves 1.17 times.
Technical scheme is as follows:
The present invention provides a kind of method of enzyme mutant for obtaining high expression, high vigor and high stability, feature exists In comprising the following steps:
Step 1: constructing function polypeptide libraries；
Step 2: by the resulting expression host cell in functional polypeptide library in step 1 using fluorescence intensity as screening criteria, Primary dcreening operation is carried out, the host cell of hyperfluorescence intensity is filtered out；
Step 3: the host cell of the hyperfluorescence intensity obtained after primary dcreening operation in step 2 being cultivated, is with fluorescence intensity Screening criteria carries out secondary screening identification, filters out the host cell with hyperfluorescence intensity again；
Step 4: the expression vector in the resulting host cell with hyperfluorescence intensity of secondary screening in step 3 is subjected to fluorescence Expression vector after missing is re-started heterogenous expression, obtains enzyme mutant by protein-encoding gene missing；
Step 5: the enzyme mutant of acquisition being subjected to Stability Determination after Overheating Treatment, is filtered out with high stability Enzyme mutant；
Functional polypeptide library in the step 1 is obtained by the host cell expression containing functional expression carrier；Institute Stating functional expression carrier includes parents' small peptide (self-assembling that different length and amino acid form Amphipathic peptide, SAP), link peptide (linker), the fluorescent protein expression base of different length and different hard and soft Cause and expression of enzymes gene to be screened.
In one embodiment of the invention, primary dcreening operation is to carry out primary dcreening operation with flow cytometer in the step 2.
The present invention provides a kind of for obtaining the functional polypeptide of the enzyme mutant of high expression, high vigor and high stability Library, the functional polypeptide library is obtained by the host cell expression containing functional expression carrier；The functional expression Carrier include different length and amino acid form parents' small peptide (self-assembling amphipathic peptide, SAP), link peptide (linker), fluorescent protein expression gene and the expression of enzymes base to be screened of different length and different hard and softs Cause.
In one embodiment of the invention, the SAP is connected to 5 ' ends of expression of enzymes gene to be screened with linker.
In one embodiment of the invention, the fluorescent protein expression Gene Fusion is in expression of enzymes gene to be screened 3 ' ends.
In one embodiment of the invention, the SAP is that the gene with sequence for SEQ ID NO.1 passes through coding institute SAP and/or using gene that sequence is SEQ ID NO.1 by encoding resulting SAP as template, carry out further length And the resulting SAPs of random mutation of amino acid.
In one embodiment of the invention, the linker includes different rigid peptide and flexible peptide assembled unit.
In one embodiment of the invention, the assembled unit amino acid sequence of the rigid peptide and flexible peptide is respectively SEQ ID NO.2 and SEQ ID NO.3.
In one embodiment of the invention, it is SEQ that the assembled unit of the rigid peptide and flexible peptide, which is respectively sequence, The gene coding of ID NO.4 and SEQ ID NO.5 is resulting.
In one embodiment of the invention, the length that rigid peptide is combined with flexible peptide in the linker is 1 to 5 Between unit.
In one embodiment of the invention, the combination of rigid peptide and flexible peptide includes table 1 in the linker Shown content.
1 link peptide sequence table of table
The present invention provides a kind of for obtaining the functional polypeptide of the enzyme mutant of high expression, high vigor and high stability The construction method in library, which is characterized in that comprise the following steps:
Step 1: introducing multiple multiple cloning sites in the multiple cloning sites of expression vector, expression vector is transformed；
Step 2: successively introducing expression of enzymes base to be screened in the multiple cloning sites of the resulting improved expression vector of step 1 Cause, SAP and fluorescent protein expression gene；
Step 3: step 2 introduce expression of enzymes gene to be screened upstream introduce linker gene so that SAP with Linker is connected with expression of enzymes gene to be screened, obtains functional expression carrier；
Step 4: the resulting functional expression carrier of step 3 being converted to expression host cell and carries out heterogenous expression, is obtained Functional polypeptide library.
In one embodiment of the invention, the expression vector skeleton is pET-22b (+).
In one embodiment of the invention, the polyclone enzyme enzyme site quantity introduced in the step 1 is four.
In one embodiment of the invention, the step 2 is 5 ' the end linker genes in enzyme gene to be screened SAP is connected, in 3 ' end fusion fluorescent protein expression genes of expression of enzymes gene to be screened.
In one embodiment of the invention, the step 2 is to be linearized expression vector with PCR method, wait sieve It selects expression of enzymes upstream area of gene to introduce SAP using degenerate primer to be mutated.
In one embodiment of the invention, the step 3 is with the primer pair linker base containing step 2 homology arm Because carrying out PCR, the recombinant plasmid to be connected to different linker expands linker as template, finally merges linker To between SAP and expression of enzymes gene to be screened SAP is connected with linker with expression of enzymes gene to be screened, obtains functional table Up to carrier.
In one embodiment of the invention, the recombinant plasmid is pMD-18T.
The present invention provides a kind of enzyme mutant, the amino acid sequence of the enzyme is SEQ ID NO.6.
The present invention provides a kind of enzyme mutant, the amino acid sequence of the enzyme is SEQ ID NO.7.
The present invention provides a kind of enzyme mutant, the amino acid sequence of the enzyme is SEQ ID NO.8.
The utility model has the advantages that
(1) enzyme mutant that the present invention obtains, compared to wild type alkaline pectase, the fusion enzyme mutant of alkaline pectase The enzymatic activities highest of body improves 15.32 times, and stability highest improves 3.86 times, and specific enzyme activity highest improves 2.55 times； Lipoxygenase fusion enzyme mutant enzyme activity highest intracellular improves 2.49 times, and stability highest improves 3.13 times, specific enzyme activity Highest improves 0.9 times；Asparaginase fusion enzyme mutant enzymatic activities highest improves 2.25 times, and stability highest improves 3.56 times, specific enzyme activity highest improves 1.34 times.
(2) present invention combines SAPs itself compositing characteristic, link peptide compositing characteristic, applied to the expression quantity of enzyme, stability Screening, catalytic activity, the screening of substrate affinity or resultant effect.
(3) the functional polypeptide library capacity that the present invention constructs can reach 106More than, forward mutation assay rate can reach 86%, And mutation species balance (such as Fig. 2).
(4) for the present invention using GFP as expression quantity selection markers, screening technique is simple, high flux screening can be carried out, in enzyme table There is extensive use up to amount and stability transformation aspect.
(5) present invention carries out high-throughput sorting to fluorecyte with flow cytometer, and selected by flow cytometry apoptosis technology has The advantage that speed is fast, precision is high, accuracy is good is one of the present age state-of-the-art cell quantitative technology.
Detailed description of the invention
Fig. 1 functional polypeptide library construction schematic diagram of the present invention.
Fig. 2-7 mutation rate phenetic analysis figure in functional polypeptide library of the present invention.
Specific embodiment
It is the embodiment of the present invention below, but the invention is not limited in any way.
Correlation fusion expression of enzymes strain culturing mode, way of purification and zymologic property measurement are as follows in the present invention.
Culture medium forms (g/L):
Seed culture medium: peptone 10, yeast extract 5, sodium chloride 5；
Fermentation medium: following component is dissolved in 0.9L water: peptone 12g, yeast extract 24g, glycerol 4mL.
High pressure sterilization after each component dissolution；Be cooled to 60 DEG C, then plus 100mL sterilizing 0.17mol/L KH2PO4、 The K of 0.72mol/L2HPO4Solution (the KH of 2.31g2PO4With the K of 12.54g2HPO4It is dissolved in enough water, makes final volume 100mL；0.22 μm of membrane filtration degerming)；
Cultural method:
Seed culture, picking engineering bacteria single colonie access in the triangular flask (250mL) that liquid amount is 25mL, cultivation temperature 37 DEG C, shaking speed 200r/min cultivates 12h；Fermented and cultured, the triangular flask that the inoculum concentration access liquid amount by 3% is 25mL In (250mL), 37 DEG C of cultivation temperature, work as OD600When reaching 0.6, IPTG induction is added, and (wherein PGL inducing amount is 0.04mM, LOX For 1mM, ASN 1mM), and adjust simultaneously culture at temperature to the optimum inducing temperature of the enzyme (PGL is 30 DEG C of culture 48h, LOX is 20 DEG C and cultivates for 24 hours that ASN is 30 DEG C of culture 12h).
Configure protein purification liquid:
A liquid: 20mM phosphate buffer, 500mMNaCl, 20mM imidazoles.
B liquid: 20mM phosphate buffer, 500mMNaCl, 500mM imidazoles.
The phosphate buffered saline method of the pH7.4 of 20mM: the NaH of 190mL20mM2PO4Add 810mL20mM's Na2HPO4。
Alkaline pectase enzyme activity and thermal stability 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 (the CaCl of 0.2mol/L, 0.44mmol/L of (substrate)2, pH9.4) and 2mL, 20 μ L of sample to be tested, Inactive enzyme solution is blank control.PGL reaction condition are as follows: reaction system is placed in water-bath 15min at 45 DEG C, with 3mL phosphoric acid Solution (0.03mol/L) terminates reaction, and absorbance value is measured at 235nm.
The enzyme solution diluted packing is placed in 60 DEG C of metal baths, is sampled every 3min and carries out remnant enzyme activity measurement, meter Calculate half-life period.
Lipoxygenase enzyme activity and thermal stability determination:
LOX enzyme activity is measured using spectrophotometry.1 unit LOX enzyme activity is defined as: catalysis substrate per minute is sub- at 25 DEG C Enzyme amount needed for oleic acid forms 1 μm of ol linoleic acid hydroperoxide (HPOD optical activity coefficient=25000L/ (mol × cm)).Enzyme activity Determination condition: using linoleic acid as substrate, using under Shimadzu UV-2450 spectrophotometer on-line determination 234nm at 25 DEG C The variation of light absorption value calculates enzyme activity with the slope of light absorption value change curve initial part.
Enzyme after purification is diluted to protein concentration with bufferA and is 100 μ g/mL and keeps the temperature at 50 DEG C that measuring space is residual Remaining enzyme activity calculates half-life period.
Asparaginase enzyme activity and thermal stability determination:
Using the enzyme activity of Nessler's reagent method measurement ASN, (1) enzyme digestion reaction: 1mL phosphate buffer (10mmol/L, pH value For 7.5), 300 μ L enzyme solutions, 100 μ L substrates are added 100 μ L trichloroacetic acids after 37 DEG C of reaction 30min and terminate reaction.After mixing 12000r/min is centrifuged 2min.(2) chromogenic reaction: 200 μ L, 3.3mL deionized water of first step enzyme digestion reaction liquid, 500 μ L Nai Shi Reagent.Light absorption value is measured after mixing at 436nm.
Unit of enzyme activity's definition: it is living to be defined as ASN for enzyme amount required for 1 μm of ol ammonia of hydrolysis altheine generation per minute Unit of force.
The drafting of standard curve: (the NH of 18mmol/L is prepared4)2SO4Standard solution, 0 μ is added in 1.5mL pipe respectively L, 50 μ L, 100 μ L, 200 μ L, 250 μ L, 300 μ L, 350 μ the L, (NH of 400 μ L4)2SO4Standard solution, with deionized water mend to Then 1mL phosphate buffer (10mmol/L, pH value 7.5) is added in 400 μ L, 100 μ L tri- are added after 37 DEG C of reaction 30min Monoxone terminates reaction, and 12000r/min after mixing carries out chromogenic reaction after being centrifuged 2min.
The enzyme solution diluted packing is placed in 55 DEG C of metal baths, is sampled every 3min and carries out remnant enzyme activity measurement, meter Calculate half-life period.
Mono- group of contrast groups of PGL, LOX and ASN, SL1-PGL, SL2-PGL, SL3-PGL, SL1- is arranged altogether in the present embodiment Tri- groups of LOX, SL2-LOX, SL3-LOX and SL1-ASN, SL2-ASN, SL3-ASN implementation groups.
Functional polypeptide library construction and screening process involved by the present embodiment are as shown in Figure 1.Involved functional polypeptide Library mutation rate phenetic analysis is as illustrated in figs. 2-7.Involved SAP and connection peptide amino acid sequence are as shown in table 2
2 SAP of table and connection peptide sequence
Embodiment 1: constructing function polypeptide libraries
According to the amino acid sequence 9S1 (AEAEAKAKAEAEAKAK) of parents' small peptide9, its gene of chemical synthesis is simultaneously connected to The gene order N-terminal of enzyme (PGL, LOX and ASN) mutually to be screened, and it is cloned into the plasmid pET-22b of target enzyme or albumen (enzyme is protein-encoding gene to be screened to (+)/enzyme, is herein NdeI the and NcoI restriction enzyme site of PGL, LOX and ASN) Between, it is configured to pET-22b (+)/9S1-enzyme plasmid.In pET-22b (+)/9S1-enzyme terminal fusion GFP expression Gene gfp constructs pET-22b (+)/9S1-enzyme-gfp plasmid.
Using pET-22b (+)/9S1-enzyme-gfp plasmid as template, degeneracy upstream primer nSAP-up and specificity under Swim primer nSAP-downenzymeThe pET-22b (+) of the SAP containing different aminoacids composition and length of PCR acquisition linearisation/ NSAP-enzyme-gfp Linearized genes segment.
The preparatory chemical synthesis of peptide gene will be connected and be connected on pMD18-T carrier, with general upstream LR-up and LF- Up, and the downstream LR-down of specificityenzymeAnd LF-downenzymePCR, which is carried out, for primer obtains link peptide genetic fragment.And with The pET-22b (+) of linearisation/nSAP-enzyme-gfp genetic fragment carries out homologous recombination.Obtain pET-22b (+)/nSAP- Linker-enzyme-gfp mixing plasmid.
Embodiment 2: primary dcreening operation
Primary dcreening operation: recombinant plasmid and recombinant plasmid library are converted to expressive host E. coli BL21 (DE3), seed Culture medium culture simultaneously induces, and continues after cultivating certain time (related with the expression of enzyme to be screened), (related bacterium training method Are as follows: work as OD600When reaching 0.6, IPTG induction is added, and (the IPTG inducing amount that wherein PGL-GFP merges enzyme is 0.04mM, LOX-GFP For 1mM, ASN-GFP 1mM), and culture at temperature to the optimum inducing temperature of the enzyme is adjusted simultaneously (PGL-GFP is 30 DEG C 5h is cultivated, LOX-GFP is that 20 DEG C of cultures 10h, ASN-GFP are 30 DEG C of culture 5h).) be diluted cell to be screened, setting stream Formula cell instrument MoFlo XDP flow cytometry (Beckman Coulter, USA) jet size is 100 μm, 20mM's The phosphate-buffered of pH7.4 is sheath fluid, cell OD600It is set as 0.1 hereinafter, cell flow rate is 5000 particle/seconds, exciting light Wavelength is 488nm, and wavelength of transmitted light is that the detection parameters of 530/40nm carry out analysis test to sample.Deduct corresponding blank pair According to.
Embodiment 3: secondary screening identification
Secondary screening identification: the hyperfluorescence Intensity Abrupt body obtained after selected by flow cytometry apoptosis is subjected to shaking flask culture and fluorescence is strong Degree measurement.Single colonie is inoculated in after seed culture medium is incubated overnight and is forwarded to fermentation medium, works as OD600When reaching 0.6, add Enter IPTG induction (the IPTG inducing amount that wherein PGL-GFP merges enzyme is 0.04mM, LOX-GFP 1mM, ASN-GFP 1mM), And culture is adjusted at temperature to the optimum inducing temperature of the enzyme simultaneously (PGL-GFP is that 30 DEG C of cultures 48h, LOX-GFP are 20 DEG C 72h is cultivated, ASN-GFP is 30 DEG C and cultivates for 24 hours).
Embodiment 4: fluorescent protein gene deletion
Using the high expression mutant plasmid of above-mentioned acquisition as template, GFP gene delection is carried out, primer is as shown in table 2.
Embodiment 5: enzyme mutant detection
Recombinant bacterium fermented and cultured, 37 DEG C of seed culture medium be incubated overnight after be forwarded to fermentation medium, work as OD600Reach 0.6 When, IPTG induction (the IPTG inducing amount that wherein PGL merges enzyme is 0.04mM, LOX 1mM, ASN 1mM) is added, and adjust simultaneously (PGL is that 30 DEG C of cultures 48h, LOX are 20 DEG C of cultures 72h, ASN 30 for culture at whole temperature to the optimum inducing temperature of the enzyme DEG C culture for 24 hours).And dilution appropriate is carried out to recombinase on 96 orifice plates, PGL recombinase is placed in 65 DEG C respectively, LOX recombination Enzyme is placed in 55 DEG C, and ASN recombinase is heat-treated under the conditions of being placed in 70 DEG C, carries out remnant enzyme activity measurement.Testing result such as 4 institute of table Show.
Table 4 merges enzyme mutant expression quantity and property representation
Enzymes Thick enzyme activity (U/mL) Half-life period (min) Specific enzyme activity (U/mg)
PGL 131.17±2.4 5.2±0.22 264.17±5.2
SL1PGL 2140.42±6.7 21.25±0.93 918.72±8.4
SL2PGL 2033.9±9.1 25.29±1.1 937.18±5.4
SL3PGL 1432.6±7.4 15.2±0.3 710.2±5.7
LOX 1.54±0.02 10.2±0.2 30.2±0.97
SL1LOX 5.38±0.03 18.2±0.5 42.7±1.5
SL2LOX 2.77±0.02 42.2±0.9 26.21±0.13
SL3LOX 3.08±0.04 30.6±0.7 57.6±1.2
ASN 3.8±0.06 12.3±0.42 17.24±0.23
SL1ASN 12.36±0.18 56.21±1.1 19.21±0.44
SL2ASN 7.12±0.2 27.18±0.4 15.29±0.8
SL3ASN 5.3±0.6 24.4±0.17 40.4±1.88
Embodiment 6: enzyme mutant purifying
Alkaline pectin enzyme purification method:
Fermented supernatant fluid is centrifuged to 15min under the conditions of 9000r/min and obtains the fermented supernatant fluid containing alkaline pectase. Ammonium sulfate precipitation is carried out on ice, and initial concentration is carried out to fermentation liquid.After dialysis desalination, 0.22 μm of filtering with microporous membrane of sample Afterwards, it is isolated and purified with 5mL cation-exchange chromatography post (HiTrapTM SP FF, GE).Purification condition are as follows: 10~15 times of columns Volume buffer A (20mmol/L Glycine-NaOH buffer, pH7.4) balances drainage column, flow velocity 2mL/min, 1mL/ After min flow velocity sample introduction 5mL, 2mL/min continue A liquid balance it is steady to curve, with B buffer (20mmol/L glycine-hydroxide Sodium buffer, 1mol/LNaCl, pH7.4) carry out linear elution.The eluent containing PGL of acquisition is dialysed in A liquid and is removed Salt, 4 DEG C of preservations.
Lipoxidase enzyme purification method:
Fermented supernatant fluid is centrifuged to 15min under the conditions of 9000r/min and obtains the fermented supernatant fluid containing alkaline pectase And the glycerol of 10% (w/v) is added, be slowly added to grinding, the saturation degree of dry ammonium sulfate powder to ammonium sulfate be 40% and after It is continuous to be slowly stirred 30min；Then sample is centrifuged 15min at 12000rpm, collects and precipitate and redissolve in containing 50mmol/ In the buffer solution A of LNaCl, the supernatant obtained after centrifugation removal precipitating is the sample after ammonium sulfate precipitation.
By the sample after ammonium sulfate precipitation be 50kDa with molecular cut off bag filter slow containing 50mmol/LNaCl It dialyses in fliud flushing A for 24 hours, desalination for 24 hours of then dialysing in buffer solution A again.
Histrap1mLFF purification column buffer solution A is balanced with the flow velocity of 1mL/min, subsequent loading, balance, and B is linear The component of recombination LOX is collected in elution.The eluent containing LOX of acquisition is dialysed desalination in A liquid, 4 DEG C of preservations.
Asparagine enzyme purification method:
Fermented supernatant fluid is centrifuged to 15min under the conditions of 9000r/min and obtains the fermented supernatant fluid containing alkaline pectase. Ammonium sulfate precipitation is carried out on ice, and initial concentration is carried out to fermentation liquid.After dialysis desalination, 0.22 μm of filtering with microporous membrane of sample Afterwards, it is redissolved with A liquid, is isolated and purified with the hydrophobic chromatography column (HiTrapTM SP FF, GE) of 5mL.Purification condition are as follows: 10~ 15 times of column volumes of buffer A (20mmol/L Glycine-NaOH buffer, pH7.4) balance drainage column, flow velocity 2mL/ After min, 1mL/min flow velocity sample introduction 5mL, 2mL/min continue A liquid balance it is steady to curve, with B buffer (the sweet ammonia of 20mmol/L Acid-sodium hydrate buffer solution, 1mol/LNaCl, pH7.4) carry out linear elution.By the eluent containing ASN of acquisition in A liquid Middle dialysis desalination, 4 DEG C of preservations.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
Sequence table
<110>Southern Yangtze University
<120>a kind of enzyme mutant
<160> 80
<170> PatentIn version 3.3
<210> 1
<211> 432
<212> DNA
<213>artificial sequence
<400> 1
gcagaagcag aagcgaaagc caaagcggag gcggaagcca aggctaaagc agaagcagaa 60
gcgaaagcca aagcggaggc ggaagccaag gctaaagcag aagcagaagc gaaagccaaa 120
gcggaggcgg aagccaaggc taaagcagaa gcagaagcga aagccaaagc ggaggcggaa 180
gccaaggcta aagcagaagc agaagcgaaa gccaaagcgg aggcggaagc caaggctaaa 240
gcagaagcag aagcgaaagc caaagcggag gcggaagcca aggctaaagc agaagcagaa 300
gcgaaagcca aagcggaggc ggaagccaag gctaaagcag aagcagaagc gaaagccaaa 360
gcggaggcgg aagccaaggc taaagcagaa gcagaagcga aagccaaagc ggaggcggaa 420
gccaaggcta aa 432
<210> 2
<211> 5
<212> PRT
<213>artificial sequence
<400> 2
Glu Ala Ala Ala Lys
1 5
<210> 3
<211> 5
<212> PRT
<213>artificial sequence
<400> 3
Gly Gly Gly Gly Ser
1 5
<210> 4
<211> 15
<212> DNA
<213>artificial sequence
<400> 4
gaagctgcgg caaaa 15
<210> 5
<211> 15
<212> DNA
<213>artificial sequence
<400> 5
ggtggtggcg gttcg 15
<210> 6
<211> 401
<212> PRT
<213>artificial sequence
<400> 6
Met Asp Ala Asp Leu Gly His Gln Thr Leu Gly Ser Asn Asp Gly Trp
1 5 10 15
Gly Ala Tyr Ser Thr Gly Thr Thr Gly Gly Ser Lys Ala Ser Ser Leu
20 25 30
Asn Val Tyr Thr Val Ser Asn Arg Asn Gln Leu Val Ser Ala Leu Gly
35 40 45
Lys Glu Thr Asn Thr Thr Pro Lys Ile Ile Tyr Ile Lys Gly Thr Ile
50 55 60
Asp Met Asn Val Asp Asp Asn Leu Lys Pro Leu Gly Leu Asn Asp Tyr
65 70 75 80
Lys Asp Pro Glu Tyr Asp Leu Asp Lys Tyr Leu Lys Ala Tyr Asp Pro
85 90 95
Ser Thr Trp Gly Lys Lys Glu Pro Ser Gly Thr Gln Glu Glu Ala Arg
100 105 110
Ala Arg Ser Gln Lys Asn Gln Lys Ala Arg Val Met Val Asp Ile Pro
115 120 125
Ala Asn Thr Thr Ile Val Gly Ser Gly Thr Asn Ala Lys Val Val Gly
130 135 140
Gly Asn Phe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg Asn Ile Glu
145 150 155 160
Phe Gln Asp Ala Tyr Asp Tyr Phe Pro Gln Trp Asp Pro Thr Asp Gly
165 170 175
Ser Ser Gly Asn Trp Asn Ser Gln Tyr Asp Asn Ile Thr Ile Asn Gly
180 185 190
Gly Thr His Ile Trp Ile Asp His Cys Thr Phe Asn Asp Gly Ser Arg
195 200 205
Pro Asp Ser Thr Ser Pro Lys Tyr Tyr Gly Arg Lys Tyr Gln His His
210 215 220
Asp Gly Gln Thr Asp Ala Ser Asn Gly Ala Asn Tyr Ile Thr Met Ser
225 230 235 240
Tyr Asn Tyr Tyr His Asp His Asp Lys Ser Ser Ile Phe Gly Ser Ser
245 250 255
Asp Ser Lys Thr Ser Asp Asp Gly Lys Leu Lys Ile Thr Leu His His
260 265 270
Asn Arg Tyr Lys Asn Ile Val Gln Arg Ala Pro Arg Val Arg Phe Gly
275 280 285
Gln Val His Val Tyr Asn Asn Tyr Tyr Glu Gly Ser Thr Ser Ser Ser
290 295 300
Ser Tyr Pro Phe Ser Tyr Ala Trp Gly Ile Gly Lys Ser Ser Lys Ile
305 310 315 320
Tyr Ala Gln Asn Asn Val Ile Asp Val Pro Gly Leu Ser Ala Ala Lys
325 330 335
Thr Ile Ser Val Phe Ser Gly Gly Thr Ala Leu Tyr Asp Ser Gly Thr
340 345 350
Leu Leu Asn Gly Thr Gln Ile Asn Ala Ser Ala Ala Asn Gly Leu Ser
355 360 365
Ser Ser Val Gly Trp Thr Pro Ser Leu His Gly Ser Ile Asp Ala Ser
370 375 380
Ala Asn Val Lys Ser Asn Val Ile Asn Gln Ala Gly Ala Gly Lys Leu
385 390 395 400
Asn
<210> 7
<211> 667
<212> PRT
<213>artificial sequence
<400> 7
Met Asn Asp Ser Ile Phe Phe Ser Pro Leu Lys Tyr Leu Gly Ala Glu
1 5 10 15
Gln Gln Arg Ser Ile Asp Ala Ser Arg Ser Leu Leu Asp Asn Leu Ile
20 25 30
Pro Pro Ser Leu Pro Gln Tyr Asp Asn Leu Ala Gly Lys Leu Ala Arg
35 40 45
Arg Ala Val Leu Thr Ser Lys Lys Leu Val Tyr Val Trp Thr Glu Asn
50 55 60
Phe Gly Asn Val Lys Gly Val Pro Met Ala Arg Ser Val Pro Leu Gly
65 70 75 80
Glu Leu Pro Asn Val Asp Trp Leu Leu Lys Thr Ala Gly Val Ile Val
85 90 95
Glu Leu Ile Val Asn Phe Val Ala Ser Leu Pro Ala Ser Ala Ala Ala
100 105 110
Gln Phe Glu Arg Ile Ala Thr Gly Leu Ser Gly Asp Leu Glu Ala Ala
115 120 125
Arg Gln Val His Glu Ala Leu Leu Glu Glu Ala Lys Asn Asp Pro Ala
130 135 140
Ala Ala Gly Ser Leu Leu Leu Arg Phe Thr Glu Leu Gln Thr Arg Val
145 150 155 160
Ile Ala Ile Leu Thr Arg Val Gly Leu Leu Val Asp Asp Ile Leu Lys
165 170 175
Ser Ala Ser Asn Leu Val Thr Gln Arg Gly Gln Gly Asp Gly Leu Asn
180 185 190
Arg Phe Arg Ala Val Phe Gly Thr Leu Arg Leu Pro Glu Val Ala Asp
195 200 205
Ser Phe Arg Asp Asp Glu Ala Phe Ala Tyr Trp Arg Val Ala Gly Pro
210 215 220
Asn Pro Leu Leu Ile Arg Arg Val Asp Ala Leu Pro Ala Asn Phe Pro
225 230 235 240
Leu Gly Glu Glu Gln Phe Arg Arg Val Met Gly Ala Asp Asp Ser Leu
245 250 255
Leu Glu Ala Ala Ala Ser Arg Arg Leu Tyr Leu Leu Asp Tyr Ala Glu
260 265 270
Leu Gly Lys Leu Ala Pro Ser Gly Ala Val Asp Lys Leu Leu Thr Gly
275 280 285
Thr Gly Phe Ala Tyr Ala Pro Ile Ala Leu Phe Ala Leu Gly Lys Asp
290 295 300
Arg Ala Arg Leu Leu Pro Val Ala Ile Gln Cys Gly Gln Asp Pro Ala
305 310 315 320
Thr His Pro Met Phe Val Arg Pro Ala Glu Ser Glu Ser Asp Leu Tyr
325 330 335
Trp Gly Trp Gln Met Ala Lys Thr Val Val Gln Val Ala Glu Glu Asn
340 345 350
Tyr His Glu Met Phe Val His Leu Ala Gln Thr His Leu Val Ser Glu
355 360 365
Ala Phe Cys Leu Ala Thr Gln Arg Thr Leu Ala Pro Ser His Pro Leu
370 375 380
His Val Leu Leu Ala Pro His Phe Glu Gly Thr Leu Phe Ile Asn Glu
385 390 395 400
Gly Ala Ala Arg Ile Leu Leu Pro Ser Ala Gly Phe Ile Asp Val Met
405 410 415
Phe Ala Ala Pro Ile Gln Asp Thr Gln Ala Thr Ala Gly Gly Asn Arg
420 425 430
Leu Gly Phe Asp Phe Tyr Arg Gly Met Leu Pro Glu Ser Leu Lys Ala
435 440 445
Arg Asn Val Asp Asp Pro Leu Ala Leu Pro Asp Tyr Pro Tyr Arg Asp
450 455 460
Asp Gly Leu Leu Val Trp Asn Ala Ile Arg Gln Trp Ala Ala Asp Tyr
465 470 475 480
Val Ala Val Tyr Tyr Ala Ser Asp Gly Asp Val Thr Ala Asp Val Glu
485 490 495
Leu Ala Ala Trp Val Gly Glu Val Ile Gly Ser Gly Lys Val Ala Gly
500 505 510
Phe Arg Pro Ile Thr Gly Arg Ser Gln Leu Val Glu Val Leu Thr Met
515 520 525
Val Ile Phe Thr Ala Ser Ala Gln His Ala Ala Val Asn Phe Pro Gln
530 535 540
Pro Ser Met Met Thr Tyr Ala Pro Ala Ile Cys Ala Met Ser Ala Ala
545 550 555 560
Pro Ala Pro Asp Ser Pro Ser Gly Lys Ser Glu Ala Asp Trp Leu Lys
565 570 575
Met Met Pro Pro Thr Leu Val Ala Leu Glu Lys Val Asn Ile Tyr His
580 585 590
Leu Leu Gly Ser Val Tyr His Gly Arg Leu Gly Asp Tyr Arg Gln Thr
595 600 605
Gly Phe Pro Tyr Ala Pro Val Phe Ser Asp Arg Arg Val Thr Ala Ser
610 615 620
Gly Gly Pro Leu Glu Arg Phe Gln Ala Arg Leu Lys Glu Val Glu Ala
625 630 635 640
Thr Ile Arg Thr Arg Asn Gln Ala Arg Arg Arg Pro Tyr Glu Tyr Leu
645 650 655
Leu Pro Ser Arg Ile Pro Ala Ser Thr Asn Ile
660 665
<210> 8
<211> 370
<212> PRT
<213>artificial sequence
<400> 8
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Met Glu Phe Phe Lys Lys Thr Ala Leu Ala
20 25 30
Ala Leu Val Met Gly Phe Ser Gly Ala Ala Leu Ala Leu Pro Asn Ile
35 40 45
Thr Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Gly Gly Asp Ser Ala
50 55 60
Thr Lys Ser Asn Tyr Thr Ala Gly Lys Val Gly Val Glu Asn Leu Val
65 70 75 80
Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val Lys Gly Glu Gln
85 90 95
Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Asp Val Trp Leu Thr
100 105 110
Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr Asp Gly Phe Val
115 120 125
Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala Tyr Phe Leu Asp
130 135 140
Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val Gly Ala Met Arg
145 150 155 160
Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn Leu Tyr Asn Ala
165 170 175
Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg Gly Val Leu Val
180 185 190
Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val Thr Lys Thr Asn
195 200 205
Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr Gly Pro Leu Gly
210 215 220
Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr Pro Ala Arg Lys
225 230 235 240
His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu Asn Glu Leu Pro
245 250 255
Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser Asp Leu Pro Ala
260 265 270
Lys Ala Leu Val Asp Ala Gly Tyr Asp Gly Ile Val Ser Ala Gly Val
275 280 285
Gly Asn Gly Asn Leu Tyr Lys Thr Val Phe Asp Thr Leu Ala Thr Ala
290 295 300
Ala Lys Asn Gly Thr Ala Val Val Arg Ser Ser Arg Val Pro Thr Gly
305 310 315 320
Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys Tyr Gly Phe Val
325 330 335
Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val Leu Leu Gln Leu
340 345 350
Val Leu Thr Gln Thr Lys Asp Pro Gln Gln Ile Gln Gln Ile Phe Asn
355 360 365
Gln Tyr
370
<210> 9
<211> 5
<212> PRT
<213>artificial sequence
<400> 9
Glu Ala Ala Ala Lys
1 5
<210> 10
<211> 5
<212> PRT
<213>artificial sequence
<400> 10
Gly Gly Gly Gly Ser
1 5
<210> 11
<211> 10
<212> PRT
<213>artificial sequence
<400> 11
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
1 5 10
<210> 12
<211> 10
<212> PRT
<213>artificial sequence
<400> 12
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 13
<211> 10
<212> PRT
<213>artificial sequence
<400> 13
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser
1 5 10
<210> 14
<211> 10
<212> PRT
<213>artificial sequence
<400> 14
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys
1 5 10
<210> 15
<211> 15
<212> PRT
<213>artificial sequence
<400> 15
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
1 5 10 15
<210> 16
<211> 15
<212> PRT
<213>artificial sequence
<400> 16
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 17
<211> 15
<212> PRT
<213>artificial sequence
<400> 17
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser
1 5 10 15
<210> 18
<211> 15
<212> PRT
<213>artificial sequence
<400> 18
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys
1 5 10 15
<210> 19
<211> 15
<212> PRT
<213>artificial sequence
<400> 19
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 20
<211> 15
<212> PRT
<213>artificial sequence
<400> 20
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
1 5 10 15
<210> 21
<211> 15
<212> PRT
<213>artificial sequence
<400> 21
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys
1 5 10 15
<210> 22
<211> 15
<212> PRT
<213>artificial sequence
<400> 22
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser
1 5 10 15
<210> 23
<211> 20
<212> PRT
<213>artificial sequence
<400> 23
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 24
<211> 20
<212> PRT
<213>artificial sequence
<400> 24
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 25
<211> 20
<212> PRT
<213>artificial sequence
<400> 25
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 26
<211> 20
<212> PRT
<213>artificial sequence
<400> 26
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 27
<211> 20
<212> PRT
<213>artificial sequence
<400> 27
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 28
<211> 20
<212> PRT
<213>artificial sequence
<400> 28
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 29
<211> 20
<212> PRT
<213>artificial sequence
<400> 29
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 30
<211> 20
<212> PRT
<213>artificial sequence
<400> 30
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 31
<211> 20
<212> PRT
<213>artificial sequence
<400> 31
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 32
<211> 20
<212> PRT
<213>artificial sequence
<400> 32
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 33
<211> 20
<212> PRT
<213>artificial sequence
<400> 33
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 34
<211> 20
<212> PRT
<213>artificial sequence
<400> 34
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 35
<211> 20
<212> PRT
<213>artificial sequence
<400> 35
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 36
<211> 20
<212> PRT
<213>artificial sequence
<400> 36
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 37
<211> 20
<212> PRT
<213>artificial sequence
<400> 37
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys
20
<210> 38
<211> 20
<212> PRT
<213>artificial sequence
<400> 38
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 39
<211> 25
<212> PRT
<213>artificial sequence
<400> 39
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 40
<211> 25
<212> PRT
<213>artificial sequence
<400> 40
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 41
<211> 25
<212> PRT
<213>artificial sequence
<400> 41
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 42
<211> 25
<212> PRT
<213>artificial sequence
<400> 42
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 43
<211> 25
<212> PRT
<213>artificial sequence
<400> 43
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 44
<211> 25
<212> PRT
<213>artificial sequence
<400> 44
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 45
<211> 25
<212> PRT
<213>artificial sequence
<400> 45
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 46
<211> 25
<212> PRT
<213>artificial sequence
<400> 46
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 47
<211> 25
<212> PRT
<213>artificial sequence
<400> 47
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 48
<211> 25
<212> PRT
<213>artificial sequence
<400> 48
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 49
<211> 25
<212> PRT
<213>artificial sequence
<400> 49
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 50
<211> 25
<212> PRT
<213>artificial sequence
<400> 50
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 51
<211> 25
<212> PRT
<213>artificial sequence
<400> 51
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 52
<211> 25
<212> PRT
<213>artificial sequence
<400> 52
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 53
<211> 25
<212> PRT
<213>artificial sequence
<400> 53
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 54
<211> 25
<212> PRT
<213>artificial sequence
<400> 54
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 55
<211> 25
<212> PRT
<213>artificial sequence
<400> 55
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 56
<211> 25
<212> PRT
<213>artificial sequence
<400> 56
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 57
<211> 25
<212> PRT
<213>artificial sequence
<400> 57
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 58
<211> 25
<212> PRT
<213>artificial sequence
<400> 58
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 59
<211> 25
<212> PRT
<213>artificial sequence
<400> 59
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 60
<211> 25
<212> PRT
<213>artificial sequence
<400> 60
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 61
<211> 25
<212> PRT
<213>artificial sequence
<400> 61
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 62
<211> 25
<212> PRT
<213>artificial sequence
<400> 62
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 63
<211> 25
<212> PRT
<213>artificial sequence
<400> 63
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 64
<211> 25
<212> PRT
<213>artificial sequence
<400> 64
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 65
<211> 25
<212> PRT
<213>artificial sequence
<400> 65
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 66
<211> 25
<212> PRT
<213>artificial sequence
<400> 66
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 67
<211> 25
<212> PRT
<213>artificial sequence
<400> 67
Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 68
<211> 25
<212> PRT
<213>artificial sequence
<400> 68
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Glu
1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 69
<211> 25
<212> PRT
<213>artificial sequence
<400> 69
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25
<210> 70
<211> 25
<212> PRT
<213>artificial sequence
<400> 70
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu
1 5 10 15
Ala Ala Ala Lys Gly Gly Gly Gly Ser
20 25
<210> 71
<211> 79
<212> PRT
<213>artificial sequence
<400> 71
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala His
1 5 10 15
Ala Glu Ala Glu Ala Lys Ala Lys Ala Asn Ala Glu Ala His Ala Lys
20 25 30
Ala Glu Ala Glu Ala Lys Ser Lys Ala Glu Ala Glu Ala Lys Ala Lys
35 40 45
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
50 55 60
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
65 70 75
<210> 72
<211> 63
<212> PRT
<213>artificial sequence
<400> 72
Ala Glu Ala Glu Ala Lys Ala Arg Ala Glu Ala Glu Ala Arg Ala Lys
1 5 10 15
Ala Glu Ala Glu Tyr Lys Ala Lys Ala Glu Ala Glu Ala His Ala Lys
20 25 30
Ala Glu Ala Glu Ala His Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
35 40 45
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
50 55 60
<210> 73
<211> 32
<212> PRT
<213>artificial sequence
<400> 73
Ala Glu Ala Glu Ala Arg Ala Lys Ala Glu Ala Glu Ala His Ala Lys
1 5 10 15
Ala Glu Cys Glu Ala Lys Ala Lys Ala Glu Tyr Glu Ala Lys Ala Lys
20 25 30
<210> 74
<211> 36
<212> PRT
<213>artificial sequence
<400> 74
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Ala Glu Ala Glu Ala Lys Ala Lys Ala Asn Ala Tyr Ala Ser Ala Lys
20 25 30
Ala Glu Ala Lys
35
<210> 75
<211> 32
<212> PRT
<213>artificial sequence
<400> 75
Ala Glu Leu Glu Gly Lys Ala Lys Ala Glu Phe Glu Ala Lys Leu Lys
1 5 10 15
Ser Glu Leu Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
20 25 30
<210> 76
<211> 24
<212> PRT
<213>artificial sequence
<400> 76
Ala Glu Ala Glu Cys Lys Ala Lys Ala Glu Phe Glu Ala Lys Ala Lys
1 5 10 15
Ala Glu Ala Glu Ala Lys Ala Lys
20
<210> 77
<211> 8
<212> PRT
<213>artificial sequence
<400> 77
Ala Glu Ala Asn Ala Lys Ala His
1 5
<210> 78
<211> 8
<212> PRT
<213>artificial sequence
<400> 78
Ala Glu Ala Glu Phe Lys Ala His
1 5
<210> 79
<211> 24
<212> PRT
<213>artificial sequence
<400> 79
Ala Glu Leu Glu Ala Lys Ala Lys Phe Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Ala Glu Ala Glu Ala Lys Ala Lys
20
<210> 80
<211> 25
<212> PRT
<213>artificial sequence
<400> 80
Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Glu Ala Ala Ala Lys
20 25

Claims (3)

1. a kind of method for the enzyme mutant for obtaining high expression, high vigor and high stability, which is characterized in that include following step It is rapid:
Step 1: constructing function polypeptide libraries；
Step 2: by the resulting expression host cell in functional polypeptide library in step 1 using fluorescence intensity as screening criteria, carrying out Primary dcreening operation filters out the host cell of hyperfluorescence intensity；
Step 3: the host cell of the hyperfluorescence intensity obtained after primary dcreening operation in step 2 being cultivated, is screening with fluorescence intensity Standard carries out secondary screening identification, filters out the host cell with hyperfluorescence intensity again；
Step 4: the expression vector in the resulting host cell with hyperfluorescence intensity of secondary screening in step 3 is subjected to fluorescin Expression vector after missing is re-started heterogenous expression, obtains enzyme mutant by expressing gene missing；
Step 5: the enzyme mutant of acquisition being subjected to Stability Determination after Overheating Treatment, it is prominent to filter out the enzyme with high stability Variant；
Functional polypeptide library in the step 1 includes host cell；The host cell contains functional expression carrier；Institute Stating functional expression carrier includes parents' small peptide (self-assembling that different length and amino acid form Amphipathic peptide, SAP), link peptide (linker), the fluorescent protein expression base of different length and different hard and soft Cause and expression of enzymes gene to be screened；
The enzyme is lipoxygenase；
The amino acid sequence of the lipoxygenase is as shown in SEQ ID NO.7.
2. a kind of enzyme mutant, which is characterized in that the enzyme mutant be by amino acid sequence as shown in SEQ ID NO.7 The N-terminal of enzyme be sequentially connected amino acid sequence link peptide as shown in SEQ ID NO.12 and amino acid sequence such as SEQ ID Parents' small peptide shown in NO.74 obtains；
Alternatively, the enzyme mutant is by being sequentially connected amino in the N-terminal of amino acid sequence enzyme as shown in SEQ ID NO.7 Acid sequence link peptide as shown in SEQ ID NO.39 and amino acid sequence parents' small peptide as shown in SEQ ID NO.75 obtain It arrives；
Alternatively, the enzyme mutant is by being sequentially connected amino in the N-terminal of amino acid sequence enzyme as shown in SEQ ID NO.7 Acid sequence link peptide as shown in SEQ ID NO.80 and amino acid sequence parents' small peptide as shown in SEQ ID NO.76 obtain It arrives.
3. a kind of enzyme mutant as claimed in claim 2, which is characterized in that the enzyme is lipoxygenase.
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CN108342407A (en) 2018-07-31
CN110373404A (en) 2019-10-25

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