CN104328102A - Trypsin mutant capable of improving enzyme activity and construction method thereof - Google Patents

Trypsin mutant capable of improving enzyme activity and construction method thereof Download PDF

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CN104328102A
CN104328102A CN201410612164.XA CN201410612164A CN104328102A CN 104328102 A CN104328102 A CN 104328102A CN 201410612164 A CN201410612164 A CN 201410612164A CN 104328102 A CN104328102 A CN 104328102A
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CN104328102B (en
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康振
陈坚
张云丰
堵国成
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Jiangnan University
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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21004—Trypsin (3.4.21.4)

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Abstract

The invention discloses a trypsin mutant capable of improving enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. According to the mutant disclosed by the invention, on the basis of an amino acid with a sequence as shown in SEQ ID NO. 2, tyrosine on the 1st site is mutated into phenylalanine, meanwhile arginine on the 123rd site is mutated into isoleucine. The mutant disclosed by the invention is expressed in pichia pastoris; the enzyme activity of amidase (BAPNA) fermented in a 3L tank is 85.3U/mL and enzyme activity of esterase (BAEE) is 5954U/mL, which are respectively improved by 3.3 times and 2.7 times in comparison with those of a strain before mutation; therefore, a problem of low extracellular enzyme activity of trypsin is solved. When applied to producing the trypsin, the mutant disclosed by the invention is high in yield, simple in process and convenient for industrial application.

Description

The trypsin mutant of a kind of enzyme raising alive and construction process thereof
Technical field
The present invention relates to trypsin mutant and the construction process thereof of the raising alive of a kind of enzyme, belong to gene engineering technology field.
Background technology
Trypsinase, as a kind of important kind being comparatively early found to apply in serine protease, is found in mammiferous enteron aisle Digestive system the earliest.Commercial trypsinase is many to be extracted from mammalian pancreatic, owing to containing abundant proteolytic enzyme in pancreas, brings difficulty to trypsinase separating-purifying.In addition, the trypsinase in Mammals source mostly is trypsin mixture, medical applications exists and endangers the immunogenicity of human body.
SGT research, Koo-Bon-Joon etc. utilize streptomycete to commonly use the conventional pWHM3 plasmid (Ermp of host Streptomyces lividans1326 and streptomycete, erythromycin promotor) obtain in the recombinant trypsin belonging to secreting, expressing in bacterium together, this plasmid is transferred to can produce in tryptic streptomyces griseus Streptomyces griseus IFO13350 and expresses by Chi-Won-Jae etc., and streptomycete trypsin is expressed in E.coli system with inclusion bodies by the people (1998) such as Daisuke Nohara.Early-stage Study have expressed the trypsinase in streptomyces griseus source in pichia spp with ripe trypsinase form.The present invention adopts simple point mutation technology, based on homology modeling methods, optimizes trypsinase molecular structure, raising enzyme is lived by selecting specific amino acids.
Heterogenous expression trypsinase distinct issues are, expressing quantity is low, trypsinase enzyme is lived low.Therefore, Fixedpoint mutation modified trypsinase, improves enzymatic activities, changes into product demand and reduce production cost significant for meeting trypsinase industry.
Summary of the invention
The technical problem to be solved in the present invention is to provide trypsin mutant and the construction process thereof of the raising alive of a kind of enzyme.
The invention provides a kind of enzyme trypsin mutant improved alive, it is characterized in that, the aminoacid sequence of described mutant is the sequence shown in SEQ ID NO.1.
The nucleotide sequence of described mutant is the sequence shown in SEQ ID NO.3.
Described mutant is on the amino acid whose basis such as shown in sequence SEQ ID NO.2, and the tyrosine of the 1st has been mutated into phenylalanine (Y1F), the arginine of the 123rd has been mutated into Isoleucine (R123I) simultaneously.
In previous research work, this research team has obtained the tryptic recombinant yeast pichia pastoris engineering bacteria of a kind of heat production stability, the trypsinase of this yeast expressed nucleotide sequence as shown in SEQ ID NO.4.
Shown in described coding SEQ ID NO.2, the nucleotide sequence of aminoacid sequence is the sequence shown in SEQ ID NO.4.
The present invention also provides a kind of genetic engineering bacterium of expressing described trypsin mutant.
The preparation method of described genetic engineering bacterium, on the basis of sequence shown in SEQ ID NO.4, the arginine of the 123rd has been mutated into Isoleucine, obtain mutant R123I, the tyrosine of the 1st is mutated into phenylalanine (representing with Y1F) simultaneously, obtain recombination, recombination is connected to expression vector and obtains recombinant plasmid, namely recombinant plasmid transformed obtains yeast gene engineering bacteria in yeast host bacterium.
Described expression vector be following any one: pGAP ZA, pAO815, pGAP α A, pPIC9K, pPIC ZB.
Described expression vector is pPIC9K in one embodiment of the invention.
Described yeast host bacterium be following any one: pichiapastoris GS115, pichiapastoris KM71, pichia pastoris X-33, pichiapastoris SMD1168.
Described yeast host bacterium is Pichiapastoris GS115 in one embodiment of the invention.
Described preparation method, specifically:
(1) arrange as template with nucleotides sequence shown in SEQ ID NO.4, F1primer (sequence is as shown in SEQ ID NO.5), R1primer (sequence is as shown in SEQ ID NO.6) are primer, carry out PCR and obtain R123I mutant;
(2) with the gene order of mutant obtained in the previous step for template, F2primer (sequence is as shown in SEQ ID NO.7), R2primer (sequence is as shown in SEQ ID NO.8) are primer, carry out PCR, namely obtain the recombination of sequence as shown in SEQ ID NO.3.
(3) by recombination sequence obtained in the previous step, be connected in pPIC9K expression vector, obtain recombinant plasmid pPIC9K-recombination, recombinant plasmid electricity transforms Pichiapastoris GS115, obtain recombination yeast engineering strain, called after FemiGS115.
The present invention is on thermostability trypsinase basis, by rite-directed mutagenesis biotechnology transformation trypsinase molecular structure, obtain a plant height and produce trypsinase Pichia yeast engineering, zymotechnique is simple, enzyme is lived high, before the trypsinase Ntn hydrolase enzyme of 3L tank fermentation recombinant bacterium FemiGS115 lives comparatively sudden change, bacterial strain improves 3.3 times, and trypsinase esterase enzyme is lived and improve 2.7 times.The substrate avidity of simultaneous mutation body enzyme ExmtI Y1F improves 1.3 times, and catalytic efficiency improves 35.7%, simultaneously raising 35.4% more alive than enzyme.R123I from the sudden change of degradation site, avoid after the outer maturation of trypsinase born of the same parents from Degradation, Y1F sudden change improves tryptic exocytosis.The invention solves trypsinase heterogenous expression enzyme to live low restricted problem, for trypsinase molecular modification provides a new thinking.
Accompanying drawing explanation
Fig. 1: Fixedpoint mutation modified trypsinase expression vector establishment figure;
The trypsinase Ntn hydrolase enzyme of Fig. 2: recombinant bacterium FemiGS115 is lived and is schemed;
Fig. 3: recombinant bacterium FemiGS115 and original strain (WT) Ntn hydrolase enzyme live comparison diagram;
The outer trypsinase SDS-PAGE of Fig. 4: recombinant bacterium FemiGS1153L tank fermentation born of the same parents schemes; Wherein 1 represent CK (control strain), 2-7 represents the situation that FemiGS115 cultivates 0h, 24h, 48h, 72h, 96h, 120h, 144h respectively.
Embodiment
Trypsinase Ntn hydrolase enzyme activity determination method: at 37 DEG C, measures 100 μ L crude enzyme liquids with 900 μ L BAPNA solution in the reaction tank of optical path 0.5cm, and the light absorption value change under 410nm in 10min, obtains A410nm/min.Enzyme is lived and is defined as: at 37 DEG C, and the enzyme amount that Δ A410nm/min raises required for 0.1 is 1 Ntn hydrolase hydrolyzing unit.(work of Ntn hydrolase enzyme is more rigorous trypsinase enzyme definition mode alive, because substrate is trypsinase specific substrate.)
Trypsinase esterase enzyme activity determination method: at 25 DEG C, measures 200 μ L crude enzyme liquids with 3mL BAEE substrate solution in the reaction tank of optical path 1cm, and the light absorption value change under 253nm in 1min, obtains Δ A253nm/min.Enzyme is lived and is defined as: at 25 DEG C, and Δ A253nm/min raises 0.001 and is tryptic 1 esterase hydrolyzed unit.
Embodiment 1 is containing the structure of the recombinant vectors of trypsin mutant
Adopt the method construction of recombinant plasmid vector shown in Fig. 1.
(1) R123I mutant obtains: with the sequence shown in SEQ ID NO.4 for template, with F1primer (sequence is as shown in SEQID NO.5), R1primer (sequence is as shown in SEQ ID NO.6) for primer, carry out PCR and obtain mutant (R123I) gene order that encoding amino acid sequence the 123rd arginine sports Isoleucine.
(2) with the gene order of mutant obtained in the previous step for template, be primer with F2primer (sequence is as shown in SEQ ID NO.7), R2primer (sequence is as shown in SEQ ID NO.8), carry out PCR, obtain the recombination of sequence as shown in SEQ ID NO.3 (sequence of Y1F mutant of namely encoding).Recombination is connected in Sample carrier T.
(3) Sample carrier T containing recombination obtained in the previous step and pPIC9K are used NotI, EcoRI double digestion respectively, after purifying, T4 ligase enzyme 16 DEG C spends the night connection.Connect product chemistry method and transform JM109 competent cell.Conversion fluid coating is dull and stereotyped containing kantlex (50mg/L) LB, extracts the recombinant plasmid that the checking of plasmid double digestion builds, called after pPIC9K-ExmtIY1F.Examining order is completed by the raw work in Shanghai.
Embodiment 2 is produced ripe trypsinase Yeast engineering bacteria and is built
The recombinant plasmid pPIC9K-ExmtIY1F Sal I linearizing that embodiment 1 is obtained, electroporated pichiapastoris GS115 competent cell, concrete grammar is as follows:
1) the pichiapastoris GS115 of YPD flat board activation is inoculated in 25mL/250mL triangular flask, 30 DEG C of incubated overnight; The above-mentioned nutrient solution of 1% inoculation is in 50mL/500mL triangular flask, and cultivating cell concentration OD600 is 1.3 ~ 1.5;
2) 5000r/min, 4 DEG C of centrifugal 10min collect thalline, use 50mL, 25mL sterilized water suspension cell respectively;
3) the resuspended above-mentioned cell of 5mL1M sorbyl alcohol, 5000r/min, 4 DEG C of centrifugal 10min collect thalline;
4) the resuspended above-mentioned cell of 500 μ L1M sorbyl alcohol, packing 80 μ L/1.5mL EP manages and is used for Electroporation-competent cells;
5) 20 μ L linearization plasmids mix with above-mentioned 80 μ L competent cells, leave standstill 15min on ice;
6) said mixture adds aseptic electricity conversion cup (0.2cm) of precooling, and 1500V, 25 μ F, 200 Ω electric shocks once, add 1mL1M sorbyl alcohol;
7) get said mixture 150 μ L and be coated with MD flat board, cultivate 3 days for 30 DEG C;
8) white colony in the above-mentioned flat board of picking, verifies correct recombinant bacterium.Respectively dibbling 1,2,3, in 4mg/mL (Geneticin) YPD flat board, select single bacterium colony in 4mg/mL Geneticin flat board for shake flask fermentation, survey trypsinase enzyme to live, select enzyme the highest recombinant bacterium alive, called after recombinant bacterium FemiGS115.
Embodiment 3 recombinant yeast pichia pastoris 3L tank is cultivated
Recombinant bacterium FemiGS115 embodiment 2 built, as production bacterial strain, activates at YPD flat board.Seed liquor is cultivated, and inoculation 50mL/250mL seed culture medium, 30 DEG C, 220r/min cultivates 24h.10% inoculation 800mL/3L fermention medium, pH5.5, cultivates for 30 DEG C: 0-17h, 500rmp/min cultivate, and DO drops to about 8% from 100%, then rises to about 60% stage by stage; 17-30h, rotating speed rises to 1000rmp/min gradually, exponential fed-batch 50% glycerine, and DO starts to drop to about 10%, rises to 70% subsequently; 30-144h, stream adds 1.8% (V/V) methanol induction and produces trypsinase.To express the tryptic Pichia yeast of nucleotide sequence as shown in SEQ ID NO.4 for contrast (bacterial strain of namely not undergoing mutation).
Seed culture medium (g/L): peptone 20, yeast extract 10, glucose 20.
Fermention medium (g/L): glycerine 40; K 2sO 418; KOH4.13; MgSO47H 2o14.9; H 3pO 427mL; CaSO40.948; Trace element ion liquid (PTM1) 4.4mL; 121 DEG C of sterilizing 15min.
PTM1 (gL – 1): CuSO45H 2o6; KI0.09; MnSO 4h 2o3; H 3bO30.02; MoNa 2o 42H 2o0.2; CoC l20.5; ZnCl220; FeSO 47H 2o65; Biotin0.2; H 2sO 45mL; 0.22 μm of filtration sterilization.
Feed supplement growth medium: the glycerine of 50% (w/v) is (containing 12mlL – 1pTM1).
In fermenting process, the enzyme of Yeast engineering bacteria is lived as shown in Figure 2.As can be seen from Figure 2, start induction at fermentation 40h and produce enzyme, enzyme work increases along with induction time, and during fermentation 133h, trypsinase Ntn hydrolase enzyme is lived and reached 85.3U/mL.And the recombinant bacterium 3L fermentation Ntn hydrolase enzyme work before sudden change is up to 19.8U/mL, enzyme is lived and is improve 3.3 times.The trypsinase esterase enzyme of Simultaneously test recombinant bacterium FemiGS115 and control strain is lived, and result shows: the esterase enzyme work of recombinant bacterium FemiGS115, up to 5954U/mL, compares the 1624U/mL of control strain, improve 2.72 times.
Analyze recombinant trypsin (ExmtI Y1F) zymologic property after purifying, as table 1, ExmtI Y1F substrate avidity improves 1.3 times, and catalytic efficiency improves 35.7%, simultaneously raising 35.4% more alive than enzyme.Due to the raising of substrate avidity and catalytic efficiency, the ratio enzyme adding mutant ExmtI Y1F is lived.This shows that this invention improves tryptic zymologic property by the innovation mode of sudden change.On the other hand, because the sudden change of Y1F occurs the 1st amino acids, when 3L tank ferments, the tryptic exocytosis of mutant is expressed and is improve (as Fig. 4).ExmtI Y1F expressing quantity after sudden change is 44.55mg/L, improves 2.18 times relative to (14.03mg/L) before sudden change.
Table 1ExmtI Y1F mutant reactive kinetics parameters
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 (10)

1. the enzyme trypsin mutant improved alive, it is characterized in that, the aminoacid sequence of described mutant is the sequence shown in SEQ ID NO.1.
2. mutant according to claim 1, it is characterized in that, described mutant is on the amino acid whose basis such as shown in sequence SEQ ID NO.2, and the arginine of the 123rd has been mutated into Isoleucine, the tyrosine of the 1st has been mutated into phenylalanine simultaneously.
3. mutant according to claim 1, is characterized in that, the nucleotide sequence of described mutant is the sequence shown in SEQ ID NO.3.
4. mutant according to claim 2, is characterized in that, shown in described coding SEQ ID NO.2, the nucleotide sequence of aminoacid sequence is the sequence shown in SEQ ID NO.4.
5. the recombinant expression vector containing mutant described in claim 1.
6. express the genetic engineering bacterium of trypsin mutant described in claim 1 for one kind.
7. the preparation method of genetic engineering bacterium described in a claim 5, on the basis of sequence shown in SEQ ID NO.4, the arginine of the 123rd has been mutated into Isoleucine, the tyrosine of the 1st has been mutated into phenylalanine simultaneously, obtain recombination, recombination is connected to expression vector and obtains recombinant plasmid, namely recombinant plasmid transformed obtains yeast gene engineering bacteria in yeast host bacterium.
8. preparation method according to claim 7, it is characterized in that, described method is specifically: (1) with nucleotide sequence shown in SEQ ID NO.4 for template, the F1primer of sequence as shown in SEQ ID NO.5, the R1primer of sequence as shown in SEQ ID NO.6 are primer, carry out PCR, the 123rd amino acids namely obtaining encoding has been mutated into the R123I mutant gene sequence of Isoleucine by arginine; (2) with mutant gene sequence obtained in the previous step for template, the F2primer of sequence as shown in SEQ ID NO.7, the R2primer of sequence as shown in SEQ ID NO.8 are primer, carry out PCR, namely obtain the recombination of sequence as shown in SEQ ID NO.3; (3) by recombination sequence obtained in the previous step, be connected in pPIC9K expression vector, obtain recombinant plasmid pPIC9K-recombination, recombinant plasmid electricity transforms Pichia pastoris GS115, obtains recombination yeast engineering strain FemiGS115.
9. the preparation method according to claim 7 or 8, is characterized in that, described expression vector be following any one: pGAP ZA, pAO815, pGAP α A, pPIC9K, pPIC ZB.
10. the preparation method according to claim 7 or 8, it is characterized in that, described yeast host bacterium be following any one: pichia pastoris GS115, pichia pastoris KM71, pichia pastoris X-33, pichia pastoris SMD1168.
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Cited By (4)

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CN104694522A (en) * 2015-02-16 2015-06-10 中国人民解放军军事医学科学院放射与辐射医学研究所 Preparation method and application of recombinant acetylation cationoid trypsin
CN112280770A (en) * 2020-10-30 2021-01-29 江南大学 Trypsin mutant with improved heat stability
CN113637663A (en) * 2021-08-03 2021-11-12 江南大学 Trypsin mutant with improved heat stability
CN113717965A (en) * 2021-09-16 2021-11-30 江南大学 Streptomyces trypsin specificity transformation method and application thereof

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CN108559734B (en) * 2018-01-15 2020-09-04 江南大学 L-lactate dehydrogenase mutant with improved catalytic efficiency and application thereof

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104694522A (en) * 2015-02-16 2015-06-10 中国人民解放军军事医学科学院放射与辐射医学研究所 Preparation method and application of recombinant acetylation cationoid trypsin
CN112280770A (en) * 2020-10-30 2021-01-29 江南大学 Trypsin mutant with improved heat stability
CN112280770B (en) * 2020-10-30 2022-09-06 江南大学 Trypsin mutant with improved heat stability
CN113637663A (en) * 2021-08-03 2021-11-12 江南大学 Trypsin mutant with improved heat stability
CN113717965A (en) * 2021-09-16 2021-11-30 江南大学 Streptomyces trypsin specificity transformation method and application thereof
CN113717965B (en) * 2021-09-16 2023-07-18 江南大学 Streptomyces trypsin specific transformation method and application thereof

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