CN104328102B - 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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CN104328102B
CN104328102B CN201410612164.XA CN201410612164A CN104328102B CN 104328102 B CN104328102 B CN 104328102B CN 201410612164 A CN201410612164 A CN 201410612164A CN 104328102 B CN104328102 B CN 104328102B
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sequence
seq
mutant
trypsin
enzyme activity
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CN104328102A (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)

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

Trypsin mutant and its construction method that a kind of enzyme activity improves
Technical field
The present invention relates to a kind of trypsin mutant of enzyme activity raising and its construction method, belong to technique for gene engineering neck Domain.
Background technology
Trypsin, as the relatively early a kind of important kind being found to apply in serine protease, is found in earliest In the intestinal Digestive system of mammal.The trypsin of commercialization is many to be extracted from mammalian pancreatic, abundant due to containing in pancreas Protease, difficulty is brought to trypsin separating-purifying.In addition, the trypsin of mammal source mostly is pancreatin mixing , there is the immunogenicity harm to human body in medical applications in thing.
SGT research, koo-bon-joon etc. utilizes streptomycete to commonly use host streptomyces The pwhm3 plasmid (ermp, erythromycin promoter) that lividans1326 and streptomycete are commonly used obtains secreting, expressing in belonging to bacterium together Recombinant trypsin, this plasmid is transferred to and can produce tryptic streptomyces griseuses by chi-won-jae etc. Express in streptomyces griseus ifo13350, daisuke nohara et al. (1998) is by streptomycete trypsin Expressed in e.coli system with inclusion bodies.Early-stage Study is in Pichia sp. with ripe trypsin form table Reach the trypsin in streptomyces griseuses source.The present invention adopts simple point mutation technology, based on homology modeling methods, by selecting Specific amino acids optimize trypsin molecular structure, improve enzyme activity.
Heterogenous expression trypsin distinct issues are that expressing quantity is low, trypsin enzyme activity is low.Therefore, pinpoint and dash forward Become and transform trypsin, improve enzymatic activities, trypsin industry is melted into product demand and reduction production cost has weight for meeting Want meaning.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of trypsin mutant of enzyme activity raising and its construction method.
The present invention provides the trypsin mutant that a kind of enzyme activity improves it is characterised in that the aminoacid of described mutant Sequence 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 basis of the aminoacid as shown in sequence seq id no.2, and the tyrosine of the 1st is dashed forward Become Phenylalanine (y1f), the arginine of the 123rd is mutated into isoleucine (r123i) simultaneously.
In previous research work, this research team has obtained a kind of tryptic recombinant yeast pichia pastoris work of heat production stability Journey bacterium, trypsin as shown in seq id no.4 for this yeast expressed nucleotide sequence.
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 expressing described trypsin mutant.
The preparation method of described genetic engineering bacterium, is on the basis of sequence shown in seq id no.4, by the 123rd Arginine has been mutated into isoleucine, obtains mutant r123i, simultaneously by the tyrosine of the 1st be mutated into Phenylalanine (with Y1f represents), obtain recombination, recombination is connected to expression vector and obtains recombiant plasmid, recombinant plasmid transformed is to yeast Yeast gene engineering bacteria is obtained in Host Strains.
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) arranged 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) is primer, carries out pcr and obtains r123i mutant；
(2) with the gene order of mutant obtained in the previous step as template, f2primer (sequence such as seq id no.7 institute Show), r2primer (sequence is as shown in seq id no.8) be primer, carry out pcr, that is, obtain sequence as shown in seq id no.3 Recombination.
(3) by recombination sequence obtained in the previous step, it is connected in ppic9k expression vector, obtains recombiant plasmid Ppic9k- recombination, recombiant plasmid electricity conversion pichiapastoris gs115, obtain recombination yeast engineered strain, name For femigs115.
The present invention, on the basis of heat stability trypsin, transforms trypsin molecule knot by rite-directed mutagenesises biotechnology Structure, obtains a plant height and produces trypsin Pichia yeast engineering, fermentation technology is simple, enzyme activity is high, in 3l tank fermentation recombinant bacterium The trypsin amidase enzyme activity of femigs115 is relatively mutated front bacterial strain and improves 3.3 times, and trypsin esterase enzyme activity improves 2.7 again.The substrate affinity of simultaneous mutation body enzyme exmti y1f improves 1.3 times, and catalytic efficiency improves 35.7%, simultaneously than enzyme Raising 35.4% alive.R123i is from the mutation of degradation site, it is to avoid after the extracellular maturation of trypsin from Degradation, y1f dashes forward Become and improve tryptic exocytosiss.The present invention solves that trypsin heterogenous expression enzyme activity is low must to limit sex chromosome mosaicism, is Trypsin molecular modification provides a new thinking.
Brief description
Fig. 1: Fixedpoint mutation modified trypsin expression vector establishment figure；
Fig. 2: recombinant bacterium femigs115 trypsin amidase enzyme activity figure；
Fig. 3: recombinant bacterium femigs115 with original strain (wt) amidase enzyme activity comparison diagram；
Fig. 4: recombinant bacterium femigs1153l tank fermentation extracellular trypsin sds-page figure；Wherein 1 represents ck (comparison bacterium Strain), 2-7 represents the situation that femigs115 cultivates 0h, 24h, 48h, 72h, 96h, 120h, 144h respectively.
Specific embodiment
Trypsin amidase enzyme activity determination method: at 37 DEG C, measure 100 μ l crude enzyme liquids with 900 μ l bapna solution In the reaction tank of optical path 0.5cm, extinction value changes in 10min under 410nm, obtain a410nm/min.Enzyme activity is defined as: At 37 DEG C, the enzyme amount that δ a410nm/min raises required for 0.1 is 1 amidase hydrolyzing unit.(amidase enzyme activity is tighter Careful trypsin enzyme activity definition mode, because substrate is trypsin specific substrate.)
Trypsin esterase enzyme activity determination method: at 25 DEG C, measure 200 μ l crude enzyme liquids and exist with 3ml baee substrate solution In the reaction tank of optical path 1cm, extinction value changes in 1min under 253nm, obtain δ a253nm/min.Enzyme activity is defined as: At 25 DEG C, δ a253nm/min raises 0.001 and is tryptic 1 esterase hydrolyzed unit.
Embodiment 1 contains the structure of the recombinant vector of trypsin mutant
Using the method construction of recombinant plasmid vector shown in Fig. 1.
(1) r123i mutant obtains:, as template, with f1primer, (sequence is such as the sequence shown in seq id no.4 Shown in seqid no.5), r1primer (sequence is as shown in seq id no.6) be primer, carry out pcr and obtain coded amino acid sequence Arrange mutant (r123i) gene order that the 123rd arginine sports isoleucine.
(2) with the gene order of mutant obtained in the previous step as template, with f2primer (sequence such as seq id no.7 Shown), r2primer (sequence is as shown in seq id no.8) be primer, carry out pcr, obtain sequence as shown in seq id no.3 The recombination sequence of y1f mutant (encode).Recombination is connected on sample t carrier.
(3) the sample t carrier containing recombination obtained in the previous step is used noti, ecori double with ppic9k respectively Enzyme action, after purification 16 DEG C of t4 ligase overnight connect.Connection product chemical method converts jm109 competent cell.Conversional solution is coated with Containing kanamycin (50mg/l) lb flat board, extract the recombiant plasmid that the checking of plasmid double digestion builds, be named as ppic9k- exmtiy1f.Examining order is completed by the raw work in Shanghai.
Embodiment 2 is produced ripe trypsin Yeast engineering bacteria and is built
The recombiant plasmid ppic9k-exmtiy1f sal i linearisation that embodiment 1 is obtained, electroporated Pichiapastoris gs115 competent cell, concrete grammar is as follows:
1) the pichiapastoris gs115 of inoculation ypd flat board activation, in 25ml/250ml triangular flask, overnight trains for 30 DEG C Support；1% inoculates above-mentioned culture fluid in 50ml/500ml triangular flask, and cultivating cell concentration od600 is 1.3～1.5；
2) 5000r/min, 4 DEG C of centrifugation 10min collects thallines, use 50ml, 25ml sterilized water suspension cell respectively；
3) the resuspended above-mentioned cell of 5ml1m Sorbitol, 5000r/min, 4 DEG C of centrifugation 10min collects thallines；
4) the resuspended above-mentioned cell of 500 μ l1m Sorbitol, subpackage 80 μ l/1.5ml ep manages for Electroporation-competent cells；
5) 20 μ l linearization plasmids are mixed with above-mentioned 80 μ l competent cells, stand 15min on ice；
6) said mixture adds aseptic electricity conversion cup (0.2cm) of pre-cooling, and 1500v, 25 μ f, 200 ω shock by electricity once, plus Enter 1ml1m Sorbitol；
7) take said mixture 150 μ l coating 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 1,2,3,4mg/ml (heredity Mycin) in ypd flat board, the single bacterium colony selected in 4mg/ml Geneticin flat board is used for shake flask fermentation, surveys trypsin enzyme activity, Select enzyme activity highest recombinant bacterium, be named as recombinant bacterium femigs115.
Embodiment 3 recombinant yeast pichia pastoris 3l tank is cultivated
The recombinant bacterium femigs115 that embodiment 2 is built, as producing bacterial strain, activates in ypd flat board.Seed liquor is cultivated, Inoculation 50ml/250ml seed culture medium, 30 DEG C, 220r/min cultivates 24h.10% inoculation 800ml/3l fermentation medium, Ph5.5, cultivates for 30 DEG C stage by stage: 0-17h, 500rmp/min culture, and do drops to 8% about from 100%, then go up to 60% about；17-30h, rotating speed gradually rises up to 1000rmp/min, exponential fed-batch 50% glycerol, and do begins to decline to 10% left side The right side, subsequently rises to 70%；30-144h, stream Jia 1.8%, and (v/v) methanol induction produces trypsin.To express nucleotide sequence such as Tryptic Pichia yeast shown in seq id no.4 is comparison (bacterial strain do not undergone mutation).
Seed culture medium (g/l): peptone 20, yeast extract 10, glucose 20.
Fermentation medium (g/l): glycerol 40；k2so418；koh4.13；mgso4·7h2o14.9；h3po427ml； caso40.948；Trace element ion liquid (ptm1) 4.4ml；121 DEG C of sterilizing 15min.
ptm1(g·l–1): cuso4 5h2o6；ki0.09；mnso4·h2o3；h3bo30.02；mona2o4·2h2o0.2； coc l20.5；zncl220；feso4·7h2o65；biotin0.2；h2so45ml；0.22 μm of filtration sterilization.
Glycerol (the l containing 12ml of feed supplement growth medium: 50% (w/v)–1ptm1).
In sweat, the enzyme activity of Yeast engineering bacteria is as shown in Figure 2.Figure it is seen that starting to induce in fermentation 40h Producing enzyme, enzyme activity increases with induction time, and during fermentation 133h, trypsin amidase enzyme activity reaches 85.3u/ml.And the weight before being mutated Group bacterium 3l fermentation amidase enzyme activity is up to 19.8u/ml, and enzyme activity improves 3.3 times.Determine recombinant bacterium femigs115 simultaneously With the trypsin esterase enzyme activity of control strain, result shows: the esterase enzyme activity of recombinant bacterium femigs115 is up to 5954u/ml, Compare the 1624u/ml of control strain, improve 2.72 times.
Analysis recombinant trypsin (exmti y1f) zymologic property after purification, such as table 1, exmti y1f substrate affinity Improve 1.3 times, catalytic efficiency improves 35.7%, specific enzyme activity simultaneously improves 35.4%.Due to substrate affinity and catalytic efficiency Improve, increased the specific enzyme activity of mutant exmti y1f.This shows that this invention improves Trypsin by the innovation mode of mutation The zymologic property of enzyme.On the other hand, because the 1st amino acids occur the mutation of y1f, when 3l tank ferments, the pancreas egg of mutant The exocytosiss expression of white enzyme improves (as Fig. 4).Exmti y1f expressing quantity after mutation is 44.55mg/l, with respect to Before mutation, (14.03mg/l) improves 2.18 times.
Table 1exmti y1f mutant reactive kinetics parameters
Although the present invention is open as above with preferred embodiment, it is not limited to the present invention, any is familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be by being defined that claims are defined.

Claims (10)

1. the trypsin mutant that a kind of enzyme activity improves is it is characterised in that the aminoacid sequence of described mutant is seq id Sequence shown in no.1.
2. mutant according to claim 1 is it is characterised in that described mutant is in such as sequence seq id no.2 institute On the basis of the aminoacid showing, the arginine of the 123rd is mutated into isoleucine, the tyrosine of the 1st has been mutated simultaneously Become Phenylalanine.
3. mutant according to claim 1 is it is characterised in that the nucleotide sequence of described mutant is seq id Sequence shown in no.3.
4. mutant according to claim 2 is it is characterised in that aminoacid sequence shown in described coding seq id no.2 Nucleotide sequence be the sequence shown in seq id no.4.
5. the recombinant expression carrier of the nucleotide sequence of mutant described in a kind of claim 1 containing coding.
6. the genetic engineering bacterium of trypsin mutant described in a kind of expression claim 1.
7. the preparation method of genetic engineering bacterium described in a kind of claim 6, is on the basis of sequence shown in seq id no.4, The arginine of the 123rd is mutated into isoleucine, the tyrosine of the 1st has been mutated into Phenylalanine simultaneously, obtain weight Group gene, recombination is connected to expression vector and obtains recombiant plasmid, recombinant plasmid transformed obtains in yeast host bacterium Yeast gene engineering bacteria.
8. preparation method according to claim 7 it is characterised in that methods described specifically: (1) is with seq id no.4 Shown nucleotide sequence is template, and f1primer as shown in seq id no.5 for the sequence, sequence are as shown in seq id no.6 R1primer is primer, carries out pcr, that is, the 123rd amino acids obtaining encoding have been mutated into isoleucine by arginine R123i mutant gene sequence；(2) with mutant gene sequence obtained in the previous step as template, sequence such as seq id no.7 institute F2primer, the r2primer as shown in seq id no.8 for the sequence showing is primer, carries out pcr, that is, obtains sequence such as seq id Recombination shown in no.3；(3) by recombination sequence obtained in the previous step, it is connected in ppic9k expression vector, obtains Recombiant plasmid ppic9k- recombination, recombiant plasmid electricity conversion pichia pastoris gs115, obtain recombination yeast engineering Bacterial strain femigs115.
9. preparation method according to claim 7 it is characterised in that described expression vector be following any one: pgap za、pao815、pgapαa、ppic9k、ppic zb.
10. preparation method according to claim 7 it is characterised in that described yeast host bacterium be following any one: pichia pastoris gs115、pichia pastoris km71、pichia pastoris x-33、pichi pastoris smd1168.
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Cited By (1)

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CN108559734A (en) * 2018-01-15 2018-09-21 江南大学 The l-lactate dehydrogenase mutant and its application that a kind of catalytic efficiency improves

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CN104694522B (en) * 2015-02-16 2018-06-12 中国人民解放军军事医学科学院放射与辐射医学研究所 A kind of preparation method and applications for recombinating acetylation cationic trypsase
CN112280770B (en) * 2020-10-30 2022-09-06 江南大学 Trypsin mutant with improved heat stability
CN113637663B (en) * 2021-08-03 2023-03-24 江南大学 Trypsin mutant with improved heat stability
CN113717965B (en) * 2021-09-16 2023-07-18 江南大学 Streptomyces trypsin specific transformation method and application thereof

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CN103173367A (en) * 2013-03-11 2013-06-26 江南大学 Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108559734A (en) * 2018-01-15 2018-09-21 江南大学 The l-lactate dehydrogenase mutant and its application that a kind of catalytic efficiency improves
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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