CN104725476B - A kind of hook formation oligopeptides and its application - Google Patents
A kind of hook formation oligopeptides and its application Download PDFInfo
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- CN104725476B CN104725476B CN201510149607.0A CN201510149607A CN104725476B CN 104725476 B CN104725476 B CN 104725476B CN 201510149607 A CN201510149607 A CN 201510149607A CN 104725476 B CN104725476 B CN 104725476B
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Abstract
The invention discloses a kind of hook formation oligopeptides for belonging to industrial biocatalytic and enzyme engineering field and its applications.The hook-shaped oligopeptides is characterized in that length is the oligopeptides that 3 12 amino acid residues are formed, and is formed from revolution hook formation by salt bridge (the ionic bond or strong hydrogen bonding) effect between oligopeptides internal amino acid residue.The hook-shaped oligopeptides label is inserted into the subunit end of enzyme, the activity and stability of enzyme significantly improve.The structure of hook-shaped oligopeptides label of the present invention and the method for improving enzymatic activity and stability can be commonly used to activity and the stability transformation of various industrial enzymes independent of the structure feature of enzyme itself.
Description
Technical field
The invention belongs to industrial biocatalytics and enzyme engineering field, and in particular to a kind of hook formation oligopeptides and its in target
Application in the activity of enzyme, stability transformation.
Background technology
Salt bridge, i.e., the Coulomb interactions between the amino acid of two kinds oppositely chargeds.Structural research shows thermophilic protein
Contain more polar amino acid residues than mesophilic homologous protein and forms more salt bridges, such as hyperthermophilic Gu bacterium
In the Pfu-GHD enzyme molecules of P.furiosus, every 10 residues just have 1.1 salt bridges, and mesophile Clostridium
In the Csy-GHD enzyme molecules of symbisun, every 10 residues only have 0.6 salt bridge.Either archeobacteria or bacterium albuminoid
The ratio of matter, total salt bridge content and salt bridge network all increases with the increase of heat resistance.On the other hand, in entire albumen texture
As in, the conformation of the free terminal of peptide chain is usually most unstable.By taking two kinds of thermophilic nitrile hydratases as an example, caused by the heat inactivation of enzyme
Conformation stretching, extension dissociation first occurs at peptide chain terminal residue, and the amino acid that its unstable degree is significantly larger than other regions is residual
Base (Liu J. etc., Journal ofMolecular Graphics and Modelling, 2008,27 (4)：529–535).It is right
The protein sequence of mesophilic nitrile hydratase carries out rite-directed mutagenesis, is inserted into comes from thermophilic nitrile respectively with subunit end inside its subunit
The salt bridge structure of hydrase, as a result, it has been found that, the mutant enzyme being transformed to nitrile hydratase salt bridge end is steady with significantly improving
Qualitative (Chen J. etc., Journal ofBiotechnology 2012,164：354–362).
In conventional genetic engineering and protein engineering research, the method being transformed to enzymatic activity and stability is usually
Carry out heterologous gene recombinant expression and gene site-directed/random mutation.These methods all rely on specific gene and protein sequence
Row.For example, Mitsubishi Li Yang Co., Ltd. discloses patent《Improved nitrile hydratase》(publication number：CN 1961072A), it is right
Nitrile hydratase β subunits the 93rd, the 167th and 219 amino acids residues carry out rite-directed mutagenesis, and the heat for improving nitrile hydratase is steady
It is qualitative.Tsinghua University constructs the nitrile hydratase of α subunit initiation codons mutation, and high activity table is realized in Escherichia coli
Up to (the patent No.：ZL200410042576.0, " a kind of nitrile hydratase and its encoding gene and application ")；Tsinghua University is also in China
Red (red) Rhodococcus sp Rhodococcus ruber are disclosed in patent of invention " a kind of nitrile hydratase gene cluster and its application "
In TH relevant structural gene and the controlling gene sequence (patent No. are expressed with nitrile hydratase height：ZL 200910076710.1)；
In Chinese invention patent " a kind of mutant nitrile hydratase ", it is resistance to disclose a kind of subunit end transformation nitrile hydratase of rite-directed mutagenesis enzyme
Method (the patent No. of hot, product tolerance and ultrasonic stability：ZL201110415465.X).Mitsubishi is melted into strain formula meeting
Society is to nitrile hydratase gene and albumen application patent from rhizobium《Novel protein with nitrile hydratase activity and volume
The gene of the code albumen》(application number：93106122.9)；Mitsui Chemicals, Inc is to coming from thermophilic Selective medium
The albumen of the nitrile hydratase of JCM3095 and encode its gene application patent《Participate in activation of nitrile hydratase albumen and
Encode its gene》(the patent No.：CN1250568C)；《Novel nitrile hydratase》The gene is had studied in recombination bacillus coli
Express (the patent No.：CN1243825C)；German Degussa applies《The nitrile hydratase of Rhod》(the patent No.：
CN1930299B)。
Invention content
One of the objects of the present invention is to provide a kind of hook formation oligopeptides (or referred to as oligopeptides labels).
The hook formation oligopeptides, is made of 3-12 amino acid residue, wherein at least containing there are one positive electricity amino acid
Residue and a negative electricity amino acid residue, and it is residual that 1 amino acid is at least spaced between the positive electricity amino acid and negative electricity amino acid
Positive negative ion pair is formed between base and positive electricity amino acid and negative electricity amino acid.
Preferably, above-mentioned hook formation oligopeptides, is made of 5-12 amino acid residue, wherein at least containing there are one positive electricity
Amino acid residue and a negative electricity amino acid residue, and 1 ammonia is at least spaced between the positive electricity amino acid and negative electricity amino acid
Positive negative ion pair is formed between base acid residue and positive electricity amino acid and negative electricity amino acid.
Preferably, above-mentioned hook formation oligopeptides, it is glycine to have 5 amino acid residue GXPYG, wherein G, and P is
Proline, X and Y are respectively the positive electricity amino acid and negative electricity amino acid of oppositely charged.
The positive electricity amino acid is arginine Arg (R) or lysine Lys (K)；The negatively charged amino acid is aspartic acid
Asp (D) or glutamic acid Glu (E).
Above-mentioned hook formation oligopeptides, the amino acid residue GXPYG are GRPEG, GRPDG, GKPEG or GKPDG.
Amino acid sequence with above-mentioned GX ... P ... YG, amino acid residue are 5-12, such as amino acid residue is 6
It is a or 7 or 9 or 11, increase and decrease through amino acid, can also form the oligopeptide structure sequence of " hook " structure.
It, can be independent of mesh by designing, building with unique from the oligopeptide structure sequence for turning round " hook " structure
Mark enzyme gene order and protein sequence, by hook-shaped oligopeptides module as label " patch " the ends N- of different protease,
The ends C- or the ends N/C-, the riveting by rigid salt bridge structure reduce surely peptide chain end the Degree of Structure Freedom and its it is heated/
Stretching, extension inactivation under the adverse circumstances such as organic solvent, to quickly, efficiently, universality promote stability or the work of various enzymes
Property.
An object of the present invention, which also resides in, provides a kind of method improving enzyme resistance and a kind of side for improving enzymatic activity
Method.
Any of the above-described hook-shaped oligopeptide sequence, is inserted into the subunit of target enzyme by the method for the raising enzyme resistance
End.
Any of the above-described hook-shaped oligopeptide sequence is inserted into the subunit end of target enzyme by the method for the raising enzymatic activity
End.
The target enzyme is nitrile hydratase or its mutant or nitrilase or its mutant.
It can be expanded by One_step PCR by introducing hook formation oligopeptide sequence in PCR (PCR) primer
It can be inserted into hook-shaped oligopeptides label gene in the ends N- of target enzyme or the ends C-, obtain stable enzyme.
An object of the present invention, which also resides in, provides a kind of enzyme.
A kind of enzyme is formed the subunit end that any of the above-described hook-shaped oligopeptide sequence is inserted into target enzyme, the target
Enzyme is nitrile hydratase or its mutant or nitrilase or its mutant.
Particularly, an object of the present invention, which also resides in, provides a kind of nitrile hydratase.
A kind of nitrile hydratase, the subunit end that the hook-shaped oligopeptide sequence described in above-mentioned one is inserted into target enzyme is formed, described
Target enzyme is nitrile hydratase or its mutant；The subunit end be it is following more than one：The N-terminal of α subunits, the ends C of α subunits
It holds, the C-terminal of the N-terminal of β subunits or β subunits.It is 1-3 that hook formation oligopeptide sequence, which is inserted into total quantity,.
The purpose of the present invention lie also in provide the gene for encoding above-mentioned nitrile hydratase, the expression vector containing the gene,
Transformant containing the gene.
The transformant is Escherichia coli or Rhodococcus sp.
Calcium Chloride Method or Electroporation Transformation method (Sambrook J etc. can be used in the construction method of above-mentioned transformant
.Molecular Cloning:A Laboratory manual.Cold Spring Harbor,NY:Cold Spring
Harbor Laboratory Press.1989) by vector introduction recipient bacterium.Improvement nitrile hydratase gene can also directly be inserted
Enter the chromosome of transformant.
When the target enzyme is double subunit nitrile hydratases, construction method is to nitrile hydratase NHaseM(it is abbreviated as NHM, sequence
Row are shown in the SEQ ID NO of " a kind of mutant nitrile hydratase (CN102517271A) "：Amino acid sequence shown in 1) and mutation nitrile
Hydrase SBM (the recombination nitrile hydratase catalytic kinetics Journal of Chemical Industry and Engineering of Chen Jie etc., coupled ends salt bridge and rite-directed mutagenesis,
2014,65 (7)：α subunits N-terminal in amino acid sequence 2821-2828) or C-terminal, β subunits N-terminal or C-terminal difference
4 kinds of salt bridge labels are inserted into, it is 1 pair, 2 pairs or 3 pairs that salt bridge, which is inserted into total quantity,.
Beneficial effects of the present invention：The hook formation oligopeptide sequence based on salt bridge interaction of design construction of the present invention,
It can be inserted directly into the subunit end of different industrial enzymes independent of the amino acid sequence of target enzyme, by limiting subunit end
Stretching, extension degree of freedom, significantly improve the resistance of enzyme.By taking nitrile hydratase as an example, when the α subunits C-terminal or β in nitrile hydratase NHM
When subunit C-terminal is inserted into oligopeptides label, the activity of mutant nitrile hydratase improves 30% or more；42 DEG C of preservation 3h, mutation nitrile hydration
The enzyme activity retention rate of enzyme is increased to 23% from the 15% of control enzyme；10% acrylamide impregnates 20min, the enzyme of mutant nitrile hydratase
Retention rate living is increased to 45% from the 27% of control enzyme.When the β subunit C-terminals of nitrile hydratase SBM are inserted into oligopeptides label, enzyme
It is living to improve 30% or so；The enzyme activity retention rate of 42 DEG C of preservation 3h, mutant nitrile hydratase are promoted from the 18% of control enzyme to 24%；
10% acrylamide impregnates 20min, and the enzyme activity retention rate of mutant nitrile hydratase is promoted from the 25% of control enzyme to 35%.The present invention
It is a kind of industrial enzyme resistance remodeling method of universality, there is important application value and good prospects for commercial application.
Description of the drawings
The present invention is further illustrated with specific example below in conjunction with the accompanying drawings.
Fig. 1 is mutant enzyme enzyme activity schematic diagram obtained by the α subunit C-terminals of different length oligopeptides label insertion NHM, wherein
SB1, SB2, SB3 respectively represent amino acid sequence GRPEG, GRGPGEG, GRGGPGGEG；
Fig. 2 is the α subunits N, C-terminal and β subunits N, C-terminal that oligopeptides label G RPEG (being abbreviated as RE, similarly hereinafter) is inserted into NHM
Afterwards, the four mutant enzyme α N/ α C/ β N/ β C enzyme activity schematic diagrames obtained；
Fig. 3 is oligopeptides label G RPEG (RE), GRPDG (RD), GKPEG (KE), GKPDG (KD) are inserted into the Asias β of NHM respectively
After base C-terminal, obtained four mutant enzyme enzyme activity schematic diagrames；
Fig. 4 is oligopeptides label G RPEG (RE), GRPDG (RD), GKPEG (KE), GKPDG (KD) are inserted into the Asias α of NHM respectively
After base C-terminal, obtained four mutant enzyme enzyme activity schematic diagrames；
Fig. 5 is the α subunit C-terminals that oligopeptides label RD is inserted into β subunits C-terminal and RE/RD/KE/KD is inserted into NHM respectively
Afterwards, the four duoupoly peptide tag mutant enzymes and RE that obtain while tri- ends NHM are inserted into, an obtained three oligopeptides labels
Mutant enzyme enzyme activity schematic diagram；
Fig. 6 is 3 NHM oligopeptides mutant enzymes and the enzyme activity and stability contrast schematic diagram that compare NHM；
Fig. 7 is 3 SBM oligopeptides mutant enzymes and the enzyme activity and stability contrast schematic diagram that compare SBM.
Specific implementation mode
Embodiment 1 improves nitrile hydratase NHM gene mutations and its transformant structure
(1) method according to Chinese invention patent CN102517271A (a kind of mutant nitrile hydratase), is contained
The recombinant plasmid pET-NHM of nitrile hydratase gene segment.
Using plasmid pET-NHM as template, gene mutation is carried out using inverse PCR (PCR) method.Design
Primer is as shown in table 1, and wherein SB1, SB2, SB3 respectively represent amino acid sequence GRPEG, GRGPGEG, GRGGPGGEG；Table 2 is
The energy balane of several different length peptide fragments；Oligopeptides label GRPEG (is abbreviated as RE, similarly hereinafter), GRPDG(RD)、GKPEG(KE)、
GKPDG(KD)。
Table 1 is inserted into the primer designed by oligopeptides label
Mutation is purchased from Takara companies (Dalian) with kit Mutan BEST, and the reaction system of PCR is：
Reaction condition is：94 DEG C, 5min；94 DEG C of 0.5min, 60 DEG C of 0.5min, 72 DEG C of 8min are recycled 30 times；Last 72 DEG C
15min.Amplified production is attached by kit method, and a pair of of end salt can be expressed by obtaining one hook-shaped oligopeptides of insertion
The plasmid pET-NHM-M1 of bridge label, being attached by kit method again by amplified production as template using this plasmid can be with
Obtain the plasmid pET-NHM-M2 for being inserted into two hook-shaped oligopeptides two to end salt bridge label.It can similarly obtain being inserted into three hook strips
Plasmid pET-NHM-M3 of the hook-shaped oligopeptides three of shape to end salt bridge label.Above-mentioned plasmid is transferred to Escherichia coli with Calcium Chloride Method
E.coliTop10 enters E. coli BL21 (DE3), solid LB trainings after sequence verification is correct with Calcium Chloride Method conversion
37 DEG C of base (kalamycin resistance) is supported to be incubated overnight to growing the single bacterium colony being of moderate size to get transformant BL21 (DE3)/pET-
NHM-M1/M2/M3。
Nitrile hydratase SBM can similarly be transformed, and (the recombination nitrile hydratase of Chen Jie etc., coupled ends salt bridge and rite-directed mutagenesis is urged
Change dynamics Journal of Chemical Industry and Engineering, 2014,65 (7)：2821-2828), transformant BL21 (DE3)/pET-SBM-M1/M2/M3 is obtained.
The energy balane of 2 several different length peptide fragments of table
Wherein, M1 indicates a pair of of salt bridge mutation, including：(α subunit N-terminals are inserted into 5 amino acid salt bridges to α C-SB1
GRPEG), α C-SB2 (α subunit N-terminals are inserted into 7 amino acid salt bridge GRGPGEG), (α subunit N-terminals are inserted into 9 ammonia to α C-SB3
Base hydrochlorate bridge GRGGPGGEG), α N-RE (α subunit N-terminals are inserted into GRPEG salt bridges), α C-RE, β N-RE, β C-RE, β C-RD, β C-
KE, β C-KD；M2 indicates two pairs of salt bridge mutation, including：β C-RE＆ α N-RE, β C-RE＆ α C-RE, α N-RE＆ α C-RE；M3 indicates three
Salt bridge is mutated, including β C-RE＆ α N-RE＆ α C-RE.
Expression of 2 present invention improvement nitrile hydratase of embodiment in transformant
The salt bridge mutant nitrile hydratase in end of the present invention that embodiment 1 is obtained is in inducible expression bacterial strain E.coli BL21
(DE3) shaking flask culture is carried out in/pET-NHM-M1/M2/M3 and E.coli BL21 (DE3)/pET-SBM-M1/M2/M3.First
(group becomes in the LB liquid medium of the kanamycins containing 50mg/L：50ml/300ml shaking flasks, peptone 10g/L, yeast powder
5g/L, sodium chloride 10g/L, pH 7.0) inoculation single bacterium colony, 37 DEG C, 200rpm culture 12h make kind of a bottle.
Culture in the LB liquid medium (50ml) of the kanamycins containing 50mg/L is transferred to according to 1% inoculum concentration from kind of bottle
2.5h or so to OD600=2.2% 0.5mol/L lactose, 0.2% 0.2mol/L CoCl is added2It is induced as derivant
Nitrile hydratase is expressed.28 DEG C culture 8 hours after harvest cell carry out enzyme activity determination.
Enzyme activity determination is using acrylonitrile as substrate, using gas chromatography.Take the phosphate PBS of 3.7ml 50mM pH7.0 slow
Fliud flushing (50mM disodium hydrogen phosphates and 50mM potassium dihydrogen phosphates) and the bacterium solution of 1ml are fitted into 7ml Eppendorf pipes, constant temperature to 20
DEG C, rapid mixing after 100 μ l acrylonitrile is added, at the same time, presses manual time-keeping, after five minutes, 200 μ l are added in accurate response
2.5M HCl terminate reaction.After reaction solution is centrifuged, mixed in equal volume with 0.4% acetamide (internal standard) solution, using gas phase
Chromatograph GC-2010 (SHIMADZU, Japan) internal standard method measures acrylamide concentration.190 DEG C of column temperature detects 260 DEG C of temperature, nitrogen
Gas velocity, 25cm/min.Enzyme-activity unit is defined as the enzyme amount needed for 1 μm of ol acrylamide of catalysis generation per minute.
The result shows that different length oligopeptides label is inserted into salt bridge end (Fig. 1), obtained recombinant bacterium E.coli
In BL21 (DE3)/pET-NHM-SB1/SB2/SB3, NHM-SB1 enzyme activity is suitable with control NHM, illustrates the oligopeptides of 5 amino acid
Label influences minimum for enzyme activity, this also matches with energy computation results (table 2)；Recombinant bacterium E.coli BL21 (DE3)/
In pET-NHM- α N/ α C/ β N/ β C (Fig. 2), the relatively control of NHM- α C and NHM- β C enzyme activity is slightly promoted, and is illustrated in a pair of of oligopeptides mark
It is maximally efficient in the transformation of subunit α C and β C-terminals in label mutation transformation, and it can be seen that the ends subunit β-N from protein electrophorese figure
Oligopeptides label is inserted at end influences the expression of enzyme；In recombinant bacterium E.coli BL21 (DE3)/pET-NHM- β RE/ β RD/ β KE/ β KD
In (Fig. 3) and α RE/ α RD/ α KE/ α KD (Fig. 4), the enzyme activity relatively control NHM bacterium of mutant nitrile hydratase promote 30-35%；At two pairs
In oligopeptides label recombinant bacterium E.coli BL21 (DE3)/pET-NHM-RD/RE, RD/RD, RD/KE, RD/KD, NHM-RD/RE enzymes
Promotion effect living is best, but and be not as high (Fig. 5) as the mutant enzyme enzyme activity of a pair of of oligopeptides label.And it is prominent in end salt bridge coupling fixed point
Become in the SBM applications of transformation (Fig. 7), the mutant enzyme enzyme activity that β C-terminals are inserted into oligopeptides label RE improves 28%.As it can be seen that end is few
Peptide tag can promote the activity of enzyme to a certain extent.
The resistance assessment of 3 present invention improvement nitrile hydratase of embodiment
50ml recombinant bacterium E.coli BL21 (the DE3)/pET-NHM-M1/M2/M3 and E.coli that embodiment 2 is harvested
BL21 (DE3)/pET-SBM-M1/M2/M3 cells (expression end oligopeptides mutant nitrile hydratase) and control strain E.coli BL21
(DE3) bodies such as/pET-NHM and E.coli BL21 (DE3)/pET-SBM cells (nitrile hydratase of the expression without oligopeptides label) use
Long-pending sterile water centrifuge washing is primary, then is resuspended in spare in the PBS buffer solution of isometric 50mM pH 7.0.
In thermal stability experiment, it is small to place 3 in 42 DEG C of water-baths for the recombinant cell for respectively taking 5ml to be resuspended in PBS buffer solution
When.The residual enzyme activity of horizontal survey end oligopeptides mutant nitrile hydratase and former nitrile hydratase, the results showed that, 42 DEG C of preservation 3h dash forward
The enzyme activity retention rate for becoming nitrile hydratase NHM- β RE is increased to 23% from the 15% of control enzyme；Mutant nitrile hydratase SBM- β RD enzyme activity
Retention rate is promoted from the 18% of control enzyme to 24%.As it can be seen that the heat that end oligopeptides label can promote enzyme to a certain extent is steady
It is qualitative.
In product tolerance test, 60% acrylamide is added in the recombinant cell for respectively taking 1ml to be resuspended in PBS buffer solution
200 μ L of solution impregnate 20min, are then centrifuged for supernatant and are cleaned twice with PBS buffer solution.The oligopeptides mutation of horizontal survey end
The residual enzyme activity of nitrile hydratase and former nitrile hydratase, the results showed that, 10% acrylamide impregnates 20min, mutant nitrile hydratase
The enzyme activity retention rate of NHM- β RD is increased to 45% from the 27% of control enzyme；The enzyme activity retention rate of mutant nitrile hydratase SBM- β KE from
Compare enzyme 25% is promoted to 35%.As it can be seen that end oligopeptides label can promote the product tolerance of enzyme to a certain extent.
Claims (6)
1. a kind of method improving enzyme resistance, which is characterized in that hook-shaped oligopeptide sequence is inserted into the subunit end of target enzyme, institute
It states hook formation oligopeptides to be made of 5 amino acid residue GXPYG, the amino acid residue GXPYG is GRPEG, GRPDG, GKPEG
Or GKPDG；The target enzyme is nitrile hydratase.
2. a kind of method improving enzymatic activity, which is characterized in that hook-shaped oligopeptide sequence is inserted into the subunit end of target enzyme, it is described
Target enzyme is nitrile hydratase, and the hook formation oligopeptides is made of 5 amino acid residue GXPYG, the amino acid residue GXPYG
For GRPEG, GRPDG, GKPEG or GKPDG.
3. a kind of enzyme, which is characterized in that the subunit end that hook-shaped oligopeptide sequence is inserted into target enzyme is formed, and the target enzyme is nitrile
Hydrase, the hook formation oligopeptides are made of 5 amino acid residue GXPYG, the amino acid residue GXPYG be GRPEG,
GRPDG, GKPEG or GKPDG.
4. enzyme according to claim 3, which is characterized in that the target enzyme is nitrile hydratase；The subunit end be with
Descend more than one：The C-terminal of the N-terminal of α subunits, the C-terminal of α subunits, the N-terminal of β subunits or β subunits.
5. enzyme according to claim 3 or 4, which is characterized in that it is 1-3 that hook formation oligopeptide sequence, which is inserted into total quantity,.
6. the expression vector or transformant of the gene containing any one of the claim 3-5 enzymes.
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| WO2001046125A2 (en) * | 1999-12-21 | 2001-06-28 | Astrazeneca Ab | Cd45 inhibitors |
| CN1584024A (en) * | 2004-05-24 | 2005-02-23 | 清华大学 | Nitrile hydratase and its coding gene and use |
| CN1729288A (en) * | 2002-12-19 | 2006-02-01 | 三井化学株式会社 | Novel nitrile hydratase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001046125A2 (en) * | 1999-12-21 | 2001-06-28 | Astrazeneca Ab | Cd45 inhibitors |
| CN1729288A (en) * | 2002-12-19 | 2006-02-01 | 三井化学株式会社 | Novel nitrile hydratase |
| CN1584024A (en) * | 2004-05-24 | 2005-02-23 | 清华大学 | Nitrile hydratase and its coding gene and use |
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