CN106085975B - A kind of efficient general policies for improving enzyme thermodynamic stability - Google Patents
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Abstract
The invention discloses a kind of general policies for efficiently improving enzyme thermodynamic stability；The specific implementation route of the strategy are as follows: centered on the catalytic residue of zymophore, surrounding is selected according to the B-factor value in the enzyme crystal structure Within high flexibility residue be target site, pass through screen mutation, obtain enzyme thermodynamic stability improve mutant.Under the guidance of the strategy, the thermal stability of the fold lipase from candida sp 1 of high level of architectural complexity is significantly improved.The stability change of the albumen (CalB) of minimum albumen (LipA) and moderate complexity is as a result, detective distance catalytic residue on integrated structureThe prominent success rate that can significantly improve enzyme mutant of neighbouring high B-factor residue.Meanwhile it will lay a good foundation by the emic network analysis of mutant structure-for further investigation enzyme regional stability law, development enzyme stabilization technology.
Description
Technical field
The present invention relates to a kind of general policies for efficiently improving enzyme thermodynamic stability.Pass through Pymol and B-FITTER
Software enzyme analysis catalytic residueWithin high flexibility amino acid；Thermodynamic stability is filtered out by saturation mutation library to mention
High mutant.The structure and functional analysis of mutant and mutant more particularly to fold lipase from candida sp 1.
Background technique
Enzyme is a kind of efficient biocatalyst, compared with chemical catalyst, enzyme molecule have high efficiency, high specificity and
The advantages that reaction condition is mildly pollution-free has important application value in scientific research and industrial production.However in high pressure, height
Under the mal-conditions such as warm, extreme pH, enzyme is easy unwinding and loses activity, and this severely limits the extensive uses of enzyme, therefore, how
The stability for improving enzyme becomes an important topic of current research.
With the development of structure biology, more and more enzymatic structures are parsed.Have more at present based on single enzyme
Structural analysis carries out the report that design and rational improves enzyme stability.Such as Cerdobbel (Cerdobbel, A.et
al.Increasing the thermostability of sucrose phosphorylase by a combination
of sequence and structure based mutagenesis[J].Protein Engineering Design and
Selection, 2011,24 (11): 829-834.) et al. by mutating acid generate electrostatic interaction, improve sucrose
2.6 times of the half-life period that 60 DEG C of phosphorylase；Wang(Wang.Y.et al.Improved thermal performance of
Thermomyces lanuginosus GH11xylanase by engineering of an N-terminal
Disulfide bridge [J] .Bioresource Technology, 2012,112:275-279.) et al. by introducing two sulphur
Key makes thermophilic fungal zytase improve 20 times in 70 DEG C of half-life period；Gallardo (Gallardo,et
al.Structural insights into the specificity of xynl0B from Paenibacillus
barcinonensis and its improved stability by torced protein evolution[J]
.Journal of Biological Chemistry, 2010,285 (4): 2721-2733.) et al. by increasing protein
Hydrophobic interaction improves series bacillus zytase Xyn10B in 20 times of half-life period of 50 DEG C.These achievements are mostly
Example analysis, there is no rule that can follow.With the introducing of proteomics concept, enzyme stabilization strategy has new development.By right
The phytase multisequencing of separate sources is analyzed, and wherein consistency site guidance mutation is found, successfully by its TmValue promotes 22 DEG C
(Lehmann, M.et al.From DNA sequence to improved functionality:Using protein
sequence comparisons to rapidly design a thermostable consensus phytase[J]
.Protein Engineering, 2000,13 (1): 49-57.)；Biostatistics analysis is carried out to multiple lactamase sequences,
Its ancestral gene is speculated, successfully by its TmValue promotes 7 DEG C of (Bershtein, S.et al.Intense neutral drifts
yield robust and evolvable consensus proteins[J].Journal of Molecular
Biology, 2008,379 (5): 1029-1044.)；The enzyme recombinant technique instructed using structural database, successfully by cellulase
Half-life period at 63 DEG C improves 30 times of (Heinzelman, et al.A family of thermostable fungal
cellulases created by structure-guided recombination[J].Proceedings of the
National Academy of Sciences, 2009,106 (14): 5610-5615.).Network analysis to enzyme system, effectively mentions
Rise the efficiency of enzyme stability transformation.But this method needs a large amount of data for statistical analysis, the deficiency of data volume also limits
The accuracy and application range of this method are made.Therefore, if key position relevant to enzyme stability can be absorbed in, establish letter
Single easy, rapidly and efficiently, versatile enzyme stabilization new strategy is the emphasis that the present invention is paid close attention to.
In order to understand the stabilizing mechanism of enzyme in depth, scholars compare the structure of Zimadzhunt L 340 Yu medium temperature enzyme, find the former
There is stronger rigidity in some regions, so that the climax of enzyme stability regionization research is brought, especially in enzymatic structure
Flexible region.In protein structures, B-factor becomes the important indicator of identification flexible amino acid.B-factor refers to
For the electron density of each composition amino acid atom in the space " fuzziness " of point of equilibrium, i.e. atom is to vibrate in zymoprotein
In the middle, B-factor reflects the Oscillation Amplitude of atom, to be converted into the flexibility of amino acid；B-factor is bigger, then amino
It is sour flexible bigger.Around this principle, well-known scholar Reetz (Reetz, the M.T.et al.Iterative of Germany
saturation mutagenesis on the basis of B-factors as a strategy for increasing
Protein thermostability [J] .Angewandte Chemie International Edition, 2006,45
It (46): 7745-7751. is) research pair with bacillus subtilis lipase the smallest in nature (LipA:181 amino acid)
As having chosen maximum 10 residues of B-factor in entire enzyme molecule as target site, constructing iteration saturation mutation library sieve
Select stability-enhanced mutant.This 10 target sites are entirely located in enzyme molecule surface.Screening through excessively taking turns, optimal mutation
Body is promoted to 16h from 2min in 55 DEG C of half-life period, as a result very significant.After document report, it is extensive academia has been obtained
Concern there is now more document utilization B-factor analyses and be transformed to enzyme stability.But when research object is streptomycete
Phosphatidase (PLD:509 amino acid) (J.et al.Improving thermostability of
phosphatidylinositol synthesizing Streptomyces phospholipase D[J].Protein
Engineering Design and Selection, 2012,25 (8): 415-424.) when, it is highest to enzyme B-factor residual
Base is mutated, and enzyme stability promotes amplitude and is not obvious, and is promoted to 34.6min by 25.9min in 65 DEG C of half-life period.To
Illustrate that there are still defects for the modification of the surface region albumen increasingly complex for molecule.
Candida rugosa lipasel (LIP1) has biggish molecular weight and complicated structure (534 amino acid
Composition).Moreover, LIP1 is a kind of important industrial applicability lipase again, there is the substrate of high catalytic efficiency and wide scope
Selectivity (Akoh, C.C.et al.Protein engineering and applications of Candida rugosa
Lipase isoforms [J] .Lipids, 2004,39 (6): 513-526.)；In medical applications field, its chirality is usually used in
Important medical product is split, such as: naproxen, Ketoprofen and brufen (Chavez-Flores, et al.Facile
Conversion of racemic ibuprofen to (s)-ibuprofen [J] .Tetrahedron:Asymmetry,
2012,23 (3): 237-239；Lee, E.G., et al.Enantioselective hydrolysis of racemic
naproxen methyl ester by two-step acetone treated Candida rugosa lipase[J]
.Process Biochemistry, 2001,37 (3): 293-298；Rangheard, M.S., et al.Multi-
Competitive enzymatic reactions in organic media:A simple test for the
determination of lipase fatty acid specificity[J].Biochimica et Biophysica
Acta (BBA)-Lipids and Lipid Metabolism, 1989,1004 (1): 20-28；Tsai, S.W.et
al.Surfactant enhancement of(s)-naproxen ester productivity from racemic
Naproxen by lipase in isooctane [J] .Biotechnology and Bioengineering, 1996,51
(2): 148-156；L ó pez, N.et al.Reactivity of pure Candida rugosa lipase isoenzymes
(LIP1, LIP2, and LIP3) in aqueous and organic media.Influence of the
isoenzymatic profile on the lipase performance in organic media[J]
.Biotechnology Progress, 2004,20 (1): 65-73.).But LIP1 weak stability limits it in high temperature, height
Application (Chang, S.W.et al.Codon optimization of Candida rugosa under the conditions of the commercial conversions such as pressure
LIP1 gene for improving expression in Pichia pastoris and biochemical
characterization of the purified recombinant LIP1 lipase[J].Journal of
Agricultural and Food Chemistry, 2006,54 (3): 815-822.).The LIP1 of stability difference is easy inactivation and becomes
Property, lose catalysis response function.Therefore, the stability for improving LIP1 also has great significance in industrial application.
The enzyme of high stability not only shows efficient catalysis activity under high temperature environment, while to reactor cooling system
It is required that it is lower, reduce energy consumption；The advantages that purity of reaction product can also be improved, reduce pollution of the bacterium to converted product.Mesh
Before, immobilised enzymes (Knezevic, the Z.et of different carriers preparation are mostly come from for LIP1 THERMAL STABILITY
al.Immobilization of lipase from Candida rugosa onC supports by
Covalent attachment [J] .Biochemical Engineering Journal, 2006,30 (3): 269-278；G., Kaya, B., Ar1ca, M.Y.Immobilization of Candida rugosa lipase onto
Spacer-arm attached poly (GMA-HEMA-EGDMA) microspheres [J] .Food Chemistry, 2005,
92 (2): 261-268.).Its relationship in structure and function also will be disclosed further to the transformation of LIP1 thermal stability.
Summary of the invention
Present invention aims to overcome that above-mentioned the shortcomings of the prior art, it is steady to provide a kind of efficiently raising enzyme thermokinetics
Qualitative general policies (method)；The thermodynamic stability of highly complex albumen LIP1 in structure is significantly improved, goes forward side by side one
Step explains enzyme active center Structure and Function.Specifically, the present invention is centered on the catalytic residue Ser209 of LIP1,
Around having selectedWithin 18 with highest B-factor residues carry out saturation mutations.In the analysis of three level screen method
Under, the mutant of five single-point thermal stability raising is obtained, and vigor also has different degrees of promotion.By means of orderly superposition group
Mutation is closed, the synergistic effect of simple point mutation has been fast implemented.
The purpose of the present invention is achieved through the following technical solutions:
The present invention relates to a kind of universal methods for improving enzyme thermodynamic stability, are with the catalytic residue of zymophore
Center selects surrounding according to the B-factor value in the enzyme crystal structureWithin high flexibility residue be target site,
By screen mutation, the mutant that enzyme thermodynamic stability improves is obtained.The activated centre refers near enzymatic residue
Structural region is different from the surface residue of labyrinth.And residue size flexible is mainly according to the B- in crystal structure
Factor value judges；B-factor is bigger, then flexible higher；B-factor is smaller, and rigidity is stronger.
Preferably, the screening is three level screen, primary dcreening operation including Fluorescence Plate and the twice secondary screening of 96 orifice plates.
Preferably, the primary dcreening operation of the Fluorescence Plate includes 65 DEG C of heat treatment 40min of mutant monoclonal, and room temperature is cooling
Cultivate 5h after 20min at 30 DEG C in flexible glue culture medium, taking-up observes fluorescence under ultraviolet light, shows biggish fluorescent ring i.e.
For high thermal stability and active candidate strain；The secondary screening of 96 orifice plates twice includes: the edge of first 96 orifice plate in
Between respectively take hole culture wild type LIP1 as control, in PCR instrument after 58 DEG C of heat treatment 15min, measurement is hydrolyzed to nitre
The vigor of base phenol butyrate (pNP-C4) is used as screening criteria, the clone screened from inactivation rate higher than wild type 20%
Into 96 orifice plates screening in next step；Each positive colony is transferred in 4 holes of a column and cultivates, the inactivation rate after analysis heat treatment；Very
The inactivation rate that the positive positive is then shown in 4 holes will be higher than wildness, and increase rate is close.
Preferably, the method also includes the mutant that the enzyme thermodynamic stability for going out screen mutation improves, pass through
Orderly stack combinations mutation obtains the step of enzyme thermodynamic stability higher mutant.
Preferably, the orderly stack combinations are mutated specific implementation process are as follows: improve width according to single-point mutants stability
The sequence of degree from big to small, is combined, step by step until all mutational sites are all introduced in a mutant.In the present invention, have
Sequence stack combinations mutation scheme is by quickly obtaining with stability increase rate maximum and time big simple point mutation stack combinations step by step
It obtained stability and improves most significant mutant.
Preferably, the enzyme is selected from CalB, LipA or the fold vacation silk with the amino acid sequence as shown in SEQ ID NO:1
Yeast-lipase LIP1.The corresponding nucleotide sequence of LIP1 enzyme is as shown in SEQ ID NO:9.
Preferably, distance LipA, CalB or the catalytic residue of LIP1 are selectedWith opposite B-factor value in 60-
100 region is mutation hot-zone.The present invention is sent out newly from the mutation result of tri- kinds of different structure complexities of LipA, CalB and LIP1
Rule: apart from catalytic residueIt can be improved prominent in the region of 60-100 to be mutated hot-zone with opposite B-factor value
Become efficiency.Wherein, opposite B-factor refers to: catalytic residueWithin, highest B-factor value is control, remaining residue
Percentage of the B-factor compared with it.
Preferably, centered on the catalytic residue Ser209 of LIP1, distance Ser209 has been selectedNeighbouring has
The residue of highest B-factor value is mutation hot-zone, carries out saturation mutation.The present invention is apart from catalytic residueNeighbouring
High B-factor residue (B-factor relative value is in 60-100) is mutation hot-zone, and the success rate of enzyme mutant can be improved.It is preferred that
, when the enzyme selects LIP1, using the sequence as shown in SEQ ID NO:1 as reference sequences, the mutation hot-zone chosen is corresponding
Site includes 344,434,133,121.
Preferably, simple point mutation occurs at site 344,434,133 or 121；Wherein, the phenylalanine in 344 sites is prominent
Become isoleucine or methionine, the phenylalanine in 434,133 and 121 sites sports tyrosine.The mutation of corresponding LIP1
Body be respectively Phe344Ile (F344I), Phe344Met (F344M), Phe434Tyr (F434Y), Phe133Tyr (F133Y),
Phe121Tyr (F121Y), corresponding amino acid sequence are corresponding successively as shown in SEQ ID NO:2~SEQ ID NO:6
Nucleotide sequence is successively as shown in SEQ ID NO:10~SEQ ID NO:14.
It is above-mentioned that the primer sequence of mutation use is carried out as shown in SEQ ID NO:17, SEQ ID NO:18 to 344 sites；It is right
133 sites carry out the primer sequence of mutation use as shown in SEQ IDNO:21, SEQ IDNO:22；121 sites are mutated
The primer sequence of use is as shown in SEQ ID NO:27, SEQ ID NO:28；434 sites are carried out with the primer sequence of mutation use
As shown in SEQ ID NO:37, SEQ ID NO:38.
Preferably, prominent by the way that the orderly stack combinations of simple point mutation progress occur at site 344,434,133 and 121 simultaneously
Become；Wherein, the phenylalanine in 344 sites sports isoleucine or methionine, and the phenylalanine in 434,133 and 121 sites is equal
Sport tyrosine.The mutant of corresponding LIP1 is Phe344Ile/Phe434Tyr/Phe133Tyr/Phe121Tyr respectively
(F344I/F434Y/F133Y/F121Y)、Phe344Met/Phe434Tyr/Phe133Tyr/Phe121Tyr(F344M/
F434Y/F133Y/F121Y), corresponding amino acid sequence is corresponding successively as shown in SEQ ID NO:7, SEQ ID NO:8
Nucleotide sequence is successively as shown in SEQ ID NO:15, SEQ ID NO:16.The present invention by orderly stack combinations mutation scheme,
Obtain the maximum two mutant VarA3 (F344M/F434Y/F133Y/F121Y) of thermal stability increase rate and VarB3
(F344I/F434Y/F133Y/F121Y)。
The principle of the invention lies in:
For complicated protein, B-factor is combined with other design and rational thinkings, it may be preferably
Enzyme stability is instructed to be transformed.
Activated centre is the most key position in the structure of enzyme, and the appropriate flexibility of local conformation helps preferably to combine bottom
Object promotes the efficient catalytic of enzyme.However the activated centre of enzyme has certain fragility, a large amount of inhomogeneities in total
The denaturation of type enzyme goes out as the result is shown, and the inactivation of enzyme is frequently experienced in front of detectable protein entirety conformation change, i.e., " first loses
It is living, rear to be denaturalized ".In addition, in the research of lipase CalB, finding enzyme active center region laboratory early period where inventor
Mutation can also significantly improve the stability of enzyme other than influencing enzymatic.Therefore, the invention proposes " enzyme active centers
The tactful stability enzyme is transformed of stabilisation " is particularly directed to the stability transformation of increasingly complex enzyme in those structures, has emphatically
The directive significance wanted.
The operating process of " enzyme active center stabilisation " strategy is: centered on the catalytic residue of enzyme, passing through Pymol and B-
Near FITTER software is chosenWithin high flexibility amino acid be target.It carries out fixed point saturation mutation and constructs mutation library, utilize
Suitable screening technique obtains stability-enhanced mutant.
Compared with prior art, the invention has the following beneficial effects:
1, the present invention, by protein engineering, successfully obtains under the guidance of " enzyme active center stabilisation " strategy
Mutant Phe344Ile, Phe344Met, Phe434Tyr, Phe133Tyr, Phe121Tyr that thermal stability significantly improves.Its
Middle Phe344Ile and Phe344Met variation is maximum, they7.7 DEG C and 7.9 DEG C, while hydrolysis pair are improved than wild type
Catalytic efficiency (the k of nitrophenol caprylate (pNP-C8)cat/Km) it is respectively 0.94 and 1.23 times of wild type, i.e. enzyme stability
Catalytic efficiency is not by too big influence while raising.
2, it is mutated by orderly stack combinations, has been quickly obtained the mutant VarB3 that stability further increases, most thermophilic
Degree improves 15 DEG C,Value also improves 13 DEG C than wild type, and half-life period improves about 40 times, the T of thermodynamic stabilitymValue mentions
13 DEG C high, catalytic efficiency is identical as wild type.
3, the present invention analyzes the lipase (LipA, CalB and LIP1) of three typical α/β hydrolases simultaneously, they
Molecular weight has from small to large, has in structure from simple to complicated feature；In conjunction with them in activated centre high flexibility residue
Mutation significantly improve the stability of enzyme, imply the versatility of " enzyme active center stabilisation " strategy；According to these three typical cases
The mutation of enzyme is as a result, statistical is precipitated: apart from catalytic residueNeighbouring high B-factor residue (B-factor relative value
In 60-100) it is mutation hot-zone, the success rate of enzyme mutant can be improved.This brings important theory to refer to for the transformation of other enzymes
It leads.
Detailed description of the invention
Fig. 1 is the schematic diagram of enzyme active center stabilization strategy；
Fig. 2 is target site schematic diagram selected by activated centre, wherein A is " lid " Unclosing structure figure of LIP1, B
For " lid " closing structure figure of LIP1；
Fig. 3 is the Tertiary screening setup schematic diagram of high thermal stability and high catalysis activity mutant；
Fig. 4 is the program flow chart of orderly stack combinations；
Fig. 5 is the substrate selectivity schematic diagram of lipase LIP1 and mutant；
Fig. 6 is the thermodynamic stability schematic diagram of lipase LIP1 and mutant；
Fig. 7 is the molecular force analysis chart inside wild type LIP1 and mutant VarB3, wherein A is mutational site
The structure change of 121 and 133 peripheral regions, B are the structure change of 344 peripheral region of mutational site, and C is mutational site 434 weeks
Enclose the structure change in region；
Fig. 8 is the topology diagram of different molecular weight lipase, wherein A is minimal structure enzyme LipA, and B is that moderate is complicated
Structure enzyme CalB, C are the enzyme LIP1 of high level of architectural complexity；
Fig. 9 is the spatial distribution of activated centre hot spot amino acid.
Specific embodiment
The present invention relates to a kind of general and efficient strategies for improving enzyme thermodynamic stability.The specific implementation road of the strategy
Line are as follows: centered on the catalytic residue of zymophore, surrounding is selected according to the B-factor value in the enzyme crystal structure Within high flexibility residue (high B-factor) be target site, by construct saturation mutation library, screen thermokinetics and mention
High mutant (see Fig. 1).Under the guidance of the strategy, centered on catalytic residue Ser209, structure height is significantly improved
The thermal stability of complicated fold lipase from candida sp 1 (Candida rugosa lipase1:LIP1).On integrated structure most
The stability change of the albumen (CalB) of little albumen (LipA) and moderate complexity is as a result, detective distance catalytic residueNear
High B-factor residue (B-factor relative value is in 60-100) be mutation hot-zone, the success rate of enzyme mutant can be improved.Together
When, it will be further investigation enzyme regional stability law, development enzyme stabilization by the emic network analysis of mutant structure-
Technology is laid a good foundation.
Invention is described further with reference to the accompanying drawings and detailed description:
The selection of embodiment 1, mutational site
Wild type (WT) amino acid sequence of the lipase 1 of Candida rugosa according to the present invention is SEQ ID
Shown in NO:1, nucleotides sequence is classified as shown in SEQ ID NO:9.
The crystal structure that LIP1 is searched in PDB database, selects the highest structure of resolution ratio as research object.Due to
LIP1 has the spiral fragment for being known as " lid " above active pocket, and causing LIP1 to have in the solution, there are two types of structures:
Open and Closed, therefore two kinds of structures are analyzed simultaneously respectively when selecting target site.It is all residual to be catalyzed respectively
Centered on base Ser209, around analysisWithin all amino acid (with PyMOL order: PyMOL- > select AA,
Polymer within 10 of Ser209 is searched).Reuse the B-factor that B-FITTER software analyzes these amino acid
Value arranges according to the sequence of value from big to small, chooses preceding 11 maximums B-factor amino acid in each structure as target
Site (is shown in Table 1).There are 3 to duplicate selection in these residues, therefore 18 residues are finally had chosen in total to two kinds of structures
As target site (see Fig. 2).Here is the specific location of these target sites in the structure: Phe344 and Phe345 are all located at
In 12 spiral of α；Phe434 is located at the folding of β 13；Ser84, Lys85, Phe87 and Glu88 are all located on the loop between α 1 and β 5；
Phe121, Gly122, Gly123, Gly124, Phe125, Glu126, Val127 and Phe133 are all located between β 6 and α 3
On loop；Phe296 and Leu302 is on the loop between α 10 and α 11；Corner of the Gly414 between α 15 and α 16.
The B-factor value of 1 activated centre target site of table and distance to catalytic residue Ser209
For significantly more efficient carry out saturation mutation, the combination of amino acids of adjacent sites is constructed mutation library together, tied
Fruit constructs following 12 mutation libraries: LibraryA (Phe344, Phe345), Library B (Leu302), Library C
(Phe133), Library D (Gly124, Phe125), Library E (Glu126, Val127), Library F (Phe121),
LibraryG (Phe87, Glu88), LibraryH (Gly122, Gly123), LibraryI (Ser84, Lys85), Library J
(Phe296),Library K(Phe434),Library L(Gly414).All mutation libraries are shown in Fig. 2 with ball-and-stick model.
Embodiment 2, fixed point saturation mutation construct gene mutation library
The primer in building saturation mutation library is shown in Table 2,
Table 2 is used to construct the primer in saturation mutation library
Under the action of PrimerSTAR max archaeal dna polymerase (TaKaRa company), using the wild type gene of LIP1 as mould
Plate, full plasmid amplification.PCR reaction condition: 98 DEG C of initial denaturation 5min, 98 DEG C of each circulation denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C
Extend 2.5min, totally 30 circulations；Last 72 DEG C of extensions 5min.PCR product is directly transferred to bacillus coli DH 5 alpha sense after purification
By state, 42 DEG C of thermal shocks 90s, 37 DEG C of incubation 1h.Be coated with later with the LB plate containing 25 μ g/ml Zeocin resistances on, to
After clone grows, all clones are eluted with sterile water from plate directly, is collected into a test tube, is mentioned with kit
Take mixing plasmid.The mixing plasmid extracted is linearized with restriction enzyme enzyme cutting AvrII, after product kits
Directly electric shock is transferred to Pichia pastoris GS115 competent cell, 30 DEG C of trainings on the YPD plate containing 25 μ g/ml Zeocin resistances
3d is supported, the bacterial strain containing mutated gene is obtained.
The preparation of embodiment 3, Pichia pastoris competent cell
It from picking Pichia pastoris single colonie on YPD plate, is inoculated in 4ml YPD fluid nutrient medium, training is vibrated at 30 DEG C
It supports overnight, about 24-48h or so, OD about 6-8.Culture solution is dispensed into 1.5ml EP pipe, 4 DEG C, 4000rpm, centrifugation 5min,
Abandon supernatant.Ice-cold sterile water washing is precipitated, 4 DEG C, 4000rpm, centrifugation 5min abandon supernatant.Precipitating 1ml lithium acetate solution
Processing after precipitating is resuspended, places 30min at 30 DEG C.4 DEG C later, 4000rpm, centrifugation 5min, abandon supernatant.(lithium acetate solution:
10mM pH7.5Tris-HCL；10mM DTT；100mM LiAC) precipitate ice-cold sterile water washing, 4 DEG C, 4000rpm, from
Heart 5min abandons supernatant.Precipitating is washed with the D-glucitol of ice-cold 1M, and 4 DEG C, 4000rpm, centrifugation 5min abandon supernatant.Thoroughly fall
Supernatant out adds the 50 μ l of D-glucitol of 1M, is prepared into competent cell, prepares to convert in next step.
(note: competence will now do it is current, the DTT in treatment fluid be use preceding addition, it is other prepare after in 4 DEG C protect
It deposits, DTT is saved separately from -20 DEG C, can be made into 100 times of stock solution.)
Embodiment 4, three level screen saturation mutation library
Three level screen method is as shown in figure 3, the mainly primary dcreening operation including Fluorescence Plate and the twice secondary screening of 96 orifice plates；Fluorescence is flat
The method of plate primary dcreening operation: the monoclonal that will be grown in YPD_Zeo resistant panel, by photocopy cellulose membrane be transferred to two it is new flat
On plate；30 DEG C are cultivated two days, and a plate is stored in refrigerator, and another plate is transferred in 65 DEG C of incubator；40min is heat-treated,
The cooling 20min of room temperature pours into the flexible glue culture medium 10ml of dissolution on the plate after taking-up；5h is cultivated at 30 DEG C again, in purple
Fluorescence is observed under outer light, high thermal stability and active mutant show biggish fluorescent ring.It can be with by fluorescent ring screening
Effectively eliminate the clone for occurring negative mutation in activity and stability.Followed by 96 orifice plate secondary screenings, the edge in each 96 hole
It respectively takes a hole culture wild type LIP1 as control with centre, cultivates 72h under 30 DEG C, 220rpm with YPD fluid nutrient medium.
4000rpm is centrifuged 96 orifice plate 30min, turns culture supernatant into another orifice plate, will each aerial sample dilution 4 with sterile water
Times.Enzyme solution after dilution is divided into two parts, and a part directly takes 10 μ l to measure the vigor of hydrolysis pNP-C4 in microplate reader, saves
Data, as the vigor before heat inactivation.Another part takes 50 μ l in 96 hole PCR plates, 58 DEG C of heat treatment 15min in PCR instrument.
It is centrifuged off precipitating later, takes centrifugation supernatant to measure remaining vigor in microplate reader, as data after heat inactivation；To data twice
It compares, calculates inactivation rate, the screening criteria that is used as of wild type 20% is higher than from inactivation rate, the clone screened enters next step 96
Hole screening.In order to exclude the false positive occurred in primary dcreening operation, obtained single colonie is transferred in next 96 new deep-well plates, often
A positive colony is transferred in 4 holes of a column and cultivates, the inactivation rate after analysis heat treatment；Really the positive is then shown in 4 holes
Inactivation rate will be higher than wildness, and increase rate is close.
Embodiment 5, the mutation of orderly stack combinations
In order to be quickly obtained the cumulative effect of beneficial mutation bacterial strain, orderly stack combinations mutation scheme is taken, is used every time
Best mutant combinations mutant second-best.This combinatorial mutagenesis scheme more can be quickly obtained combinatorial mutagenesis as a result,
Specific anabolic process is shown in Fig. 4.Since two single-point mutants at site 344 have mentioning for highest amplitude in all mutant
Height, so having separately designed two orderly stack combinations paths: from F344M to F121Y and F344I to F121Y.This two paths
In addition to other than starting strain is different (being F344M and F344I respectively), remaining anabolic process is just the same.Group in two approach
Mutant is closed to be replaced with new name respectively:
VarA1(F344M/F434Y)、VarA2(F344M/F434Y/F133Y)、VarA3(F344M/F434Y/F133Y/
F121Y)、VarB1(F344I/F434Y)、VarB2(F344I/F434Y/F133Y)、VarB3(F344I/F434Y/F133Y/
F121Y).It is shown from the combined result of Fig. 4, each combination can make stability have different degrees of raising.These results
It absolutely proves that there is cumulative effects between four single-point mutants, and confirms that orderly stack combinations mutation scheme is a kind of
It is quickly obtained the effective way of more preferable result.
The expression and purifying of embodiment 6,1 wild type of fold lipase from candida sp and mutant
It is cultivated from the YPD culture medium that picking single bacterium falls within 4ml on YPD_Zeo plate, works as OD600When reaching 2, by 1%
Inoculum concentration is transferred in new 200ml YPD and expresses, every 12h sample detection vigor, until culture terminates.
It, can be directly pure using the method for Ni- affinity chromatography since the N-terminal of target gene devises the label of 6 × His
Change.LIP1 is with secretion expression in Pichia pastoris, and final expression product needs to carry out culture solution mainly in culture solution
Concentration collect.Therefore, the purifying of entire LIP1 is divided into two steps of ultrafiltration concentration and Ni- affinity chromatography:
(1) it is concentrated by ultrafiltration
Yeast expression culture solution is centrifuged 20min under the revolving speed of 8000rpm, collects supernatant；Pass through 0.22 μm of fiber
After film filtering, it is transferred in the ultrafiltration apparatus of Millipore；Desalination, decoloration and concentration are carried out with the ultrafiltration membrane packet of 10kD, dense
Constantly with addition ddH in compression process2O, until concentrate is close to colourless；It is again that albumen is dense with the Tris-HCl 200ml of 20mM
Contracting is transferred to affinity chromatography in 100ml or so, and samples and carry out electroresis appraisal and concentration mensuration.
(2) affinity chromatography
After 0.22 μm of membrane filtration of water, directly add 5ml to the complete rear addition 5ml strip of nickel column top → stream
Buffer (100mM EDTA, 0.5M NaCl, 50mM Tris-HCl, pH 7.5), by Ni2+With foreign protein from carrier NTA agar
Washed on sugar → pillar add 5ml water punching do → flow afterwards addition 5ml charge buffer (100mM NiSO4) → flowed
Afterwards plus water rinses → is added 10ml binding buffer (50mM Tris-HCl, pH 7.5,20mM imidazoles, 0.5M NaCl)
→ flow the complete rear complete rear addition 5ml of addition 10ml binding buffer → stream of enzyme solution → stream after rear addition is concentrated by ultrafiltration
Wash buffer (200mM imidazoles, 0.5M NaCl, 50mM Tris-HCl, pH 7.5) gradient elution collects efflux → column
Son plus water rinse, and are stored in 4 DEG C of refrigerators with 20% ethyl alcohol.
Eluent is collected together and is transferred to bag filter, 4 DEG C of dialysis in 50mM Tris-HCl, 7.5 buffer of pH
For 24 hours, dialyzate twice is changed in centre.Albumen is finally collected, is saved spare with -80 DEG C of refrigerators.
Obtain respectively in aforementioned manners the mutant Phe344Ile (F344I) of LIP1, Phe344Met (F344M),
Phe434Tyr(F434Y)、Phe133Tyr(F133Y)、Phe121Tyr(F121Y)、Phe344Ile/Phe434Tyr/
Phe133Tyr/Phe121Tyr(F344I/F434Y/F133Y/F121Y)、Phe344Met/Phe434Tyr/Phe133Tyr/
The mutant of Phe121Tyr (F344M/F434Y/F133Y/F121Y).And by sequencing, the amino acid sequence of above-mentioned mutant enzyme
Leie time is as shown in SEQ ID NO:2~SEQ ID NO:8；Corresponding nucleotide sequence is successively such as SEQ ID NO:10~SEQ
Shown in ID NO:16.
Embodiment 7, fold lipase from candida sp vitality test and substrate are selectively analyzed
It uses p-nitrophenyl to divide caprylate (pNP-C8) as substrate, the concentration of 10mM is configured to acetonitrile.With the pH of 50mM
8.0Tris-HCl is as buffer, and the NaTDC containing 1% (w/v) Arabic gum and 2% (w/v) is (as emulsification
Agent).In 40 DEG C of water-baths, the absorption value of light in the analytical unit time of ultraviolet specrophotometer is utilized.Specific reaction system are as follows:
The substrate of 20 μ l is taken to be dissolved in the buffer of 970 μ l, 40 DEG C of preheating 2min are added 10 μ l enzyme solutions, blow even reaction with liquid-transfering gun
System, start recording reaction process stop reaction after about 2min, the vigor of enzyme are calculated according to following formula (formula 1).1
A enzyme activity unit (U) are as follows: enzyme amount required for 1 μm of ol substrate of catalyzing hydrolysis per minute.
A (U/mg): the Rate activity of enzyme；ΔE(min-1): the changing value of light absorption 405nm in the reaction time；vr(1): reaction
Volume；ε(M-1·em-1): the molar absorption coefficient (1.6 × 10 of p-nitrophenol4)；D (cm): the path length that light passes through；ve
(1): reaction total volume；ce(mg/l): the concentration of enzyme；Experimental data is to be averaged three times.
For measuring the vigor of the pNP substrate of different chain length, method is same as described above, as a result sees Fig. 5.
Embodiment 8, the thermal stability of fold lipase from candida sp 1 and mutant and kinetic parameter analysis
In order to analyze the thermal stability variation of mutant, the present invention utilizes final concentration 0.2mM p-nitrophenol caprylate, point
It is other to thermal stability Common Parameters t1/2(half-life period), Topt(optimum temperature) and(Enzyme at different temperatures, is heat-treated
15min, vigor temperature corresponding when losing half) it is determined, measure the method such as embodiment 6 of enzyme activity.
Within the temperature range of 30~65 DEG C, the hydrolysis reaction for analyzing pNP-C8 under different temperatures measures LIP1 most
Thermophilic degree.Heat stability test is placed by the way that the every 50 μ l of purified LIP1 (0.1mg/ml) to be divided in multiple centrifuge tubes
It is kept the temperature at 60 DEG C, sampling in interval in different times measures remaining vigor.According to formula 2, can converse at this temperature
Half-life period.
t1/2=ln2/kd(formula 2)
Thermal stability parameterMeasuring method: by the way that the every 50 μ l of purified LIP1 (0.1mg/ml) is dispensed into 96 holes
In PCR plate, it is arranged 40~80 DEG C of temperature gradient, the processing of 15min heat inactivation is carried out to enzyme；After 4 DEG C of cooling 10min, room temperature
20min is stood, 4000rpm is centrifuged 96 hole PCR plate 30min, removes precipitating；The remaining vigor of supernatant is analyzed, with relative residual
Vigor and temperature make curve graph；According to Boltzmann's model (formula 3), it is fitted, calculates under Origin8.0 software auxiliaryValue.
Y=A2+ (A1-A2)/(1+exp ((x-x0)/dx)) (formula 3)
The analysis of kinetic parameter is, using pNP-C8 as substrate, to measure bottom respectively under conditions of pH 8.0,40 DEG C of temperature
Object concentration is hydrolysis vigor corresponding to 4,10,15,20,50,100,150 and 200 μM.Using LIP1 to different concentration of substrate
Catalysis activity data fit function curve by software Origin 8.0 and Michaelis-Menten equation (formula 4), to obtain KmWith
Vmax, further according to the functional equation (formula 5) of maximum rate and enzyme concentration, kinetic constant K is calculatedm, kcatAnd kcat/Km。
V=Vmax[S]/(Km+ [S]) (formula 4)
Vmax=kcat× [E] (formula 5)
Measuring method more than, the kinetic parameter and thermal stability parameter for obtaining LIP1 and mutant are shown in Table 3.
The kinetic parameter and thermal stability of table 3 lipase LIP1 and mutant
The thermodynamic stability of embodiment 9, analysis LIP1 and mutant changes (Tm)
Scanning temperature range is set as 30~90 DEG C, scanning speed is 1 DEG C/min.The concentration control of protein sample exists
0.5mg/ml is dissolved in the phosphate buffer of 10mM；Start before scanning first in 30 DEG C of pre-equilibrations 15min, TmFor highest wave crest
Locate corresponding temperature.
Fig. 6 analysis goes out the T of all mutant as the result is shownmValue has different degrees of raising relative to wild type, most
Promotion (Δ T by a small marginm) also there are 4.5 DEG C (F434Y, 57.9 DEG C).It is worth noting that, TmIt is not to move that value, which improves maximum,
Mechanical stability improves most significant Var3 (Δ Tm=12.7 DEG C), but three Sites Combination body combination mutant VarA2
(F344M/F434Y/F133Y), its TmValue is promoted by 53.4 DEG C of wild type to 70.8 DEG C, improves nearly 17.5 DEG C.This
It is a the result shows that, dynamic stability and thermodynamic stability simultaneously non-fully correspond to direct proportionality.
Embodiment 10, the analysis stability-enhanced mechanism of stability mutant VarB3
Show that mutant VarB3 and wild type LIP1 are closely similar in structure by homologous modeling, in order to rationally solve
The mechanism for causing protein stability to improve is released, needs to carry out the residue that Normalized B-factor near catastrophe point is reduced
Intramolecular action power is deeply seen clearly.Mutant VarB3 model structure and LIP1 crystal structure are analyzed using PyMOL software
The intramolecule of (1CRL) interacts, detailed results See Figure 7.
As can be seen that site 12 1 and 133 is mutated into tyrosine from Fig. 7 A, results in 7 new hydrogen bonds and generates,
Tyr133 and hydrone Wa, hydrone Wb and Gly122 after middle mutation form 3 hydrogen bonds, deposit before this 3 hydrogen bonds and mutation
4 hydrogen bonds being between hydrone Wa, Wb and Gly122, in total 7 hydrogen bond shapes between Tyr133, Wa, Wb and Gly122
The hydrogen bond network to interact at one.And Tyrl21 and hydrone Wc, Phe128, Asn155 and Tyr156 shape after being mutated
At 4 hydrogen bonds, it is present in 2 hydrogen bonds between hydrone Wc and Phe128, Asn155 before this 4 hydrogen bonds and mutation, in total 6
Also there is the hydrogen bond network of an interaction between Tyr121, Wc, Phe128, Asn155 and Tyr156 in a hydrogen bond.Hydrogen
Key network is a key factor for improving protein stability, and the appearance of the two hydrogen bond networks will be helpful to stablize residue
The swing in the region loop between 120 and 136, to also contribute to the stabilization of whole albumen.
After being apparent that site 344 is mutated into Ile from Fig. 7 B, 12 spiral of α is obviously prolonged.This is mainly Ile
Introducing cause the side chain of Phe345 and Thr343 that slight deflection has occurred, furthered the distance between Thr343 and Gly346,
One is produced between themNew hydrogen bond；Also cause the hydrogen-bond length between Phe345 and Ser348 by original simultaneously
ComeBecomeGly346 makes its side chain closer to Ser349 under the pulling for newly forming hydrogen bond, as a result exists
Two hydrogen-bond lengths between Gly346 and Ser349 are all by originalBecomeAll these closer stackings
So that the original loop between Thr347 and Ser349 is transformed to α spiral, spiral α 12 is extended accordingly；Loop turns
Turn to the rigidity reinforced that spiral makes activated centre part.
It is found from Fig. 7 C, phenylalanine is mutated into tyrosine and forms one at site 434Hydrogen bond, this
A hydrogen bond appears between Tyr434 and hydrone Wd.It examines β 13 and folds residue and surrounding ammonia, it can be seen that on it
Different degrees of reduction all has occurred in the mutual hydrogen-bond length of base acid, they are respectively: between Tyr432 and Asp479
Hydrogen-bond length is by originalBecomeTwo hydrogen-bond lengths between Ser433 and Ile335 respectively byWithBecomeWithHydrogen-bond length between Ser433 and Met503 by BecomeLeu435 with
Hydrogen-bond length between Ile505 byBecomeAlthough the variation of these hydrogen-bond lengths is smaller, multiple minor changes
Polymerization will bring β 13 fold rigidity reinforced.After above to the detailed analysis of structure, interaction of hydrogen bond net is found
The intensive stacking of network and activated centre is the main reason for causing local stiffness to enhance, and then reflects mutant on the whole
The stability of VarB3 improves.
The versatility of embodiment 11, activated centre stabilization strategy
In order to explore the amino acids distribution feature that enzyme active center has a major impact stability, to different structure complexity
The stability of zymoprotein improve result carried out detailed analysis, especially German scholar Reetz research achievement (Reetz,
M.T.et al.Iterative saturation mutagenesis on the basis of B-factors as a
strategy for increasing protein thermostability[J].Angewandte Chemie
International Edition, 2006,45 (46): 7745-7751.).As a result, it has been found that the albumen of Reetz mutation is " enzyme activity
Property it is central stabilizing " strategy a special case.LipA is one the smallest α/β hydrolase folded protein, it only has 181 amino
Acid composition forms active site from can be seen that it only has standard in structure α spiral and β-pleated sheet in Fig. 8 A, not extra
Secondary structure is wrapped in protein surface, and LipA also has most simple feature in structure.The catalytic activity that the present invention selectes
Around residueWithin high flexibility residue almost entire albumen is all contained into for LipA, Reetz choosing
The Mutated residues selected are most of all apart from catalytic residueWithin.Compared with LipA, CalB is in molecular weight and structure
There is certain increase in complexity.Such as Fig. 8 B, CalB is made of 317 amino acid, in addition to there is standard hydrolysis in centre in structure
Outside the α/β of enzyme folds, around there are also 4 α spirals and 2 β-pleated sheets to wrap up (Xie, Y.et al.Enhanced enzyme
kinetic stability by increasing rigidity within the active site[J].Journal of
Biological Chemistry, 2014,289 (11): 7994-8006.)；In addition also have one section by 5 amino acid
The α spiral that (residues 142~146) is formed is covered on active pocket, since this section of spiral is too short, not to vigor band
Carry out too big influence；Also this section of spiral of document report is not to be regarded as lid, therefore this section of spiral can be counted as forming lid
The transition state of son.LIP1 is then structurally different from CalB, it has primary height super again on molecular weight and structure complexity
More.If Fig. 8 C, LIP1 are made of 534 amino acid, other than the α/β of inside configuration hydrolase standard folds, in zymoprotein
Around there are also 12 α spirals and 5 β-pleated sheets package；In addition have above its active pocket one section by 29 amino acid
(residues 65~94) formed α spiral as lid, this lid make it have in structure there are two types of configuration (open with
closed).To sum up analyze the structure feature of LipA, CalB and LIP1, it is found that they are zymoproteins in molecular weight and structure
On from small to large, from simple to complicated Typical Representative, their catalytic residue serinesWithin high flexibility amino acid mutation
The stability for significantly improving enzyme implies that " enzyme active center stabilisation " there are certain versatilities for strategy；The knot improved from them
Find that some potential rules may bring important guiding information for the stability transformation of other enzymes in fruit.
Embodiment 12, the distribution characteristics for analyzing activated centre hot spot amino acid
Result is improved at a distance from Mutated residues to catalytic residue, opposite B- to the stability of LipA, CalB and LIP1
Factor value is (with respective catalytic residueInterior highest B-factor value is reference, calculates remaining simple point mutation residue
B-factor detailed analysis) has been carried out.WithIndicate that all simple point mutations improve the parameter of stability, remaining negative mutation or group
It closes mutation and is all considered as stability there is no variation, obtained the result of Fig. 9 on this basis.Detective distance catalytic residueIn the range of be all improved the stability of enzyme, wherein most mutation are located atAndThe width nearby improved
It spends the most obvious；And it is lower thanBe greater thanIn the range of find no single-point amino acid mutation improve enzyme stability；
And opposite B-factor has an impact to the raising of enzyme stability in wide distribution, is concentrated mainly on opposite B-factor
Between value 60~70 and 90~103；Opposite B-factor value, which is greater than 103 and finds no simple point mutation raising enzyme less than 60, to be stablized
Property, therefore, " enzyme active center stabilisation " strategy is in selected distance catalytic residueWith opposite B-factor value 60~100
(with activated centreInterior highest B-factor be reference) between high flexibility residue be mutated, it will further increase enzyme
The success rate of stability transformation.
Claims (2)
1. a kind of mutant with high enzyme thermodynamic stability, which is characterized in that with the catalytic residue of zymophore
Centered on, it is selected according to the B-factor value in the enzyme crystal structure apart from catalytic residueWithin high flexibility residue
The mutant that enzyme thermodynamic stability improves is obtained by screen mutation for target site；It is prominent by orderly stack combinations again
Become and obtains enzyme thermodynamic stability higher mutant VarA2, VarA3, VarB2 or VarB3, i.e., it is described to have high enzyme heat dynamic
The mutant of mechanical stability；
The enzyme is the fold lipase from candida sp LIP1 of the amino acid sequence as shown in SEQ ID NO:1, the mutation chosen
The corresponding site in hot-zone includes 344,434,133,121；It is carried out by the way that simple point mutation occurs at site 344,434,133 and 121
Orderly stack combinations mutation, wherein the phenylalanine in 344 sites sports isoleucine or methionine, and 434,133 and 121
The phenylalanine of point sports tyrosine；
The orderly stack combinations path of mutant VarA2 are as follows: Phe344Met/Phe434Tyr/Phe133Tyr；
The orderly stack combinations path of mutant VarA3 are as follows: Phe344Met/Phe434Tyr/Phe133Tyr/Phe121Tyr；
The orderly stack combinations path of mutant VarB2 are as follows: Phe344Ile/Phe434Tyr/Phe133Tyr；
The orderly stack combinations path of mutant VarB3 are as follows: Phe344Ile/Phe434Tyr/Phe133Tyr/Phe121Tyr.
2. as described in claim 1 with the mutant of high enzyme thermodynamic stability, which is characterized in that mutant
The amino acid sequence of VarA3 is as shown in SEQ ID NO:8；The amino acid sequence of mutant VarB3 is as shown in SEQ ID NO:7.
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| CN107151660B (en) * | 2017-01-11 | 2019-12-10 | 广东溢多利生物科技股份有限公司 | lipase variant Ala36Ser/Asp49His/Ala52Val/Asn55Asp |
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