CN106085975A - A kind of general policies of efficient raising enzyme thermodynamic stability - Google Patents
A kind of general policies of efficient raising enzyme thermodynamic stability Download PDFInfo
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Abstract
The invention discloses the general policies of a kind of efficient raising enzyme thermodynamic stability；The route that is embodied as of this strategy is: centered on the catalytic residue of zymophore, selects around according to the B factor value in this enzyme crystal structure Within high flexibility residue be target site, pass through screen mutation, it is thus achieved that enzyme thermodynamic stability improve mutant.Under the guidance of this strategy, significantly improve the heat endurance of the fold lipase from candida sp 1 of high level of architectural complexity.The stability change result of the albumen (CalB) of minimum albumen (LipA) and moderate complexity, detective distance catalytic residue on integrated structureThe prominent success rate that can significantly improve enzyme mutant of neighbouring high B factor residue.Meanwhile, will lay a good foundation for further investigation enzyme regional stability law, development enzyme stabilization technology by the emic network analysis of mutant structure.
Description
Technical field
The present invention relates to the general policies of a kind of efficient raising enzyme thermodynamic stability.By Pymol and B-FITTER
Software analysis enzymatic residueWithin the amino acid of high flexibility；Filter out thermodynamic stability by saturation mutation storehouse to carry
High mutant.It is specifically related to the mutant of fold lipase from candida sp 1, and the structure of mutant and functional analysis.
Background technology
Enzyme is a kind of efficient biocatalyst, compared with chemical catalyst, enzyme molecule have high efficiency, high selectivity and
The advantages such as reaction condition gentleness is pollution-free, have important using value in scientific research and industrial production.But at high pressure, height
Under the mal-conditions such as warm, extreme pH, enzyme easily unwinds and loses activity, and this severely limits the extensive of enzyme and applies, therefore, how
The stability improving enzyme becomes an important topic of current research.
With the development of structure biology, increasing enzymatic structure is resolved.Have many at present based on single enzyme
Structural analysis, carries out design and rational and improves the report of 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. produce electrostatic interaction by mutating acid, improve sucrose
The half-life that phosphorylase is 60 DEG C 2.6 times；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 the half-life of 70 DEG C；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 the half-life 20 times that series bacillus zytase Xyn10B is at 50 DEG C.These achievements mostly are individual
Example is analyzed, and there is no rule can follow.With the introducing of proteomics concept, enzyme stabilization strategy has had new development.By right
The phytase multisequencing of separate sources is analyzed, and finds wherein uniformity site to instruct sudden change, 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,
Speculate its ancestral gene, 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.)；Utilize the enzyme recombinant technique that structural database instructs, successfully by cellulase
Half-life 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 carries
Rise the efficiency of enzyme stability transformation.But, this method needs substantial amounts of data to carry out statistical analysis, and the deficiency of data volume also limits
Accuracy and the range of application of the method are made.Therefore, if the key position related to enzyme stability can be absorbed in, letter is set up
Single easy, rapidly and efficiently, the enzyme stabilization New Policy of highly versatile, is the emphasis that the present invention pays close attention to.
In order to understand the stabilizing mechanism of enzyme in depth, scholars compare the structure of Zimadzhunt L 340 and middle temperature enzyme, find the former
Some region has higher rigidity, thus brings the climax of enzyme stability regionization research, especially in enzymatic structure
Flexible region.In protein structures, B-factor becomes the important indicator identifying flexible amino acid.B-factor refers to
In zymoprotein, the electron density of each composition amino acid atom is in the space " fuzziness " of point of equilibrium, i.e. atom is in vibration
In the middle of, B-factor reflects the Oscillation Amplitude of atom, thus is converted into the flexibility of amino acid；B-factor is bigger, then amino
Acid flexibility is bigger.Around this principle, German well-known scholar Reetz (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.) right with bacillus subtilis lipase (LipA:181 amino acid) minimum in nature for research
As have chosen 10 residues that in whole enzyme molecule, B-factor is maximum as target site, structure iteration saturation mutation storehouse sieve
Select stability-enhanced mutant.This 10 target sites are entirely located in enzyme molecular surface.Through the screening of too much wheel, optimum sudden change
Body is promoted to 16h the half-life of 55 DEG C from 2min, and result is very notable.After document report, obtain academia extensive
Concern, there is now many document utilization B-factor analyses and transforms 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, to the highest residual of enzyme B-factor
Base suddenlys change, and enzyme stability lifting amplitude is simultaneously inconspicuous, is promoted to 34.6min the half-life of 65 DEG C by 25.9min.Thus
Illustrate that the modification in surface region still suffers from defect for the increasingly complex albumen of molecule.
Candida rugosa lipasel (LIP1) has bigger molecular weight and complicated structure (534 amino acid
Composition).And, LIP1 is again a kind of important industrial applicability lipase, has high catalytic efficiency and the substrate of 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
Split important medical product, such as: (Chavez-Flores, the et al.Facile such as naproxen, Ketoprofen and brufen
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 at high temperature, height
Application (Chang, S.W.et al.Codon optimization of Candida rugosa under the conditions of the commercial conversion 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 poor stability easily inactivates change
Property, lose catalytic reaction function.Therefore, the stability improving LIP1 also has great significance in commercial Application.
The enzyme of high stability not only shows efficient catalysis activity in high temperature environments, simultaneously to reactor cooling system
Require relatively low, reduce energy consumption；Can also improve the purity of product, reduce bacterium to advantages such as the pollutions of converted product.Mesh
Before, immobilised enzymes (Knezevic, Z.et prepared by different carriers is 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.).Also it will be disclosed further in structure and relation functionally to the transformation of LIP1 heat endurance.
Content of the invention
Present invention aim to overcome that the deficiency that above-mentioned prior art exists, provide a kind of efficiently raising enzyme thermokinetics steady
General policies (method) qualitatively；Significantly improve the thermodynamic stability of high complexity albumen LIP1 in structure, go 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,
Have selected aroundWithin 18 residues with the highest B-factor carry out saturation mutation.Analysis in three level screen method
Under, it is thus achieved that the mutant that five single-point heat endurances improve, and vigor also has and promotes in various degree.By means of orderly superposition group
Close sudden change, quickly achieve the cooperative effect of simple point mutation.
It is an object of the invention to be achieved through the following technical solutions:
The present invention relates to a kind of universal method improving enzyme thermodynamic stability, the catalytic residue with zymophore is
Center, selects around according to the B-factor value in this enzyme crystal structureWithin high flexibility residue be target site,
Pass through screen mutation, it is thus achieved that the mutant that enzyme thermodynamic stability improves.This activated centre refers near enzymatic residue
Structural region, is different from the surface residue of labyrinth.And the size of residue flexibility is mainly according to the B-in crystal structure
Factor value judges；B-factor is bigger, then flexibility is higher；B-factor is less, and rigidity is stronger.
Preferably, described screening is three level screen, including the multiple sieve of the primary dcreening operation of Fluorescence Plate and twice 96 orifice plates.
Preferably, the primary dcreening operation of described Fluorescence Plate includes mutant monoclonal 65 DEG C heat treatment 40min, and room temperature cools down
Cultivate 5h after 20min at 30 DEG C in flexible glue culture medium, take out and observe fluorescence under ultraviolet light, demonstrate that bigger fluorescent ring is
Candidate strain for high thermal stability and activity；The multiple sieve of described 96 orifice plates twice includes: the edge of first 96 orifice plate and in
Between respectively take hole and cultivate wild type LIP1 as comparison, in PCR instrument after 58 DEG C of heat treatment 15min, measure hydrolysis to nitre
The vigor of base phenol butyrate (pNP-C4), from inactivation rate higher than wild type 20% as screening criteria, the clone screening
Enter the screening of next step 96 orifice plate；Each positive colony proceeds to cultivate in 4 holes of row, analyzes the inactivation rate after heat treatment；Very
The positive positive then shows the inactivation rate in 4 holes will be higher than wildness, and increase rate is close.
Preferably, described method also includes the mutant that the enzyme thermodynamic stability screen mutation improves, and passes through
Orderly stack combinations sudden change obtains the step of the higher mutant of enzyme thermodynamic stability.
Preferably, described orderly stack combinations sudden change specific implementation process is: improve width according to single-point mutants stability
Degree order from big to small, combines 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 by the maximum and secondary big simple point mutation stack combinations step by step of stability increase rate, quickly obtaining
Obtain stability and improve the most significant mutant.
Preferably, described enzyme is selected from CalB, LipA or has the false silk of fold of amino acid sequence as shown in SEQ ID NO:1
Yeast-lipase LIP1.This corresponding nucleotide sequence of LIP1 enzyme is as shown in SEQ ID NO:9.
Preferably, the catalytic residue of chosen distance LipA, CalB or LIP1With relative B-factor value at 60-
The region of 100 is sudden change hot-zone.The present invention sends out new from the sudden change result of tri-kinds of different structure complexities of LipA, CalB and LIP1
Rule: distance catalytic residueWith relative B-factor value in the region of 60-100 for sudden change hot-zone, it is possible to increase prominent
Become efficiency.Wherein, relative B-factor refers to: catalytic residueWithin, the highest B-factor value is comparison, remaining residue
The percentage that B-factor compares with it.
Preferably, centered on the catalytic residue Ser209 of LIP1, have selected distance Ser209Neighbouring has
The residue of the highest B-factor value is sudden change hot-zone, carries out saturation mutation.The present invention is at distance catalytic residueNear
High B-factor residue (B-factor relative value is at 60-100) is sudden change hot-zone, can improve the success rate of enzyme mutant.Preferably
, when described enzyme selects LIP1, with sequence as shown in SEQ ID NO:1 as reference sequences, the sudden change hot-zone chosen is corresponding
Site includes the 344th, the 434th, the 133rd, 121.
Preferably, site the 344th, the 434th, 133 or 121s at occur simple point mutation；Wherein, the phenylalanine in 344 sites is dashed forward
Becoming isoleucine or methionine, the 434th, the phenylalanine in 133 and 121 sites sports TYR.The sudden change of corresponding LIP1
Body be respectively Phe344Ile (F344I), Phe344Met (F344M), Phe434Tyr (F434Y), Phe133Tyr (F133Y),
Phe121Tyr (F121Y), corresponding amino acid sequence is successively as shown in SEQ ID NO:2～SEQ ID NO:6, corresponding
Nucleotide sequence is successively as shown in SEQ ID NO:10～SEQ ID NO:14.
The above-mentioned primer sequence carrying out sudden change employing to 344 sites is as shown in SEQ ID NO:17, SEQ ID NO:18；Right
133 sites carry out the primer sequence of sudden change employing as shown in SEQ IDNO:21, SEQ IDNO:22；121 sites are suddenlyd change
The primer sequence using is as shown in SEQ ID NO:27, SEQ ID NO:28；Carry out the primer sequence that sudden change uses to 434 sites
As shown in SEQ ID NO:37, SEQ ID NO:38.
Preferably, the 344th, the 434th, occur at 133 and 121 simple point mutation to carry out orderly stack combinations by site to dash forward simultaneously
Become；Wherein, the phenylalanine in 344 sites sports isoleucine or methionine, and the 434th, the phenylalanine in 133 and 121 sites is equal
Sport TYR.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 successively as shown in SEQ ID NO:7, SEQ ID NO:8, corresponding
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 two maximum mutant VarA3 (F344M/F434Y/F133Y/F121Y) of heat endurance increase rate and VarB3
(F344I/F434Y/F133Y/F121Y)。
The principle of the present invention is:
For baroque protein, B-factor is combined with other design and rational thinkings, may be preferably
Enzyme stability is instructed to transform.
Activated centre be enzyme structure in the most key position, the appropriate flexibility of local conformation contributes to preferably combining the end
Thing, promotes the efficient catalytic of enzyme.But the activated centre of enzyme has certain fragility, a large amount of inhomogeneities in total
The denaturation result of type enzyme demonstrates, before the inactivation of enzyme is frequently experienced in the overall conformation change of detectable protein, i.e. " first loses
Live, rear denaturation ".In addition, in the research that laboratory, inventor place early stage is to lipase CalB, discovery enzyme active center region
Sudden change, in addition to affecting enzymatic, can also significantly improve the stability of enzyme.Therefore, the present invention proposes " enzyme active center
Stabilisation " strategy transforms the stability of enzyme, 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, by Pymol and B-
Near FITTER software is chosenWithin high flexibility amino acid be target.Carry out fixed point saturation mutation and build mutation library, utilize
Suitable screening technique obtains stability-enhanced mutant.
Compared with prior art, there is advantages that
1st, the present invention is under the guidance of " enzyme active center stabilisation " strategy, by protein engineering, is successfully obtained
Mutant Phe344Ile, Phe344Met, Phe434Tyr, Phe133Tyr, Phe121Tyr that heat endurance significantly improves.Its
Middle Phe344Ile and Phe344Met changes maximum, theyImprove 7.7 DEG C and 7.9 DEG C than wild type, to hydrolyze right simultaneously
Catalytic efficiency (the k of nitrophenol caprylate (pNP-C8)cat/Km) be respectively 0.94 and 1.23 times of wild type, i.e. enzyme stability
While raising, catalytic efficiency is not subject to too big impact.
2nd, suddenlyd change by orderly stack combinations, quickly obtain the mutant VarB3 that stability improves further, thermophilic
Degree improves 15 DEG C,Value also improves 13 DEG C than wild type, and the half-life improves about 40 times, the T of thermodynamic stabilitymValue carries
High 13 DEG C, catalytic efficiency is identical with wild type.
3rd, the present invention analyze simultaneously three typical case α/β hydrolases lipase (LipA, CalB and LIP1), they
Molecular weight has from small to large, structure has from simple to complicated feature；In conjunction with them at activated centre high flexibility residue
Sudden change significantly improve the stability of enzyme, imply that the versatility of " enzyme active center stabilisation " strategy；According to these three typical case
The sudden change result of enzyme, statistical separates out: distance catalytic residueNeighbouring high B-factor residue (B-factor relative value
At 60-100) it is sudden change hot-zone, the success rate of enzyme mutant can be improved.This transformation being other enzymes brings important theory to refer to
Lead.
Brief description
Fig. 1 is the schematic diagram of enzyme active center stabilisation strategy；
Fig. 2 is the target site schematic diagram selected by activated centre, and wherein, A is " lid " Unclosing structure figure of LIP1, B
" lid " closing structure figure for LIP1；
Fig. 3 is high thermal stability and the Tertiary screening setup schematic diagram of high catalysis activity mutant；
Fig. 4 is the protocol procedures figure of orderly stack combinations；
Fig. 5 is lipase LIP1 and the substrate selective schematic diagram of mutant；
Fig. 6 is lipase LIP1 and the thermodynamic stability schematic diagram of mutant；
Fig. 7 is the molecular force analysis chart within wild type LIP1 and mutant VarB3, and wherein, A is mutational site
The structure change of 121 and 133 peripheral regions, B is the structure change of mutational site 344 peripheral region, and C is mutational site 434 weeks
Enclose the structure change in region；
Fig. 8 is the topology diagram of different molecular weight lipase, and 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 focus amino acid.
Detailed description of the invention
The present invention relates to the strategy of a kind of general and efficient raising enzyme thermodynamic stability.This strategy be embodied as road
Line is: centered on the catalytic residue of zymophore, selects around according to the B-factor value in this enzyme crystal structure Within high flexibility residue (high B-factor) be target site, by build saturation mutation storehouse, screen thermokinetics and carry
High mutant (see Fig. 1).Under the guidance of this strategy, centered on catalytic residue Ser209, significantly improve structure height
The heat endurance of complicated fold lipase from candida sp 1 (Candida rugosa lipase1:LIP1).On integrated structure
The stability change result of the albumen (CalB) of little albumen (LipA) and moderate complexity, detective distance catalytic residueNear
High B-factor residue (B-factor relative value is at 60-100) for sudden change hot-zone, the success rate of enzyme mutant can be improved.With
When, further investigation enzyme regional stability law, development enzyme stabilization will be by mutant structure-emic network analysis
Technology is laid a good foundation.
With detailed description of the invention, invention is described further below in conjunction with the accompanying drawings:
The selection in embodiment the 1st, mutational site
Wild type (WT) amino acid sequence of the lipase 1 of Candida rugosa involved in the present invention is SEQ ID
Shown in NO:1, its nucleotides sequence is classified as shown in SEQ ID NO:9.
Search for the crystal structure of LIP1 in PDB database, select the structure that resolution ratio is the highest as research object.Due to
LIP1 has a spiral fragment being referred to as " lid " above active pocket, causes LIP1 to have two kinds of structures in the solution:
Open and Closed, is therefore selecting the target site time-division other to be analyzed two kinds of structures simultaneously.All residual to be catalyzed respectively
Centered on base Ser209, analyze aroundWithin all amino acid (with PyMOL order: PyMOL-＞ select AA,
Polymer within 10 of Ser209 searches).Re-use the B-factor of B-FITTER these amino acid of software analysis
Value, according to value order arrangement from big to small, chooses the maximum B-factor amino acid of first 11 in each structure as target
Site (is shown in Table 1).Have 3 to duplicate in these residues to choose, therefore finally 18 residues be have chosen altogether to two kinds of structures
As target site (see Fig. 2).These target site particular location in the structure is presented herein below: Phe344 and Phe345 is all located at
On α 12 spiral；Phe434 is positioned at β 13 and folds；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 positioned on the loop between α 10 and α 11；Gly414 is positioned at the corner between α 15 and α 16.
The B-factor value in table 1 activated centre target site and the distance arriving catalytic residue Ser209
Carry out saturation mutation in order to significantly more efficient, the combination of amino acids of adjacent sites is built mutation library together, knot
Fruit constructs following 12 mutation library: 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 show in fig. 2 with ball-and-stick model.
The 2nd, embodiment pinpoints saturation mutation and builds gene mutation storehouse
The primer building saturation mutation storehouse is shown in Table 2,
Table 2 is for building the primer in saturation mutation storehouse
Under the effect of PrimerSTAR max archaeal dna polymerase (TaKaRa company), with the wild type gene of LIP1 as mould
Plate, full plasmid amplification.PCR reaction condition: 98 DEG C of denaturations 5min, each circulates 98 DEG C of denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C
Extend 2.5min, totally 30 circulations；Last 72 DEG C extend 5min.PCR primer is directly proceeded to after purification bacillus coli DH 5 alpha sense
By state, 42 DEG C of thermal shock 90s, hatch 1h for 37 DEG C.It is coated with afterwards with on the LB flat board containing 25 μ g/ml Zeocin resistances, treated
After clone grows, directly with sterilized water, all clones are eluted from flat board, collect in a test tube, carry with kit
Take mixing plasmid.With the restricted enzyme AvrII that is digested, the mixing plasmid extracting is linearized, after product kits
Directly electric shock proceeds to Pichia pastoris GS115 competent cell, 30 DEG C of trainings on the YPD flat board containing 25 μ g/ml Zeocin resistances
Support 3d, obtain the bacterial strain containing mutator.
The preparation of embodiment the 3rd, Pichia pastoris competent cell
Picking Pichia pastoris list bacterium colony from YPD flat board, is inoculated in 4ml YPD fluid nutrient medium, vibration training at 30 DEG C
Support overnight, about about 24-48h, OD about 6-8.Nutrient solution is dispensed in 1.5ml EP pipe, 4 DEG C, 4000rpm, centrifugal 5min,
Abandon supernatant.Precipitation ice-cold sterilized water washing, 4 DEG C, 4000rpm, centrifugal 5min, abandon supernatant.Precipitation 1ml lithium acetate solution
Process, precipitate resuspended after, 30min at placing 30 DEG C.4 DEG C afterwards, 4000rpm, centrifugal 5min, abandon supernatant.(lithium acetate solution:
10mM pH7.5Tris-HCL；10mM DTT；100mM LiAC) precipitation washed by ice-cold sterilized water, 4 DEG C, 4000rpm, from
Heart 5min, abandons supernatant.The precipitation D-glucitol washing of ice-cold 1M, 4 DEG C, 4000rpm, centrifugal 5min, abandon supernatant.Thoroughly fall
Go out supernatant, add the D-glucitol 50 μ l of 1M, be prepared as competent cell, prepare next step and convert.
(note: competence now to do existing use, and the DTT in treatment fluid is to add before use, and other prepare after 4 DEG C of guarantors
Depositing, DTT, separately from-20 DEG C of preservations, can be made into the stock solution of 100 times.)
Embodiment the 4th, three level screen saturation mutation storehouse
Three level screen method is as it is shown on figure 3, mainly include the primary dcreening operation of Fluorescence Plate and the multiple sieve of 96 orifice plates twice；Fluorescence is put down
The method of plate primary dcreening operation: the monoclonal that will grow in YPD_Zeo resistant panel, transfers to two new putting down by photocopy cellulose membrane
On plate；Cultivating two days for 30 DEG C, a flat board is stored in refrigerator, and another flat board proceeds in the incubator of 65 DEG C；Heat treatment 40min,
After taking-up, room temperature cooling 20min, the flexible glue culture medium 10ml that will dissolve, pours on this flat board；Again cultivate 5h at 30 DEG C, at purple
Observing fluorescence under outer light, the mutant of high thermal stability and activity demonstrates bigger fluorescent ring.Screened permissible by fluorescent ring
Effectively eliminate the clone that negative sudden change occurs in activity and stability.Sieve again followed by 96 orifice plates, the edge in each 96 hole
Respectively take hole with centre and cultivate wild type LIP1 as comparison, with YPD fluid nutrient medium at 30 DEG C, under 220rpm, cultivate 72h.
4000rpm centrifuges 96 orifice plate 30min, turns culture supernatant in another orifice plate, with sterilized water by each aerial Sample Dilution 4
Times.Enzyme liquid after dilution is divided into two parts, and a part directly takes the vigor that 10 μ l measure hydrolysis pNP-C4 on ELIASA, preserves
Data, as the vigor before heat inactivation.Another part takes 50 μ l in 96 hole PCR plate, 58 DEG C of heat treatment 15min in PCR instrument.
It is centrifuged off precipitation afterwards, take centrifugal supernatant and on ELIASA, measure remaining vigor, as data after heat inactivation；By two secondary data
Compare, calculate inactivation rate, from inactivation rate higher than wild type 20% as screening criteria, the clone screening enter next step 96
Hole sizer selects.In order to get rid of the false positive occurring in primary dcreening operation, by 96 new for single colony lift of obtaining to next one deep-well plates, often
Individual positive colony proceeds to cultivate in 4 holes of row, analyzes the inactivation rate after heat treatment；The real positive then shows in 4 holes
Inactivation rate will be higher than wildness, and increase rate is close.
Embodiment the 5th, orderly stack combinations is suddenlyd change
In order to quickly obtain the cumulative effect of beneficial mutation bacterial strain, take orderly stack combinations mutation scheme, use every time
The next best mutant of best mutant combinations.This combinatorial mutagenesis scheme can more be quickly obtained combinatorial mutagenesis result,
Concrete anabolic process is shown in Fig. 4.Owing to two single-point mutants at site 344 have carrying of high-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
Except, in addition to starting strain is different (being F344M and F344I respectively), remaining anabolic process is just the same.Article two, the group in approach
Close mutant to replace by 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).Demonstrating from the combined result of Fig. 4, each combination can make stability have to improve in various degree.These results
Absolutely prove and between four single-point mutants, there is cumulative effect, and confirm that orderly stack combinations mutation scheme is a kind of
The quick effective way obtaining more preferable result.
The expression of embodiment the 6th, fold lipase from candida sp 1 wild type and mutant and purifying
From YPD_Zeo flat board, picking list bacterium colony is cultivated in the YPD culture medium of 4ml, works as OD600When reaching 2, by 1%
Inoculum concentration proceeds to express in new 200ml YPD, and every 12h samples detection vigor, terminates until cultivating.
N end due to genes of interest devises the label of 6 × His, and the method that can directly utilize Ni-affinity chromatography is pure
Change.LIP1 is with secretion expression in Pichia pastoris, and final expression product is mainly in nutrient solution, therefore needs to carry out nutrient solution
Concentration collect.Therefore, the purifying of whole LIP1 be divided into ultrafiltration concentration and two steps of Ni-affinity chromatography:
(1) it is concentrated by ultrafiltration
Yeast expression nutrient solution centrifuges 20min under the rotating speed of 8000rpm, collects supernatant；By the fiber of 0.22 μm
After membrane filtration, proceed in the ultrafiltration apparatus of Millipore；Carry out desalination, decolouring and concentration with the milipore filter bag of 10kD, dense
Constantly with addition ddH in compression process2O, until concentrate is close to colourless；Again that albumen is dense with the Tris-HCl 200ml of 20mM
Contracting, at about 100ml, proceeds to affinity chromatography, and sampling carries out electroresis appraisal and concentration measures.
(2) affinity chromatography
By water with after the membrane filtration of 0.22 μm, directly add 5ml to above nickel post → flowed after add 5ml strip
Buffer (100mM EDTA, 0.5M NaCl, 50mM Tris-HCl, pH 7.5), by Ni2+With foreign protein from carrier NTA agar
On sugar washing get off → pillar add 5ml water punching do → flowed after addition 5ml charge buffer (100mM NiSO4) → flowed
After add water flushing → addition 10ml binding buffer (50mM Tris-HCl, pH 7.5,20mM imidazoles, 0.5M NaCl)
→ flowed after add the enzyme liquid → flowed after being concentrated by ultrafiltration after add 10ml binding buffer → flowed after add 5ml
Wash buffer (200mM imidazoles, 0.5M NaCl, 50mM Tris-HCl, pH 7.5) gradient elution, collects efflux → post
Son adds water flushing, is saved in 4 DEG C of refrigerators with 20% ethanol.
Eluent is collected together and proceeds to bag filter, 4 DEG C of dialysis in 50mM Tris-HCl, pH 7.5 buffer solution
24h, dislysate twice is changed in centre.Finally collect albumen, preserve standby 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 through order-checking, the amino acid sequence of said mutation body enzyme
Leu 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 the 7th, fold lipase from candida sp vitality test and substrate selective analysis
Divide caprylate (pNP-C8) as substrate with p-nitrophenyl, become the concentration of 10mM with acetontrile.PH with 50mM
8.0Tris-HCl is as buffer solution, 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 utilizing ultraviolet specrophotometer.Concrete reaction system is:
The substrate taking 20 μ l is dissolved in the buffer solution of 970 μ l, 40 DEG C of preheating 2min, adds 10 μ l enzyme liquid, blows even reaction with liquid-transfering gun
System, starts to record course of reaction, stops reaction, calculate the vigor of enzyme according to formula below (formula 1) after about 2min.1
Individual enzyme activity unit (U) is: the 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): in the reaction time, light absorbs the changing value of 405nm；vr(1): reaction
Volume；ε(M-1·em-1): the molar absorption coefficient (1.6 × 10 of p-nitrophenol4)；D (cm): the path that light passes through；ve
(1): reaction cumulative volume；ce(mg/l): the concentration of enzyme；Experimental data is to average for three times.
For the vigor of the pNP substrate measuring different chain length, method is same as described above, and result is shown in Fig. 5.
The heat endurance of embodiment the 8th, fold lipase from candida sp 1 and mutant and kinetic parameter analysis
Heat endurance in order to analyze mutant changes, and the present invention utilizes final concentration 0.2mM p-nitrophenol caprylate, point
Other to heat endurance Common Parameters t1/2(half-life), 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 analyzing pNP-C8 under different temperatures measures LIP1
Thermophilic degree.Heat stability test, by being divided in every for the LIP1 having purified (0.1mg/ml) 50 μ l in multiple centrifuge tube, is placed
It is incubated at 60 DEG C, samples in different time intervals, measure remaining vigor.According to formula 2, at a temperature of can being conversed this
Half-life.
t1/2=ln2/kd(formula 2)
Heat endurance parameterAssay method: by every for the LIP1 having purified (0.1mg/ml) 50 μ l are dispensed into 96 holes
In PCR plate, thermograde 40～80 DEG C is set, carries out 15min heat inactivation process to enzyme；Terminate latter 4 DEG C cooling 10min, room temperature
Standing 20min, 4000rpm centrifuges 96 hole PCR plate 30min, removes precipitation；Analyze the remaining vigor of supernatant, with relative residual
Vigor and temperature make curve map；According to Boltzmann's model (formula 3), matching under Origin8.0 software auxiliary, calculateValue.
Y=A2+ (A1-A2)/(1+exp ((x-x0)/dx)) (formula 3)
The analysis of kinetic parameter is at pH 8.0, under conditions of temperature 40 DEG C, with pNP-C8 as substrate, measures the end respectively
Substrate concentration is the 4th, the 10th, the 15th, the 20th, the 50th, the 100th, the hydrolysis vigor corresponding to 150 and 200 μM.Utilize LIP1 to different concentration of substrate
Catalysis activity data, simulate function curve by software Origin 8.0 and Michaelis-Menten equation (formula 4), thus obtain KmWith
Vmax, it further according to the functional equation (formula 5) of maximum rate and enzyme concentration, is calculated kinetic constant Km, kcatAnd kcat/Km。
V=Vmax[S]/(Km+ [S]) (formula 4)
Vmax=kcat× [E] (formula 5)
Through above assay method, it is thus achieved that kinetic parameter and the heat endurance parameter of LIP1 and mutant are shown in Table 3.
The kinetic parameter of table 3 lipase LIP1 and mutant and heat endurance
The 9th, embodiment analyzes the thermodynamic stability change (T of LIP1 and mutantm)
Set scanning temperature range as 30～90 DEG C, sweep speed is 1 DEG C/min.The concentration of protein sample controls
0.5mg/ml, is dissolved in the phosphate buffer of 10mM；First at 30 DEG C of pre-equilibration 15min, T before starting to scanmFor the highest crest
Locate corresponding temperature.
Fig. 6 analysis result demonstrates the T of all mutantmValue has had raising in various degree relative to wild type,
Lifting (Δ T by a small marginm) also there are 4.5 DEG C (F434Y, 57.9 DEG C).It should be noted that TmWhat value improved maximum is not to move
Mechanical stability improves the most significant Var3 (Δ Tm=12.7 DEG C), but three Sites Combination body combination mutant VarA2
(F344M/F434Y/F133Y), its TmValue is risen to 70.8 DEG C by the 53.4 of wild type DEG C, improves nearly 17.5 DEG C.This
Individual result shows, dynamic stability and thermodynamic stability non-fully corresponding direct proportionality.
The 10th, embodiment analyzes the stability-enhanced mechanism of stability mutant VarB3
Demonstrate that mutant VarB3 and wild type LIP1 are structurally closely similar by homology modeling, in order to rationally solve
Release the mechanism causing protein stability to improve, need the residue reducing Normalized B-factor near catastrophe point to carry out
Deeply seeing clearly of intramolecular action power.Utilize PyMOL software analysis mutant VarB3 model structure and LIP1 crystal structure
(1CRL) intramolecule interacts, detailed results See Figure 7.
It can be seen that site 12 1 and 133 is mutated into TYR from Fig. 7 A, result in 7 new hydrogen bonds and produce, its
Tyr133 after middle sudden change defines 3 hydrogen bonds with hydrone Wa, hydrone Wb and Gly122, and this 3 hydrogen bonds are deposited before sudden change
4 hydrogen bonds being between hydrone Wa, Wb and Gly122, altogether 7 hydrogen bond shapes between Tyr133, Wa, Wb and Gly122
Become a hydrogen bond network interacting.And the Tyrl21 after suddenling change and hydrone Wc, Phe128, Asn155 and Tyr156 shape
Become 4 hydrogen bonds, 2 hydrogen bonds being present between hydrone Wc and Phe128, Asn155 before this 4 hydrogen bonds and sudden change, altogether 6
Individual hydrogen bond also occurs in that a hydrogen bond network interacting between Tyr121, Wc, Phe128, Asn155 and Tyr156.Hydrogen
Key network is the key factor improving protein stability, and the appearance of the two hydrogen bond network will assist in stable residue
The swing in loop region between 120 and 136, thus also contributed to stablizing of overall albumen.
It is apparent that after site 344 is mutated into Ile, α 12 spiral is obviously prolonged from Fig. 7 B.This is mainly Ile
Introducing cause the side chain of Phe345 and Thr343 to there occurs slight deflection, furthered the distance between Thr343 and Gly346,
Create one between whichNew hydrogen bond；Also cause hydrogen-bond length between Phe345 and Ser348 by former simultaneously
ComeBecomeGly346 makes its side chain exist closer to Ser349, result under the pulling being newly formed hydrogen bond
Two hydrogen-bond length between Gly346 and Ser349 are all by originalBecomeAll these stack more closely
Make the loop being originally positioned between Thr347 and Ser349 be transformed to α spiral, extend spiral α 12 accordingly；Loop turns
Turn to spiral and make the rigidity reinforced of local, activated centre.
Finding from Fig. 7 C, at the 434s of site, phenylalanine is mutated into TYR and defines oneHydrogen bond, this
Individual hydrogen bond occurs between Tyr434 and hydrone Wd.Examine β 13 and fold it can be seen that residue on it and surrounding ammonia
Base acid hydrogen-bond length each other all there occurs reduction in various degree, they respectively: between Tyr432 and Asp479
Hydrogen-bond length is by originalBecomeTwo hydrogen-bond length between Ser433 and Ile335 respectively byWithBecomeWithHydrogen-bond length between Ser433 and Met503 by BecomeLeu435 with
Hydrogen-bond length between Ile505 byBecomeAlthough the change of these hydrogen-bond length is less, but multiple minor variations
Polymerization will bring the rigidity reinforced that β 13 folds.After analysis detailed to structure above, find interaction of hydrogen bond net
The intensive stacking of network and activated centre is to cause the enhanced main cause of local stiffness, and then reflects mutant on the whole
The stability of VarB3 improves.
The versatility of embodiment the 11st, activated centre stabilisation strategy
In order to explore the amino acids distribution feature that stability is had a major impact by enzyme active center, to different structure complexity
The stability of zymoprotein improves result and has carried out labor, especially German scholar Reetz achievement in research (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.).It is found that the albumen of Reetz sudden change is that " enzyme is lived
Property central stabilizing " strategy a special case.LipA is a minimum α/β hydrolase folded protein, and it only has 181 amino
Acid composition, from Fig. 8 A it can be seen that in structure, it only has α spiral and the β-pleated sheet formation active site of standard, and there do not have to be unnecessary
Secondary structure is wrapped in protein surface, and LipA has structurally also possessed simple feature.The catalysis activity that the present invention selectes
Around residueWithin high flexibility residue, for LipA, almost whole albumen is all contained into, Reetz select
The Mutated residues major part selected is all at distance 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 up of 317 amino acid, except there is standard hydrolysis centre in structure
Outside the α/β of enzyme folds, around also have 4 α spirals and 2 β-pleated sheet parcel (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.)；Additionally also there is one section by 5 amino acid
The α spiral that (residues 142～146) is formed covers on active pocket, owing to this section of spiral is too short, not to vigor band
Carry out too big impact；Also there is document to report that this section of spiral 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, and it has had again once highly super on molecular weight and structure complexity
More.Such as Fig. 8 C, LIP1 is made up of 534 amino acid, in addition to the α/β of inside configuration hydrolase standard folds, at zymoprotein
Surrounding also have 12 α spirals and 5 β-pleated sheets parcel；There is one section by 29 amino acid above its active pocket other
(residues 65～94) formed α spiral as lid, this lid make it structurally have two kinds of configurations (open and
closed).To sum up analyze the architectural feature of LipA, CalB and LIP1, it is found that they are that zymoprotein is 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
Significantly improve the stability of enzyme, imply that " enzyme active center stabilisation " strategy exists certain versatility；The knot improving from them
Find in Guo that some potential rules may bring important guiding information for the stability transformation of other enzymes.
The 12nd, embodiment analyzes the distribution characteristics of activated centre focus amino acid
With the stability of LIP1, result and the distance of Mutated residues to catalytic residue, relative B-are improved to LipA, CalB
Factor value is (with respective catalytic residueInside high B-factor value is reference, calculates remaining simple point mutation residue
B-factor) labor has been carried out.WithRepresent that all simple point mutations improve the parameter of stability, remaining negative sudden change or group
Conjunction sudden change is all considered as stability and does not change, and has obtained the result of Fig. 9 on this basis.Detective distance catalytic residueIn the range of be all improved the stability of enzyme, wherein many numerical mutations are positioned atAndThe neighbouring width improving
Spend the most obvious；And be less thanBe more thanIn the range of find no single-point amino acid sudden change improve enzyme stability；
It and B-factor raising to enzyme stability in wide distribution relatively all has an impact, is concentrated mainly on relative B-factor
Between value 60～70 and 90～103；It is stable that relative B-factor value finds no simple point mutation raising enzyme more than 103 with less than 60
Property, therefore, " enzyme active center stabilisation " strategy is at selected distance catalytic residueWith relative B-factor value 60～100
(with activated centreInside high B-factor is reference) between high flexibility residue suddenly change, it will further improve enzyme
The success rate of stability transformation.
Claims (10)
1. the universal method improving enzyme thermodynamic stability, it is characterised in that the catalytic residue with zymophore is
Center, carrys out chosen distance catalytic residue according to the B-factor value in this enzyme crystal structureWithin high flexibility residue be
Target site, passes through screen mutation, it is thus achieved that the mutant that enzyme thermodynamic stability improves.
2. the universal method improving enzyme thermodynamic stability as claimed in claim 1, it is characterised in that described screening is three
Level screening, including the multiple sieve of the primary dcreening operation of Fluorescence Plate and twice 96 orifice plates.
3. the universal method improving enzyme thermodynamic stability as claimed in claim 2, it is characterised in that described Fluorescence Plate
Primary dcreening operation include mutant monoclonal 65 DEG C heat treatment 40min, room temperature cooling 20min after in flexible glue culture medium at 30 DEG C cultivate
5h, takes out and observes fluorescence under ultraviolet light, demonstrates that bigger fluorescent ring is high thermal stability and the candidate strain of activity；Institute
The multiple sieve stating 96 orifice plates twice includes: the edge of first 96 orifice plate and centre respectively take hole and cultivate wild type LIP1 as right
According to, in PCR instrument after 58 DEG C of heat treatment 15min, the vigor of mensuration hydrolysis p-nitrophenol butyrate, from inactivation rate higher than open country
Raw type 20% as screening criteria, the clone screening enters the screening of next step 96 orifice plate；Each positive colony proceeds to row 4
Individual hole is cultivated, analyzes the inactivation rate after heat treatment；The real positive then shows the inactivation rate in 4 holes will compare wildness
Want height, and increase rate is close.
4. the universal method improving enzyme thermodynamic stability as claimed in claim 1, it is characterised in that described method is also wrapped
Include the mutant that the enzyme thermodynamic stability screen mutation improves, obtain enzyme heat power by the sudden change of orderly stack combinations
Learn the step of the higher mutant of stability.
5. the universal method improving enzyme thermodynamic stability as claimed in claim 4, it is characterised in that described orderly superposition
Combinatorial mutagenesis specific implementation process is: according to single-point mutants stability increase rate order from big to small, combine step by step, directly
It is all introduced in a mutant to all mutational sites.
6. the universal method improving enzyme thermodynamic stability as claimed in claim 1, it is characterised in that described enzyme is selected from
LipA, Ca1B or the fold lipase from candida sp LIP1 with amino acid sequence as shown in SEQ ID NO:1.
7. the universal method improving enzyme thermodynamic stability as claimed in claim 6, it is characterised in that chosen distance
The catalytic residue of LipA, Ca1B or LIP1With relative B-factor value in the region of 60-100 for sudden change hot-zone.
8. the universal method improving enzyme thermodynamic stability as claimed in claim 6, it is characterised in that described enzyme is Lip1
When, with sequence as shown in SEQ ID NO:1 as reference sequences, the 434th, the 344th, the corresponding site in sudden change hot-zone chosen include
133、121。
9. the universal method improving enzyme thermodynamic stability as claimed in claim 8, it is characterised in that in site the 344th,
434th, there is simple point mutation at 133 or 121；Wherein, the phenylalanine in 344 sites sports isoleucine or methionine, the 434th,
The phenylalanine in 133 and 121 sites sports TYR.
10. the universal method improving enzyme thermodynamic stability as claimed in claim 8, it is characterised in that pass through site
344th, the 434th, simple point mutation is occurred at 133 and 121 to carry out orderly stack combinations sudden change；Wherein, the phenylalanine sudden change in 344 sites
For isoleucine or methionine, the 434th, the phenylalanine in 133 and 121 sites all sports TYR.
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| CN111286497A (en) * | 2020-02-19 | 2020-06-16 | 江南大学 | Lipase with improved catalytic performance and application thereof |
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| CN108118065A (en) * | 2016-11-26 | 2018-06-05 | 华中科技大学 | A kind of method for improving enzyme heat stability |
| CN108118043A (en) * | 2016-11-26 | 2018-06-05 | 华中科技大学 | The lipase mutant that a kind of thermal stability improves |
| CN108118043B (en) * | 2016-11-26 | 2020-05-19 | 华中科技大学 | Lipase mutant with improved heat stability |
| CN108118065B (en) * | 2016-11-26 | 2020-05-19 | 华中科技大学 | Method for improving thermal stability of enzyme |
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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 |
| EP3677682A4 (en) * | 2017-09-01 | 2021-05-05 | Amano Enzyme Inc. | Modified lipase and use thereof |
| EP4282960A3 (en) * | 2017-09-01 | 2024-01-24 | Amano Enzyme Inc. | Modified lipase and use thereof |
| CN111032872A (en) * | 2017-09-01 | 2020-04-17 | 天野酶制品株式会社 | Modified lipase and use thereof |
| US11299722B2 (en) | 2017-09-01 | 2022-04-12 | Amano Enzyme Inc. | Modified lipase and use thereof |
| CN108841801A (en) * | 2018-05-29 | 2018-11-20 | 江南大学 | A kind of method of amino acid residue relevant to enzyme activity in screening enzyme |
| CN110982801A (en) * | 2019-12-27 | 2020-04-10 | 中国科学院苏州生物医学工程技术研究所 | Transaminase mutant and construction method and application thereof |
| CN111286497B (en) * | 2020-02-19 | 2021-08-24 | 江南大学 | Lipase with improved catalytic performance and application thereof |
| CN111286497A (en) * | 2020-02-19 | 2020-06-16 | 江南大学 | Lipase with improved catalytic performance and application thereof |
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