CN108342372A - A kind of marine source high temperature lipase and its crystal and preparation method - Google Patents
A kind of marine source high temperature lipase and its crystal and preparation method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention belongs to technical field of enzyme engineering, a kind of marine source high temperature lipase and its crystal and preparation method are disclosed.The amino acid sequence of marine source high temperature lipase such as SEQ ID NO：Shown in 1.The preparation method of the marine source high temperature lipase, the sequence of marine source high temperature lipase is cloned into pET22b (+) prokaryotic expression carrier and converts the carrier to competent escherichia coli cell Rosetta (DE3) and is expressed, it is purified by affinity chromatography and exclusion Gel Chromatography, obtains marine source high temperature lipase aqueous solution.Lipase of the present invention has good high temperature and organic solvent tolerance, esterification efficient or even more preferable than the effect of commercial lipases 435 (Novi's letter) lactate synthesis triglyceride with hydrolyze lipid.
Description
Technical field
The present invention relates to two kinds of marine source high temperature lipase SL1 crystal and preparation methods, belong to technical field of enzyme engineering.
Background technology
Lipase is a kind of α/β hydrolase for being widely present in nature and having essential industry application value, can be used
In reactions such as catalysis esterlysis (hydrolysis, alcoholysis and acidolysis), Lipase absobed, transesterifications, it is widely used in fats and oils processing, food, makes
The industrial circles such as paper, detergent, indicator, biodiesel, fine chemistry industry, leather processing.Currently, in US National biological information
The lipase reported in the heart (NCBI) is more than 6000, and RCSB Protein Data Banks (PDB) have included nearly 700 fat
The three-dimensional structure of fat enzyme, and these numbers are also rapidly increasing.
Although the catalyst mechanism of lipase is resolved already.But it is still shown not between various types of lipase
The same biochemistry and catalysis characteristics, such as optimal reaction environment, temperature and solvent tolerance, substrate selective, kinetics spy
Sign, the difference of interfacial effect etc..This is primarily due between each lipase that there are more or less sequence difference and structure are poor
It is anisotropic.Although the present mankind have had certain accumulation to the excavation of lipase resource, but still cannot fully meet production
Demand in practice.Therefore, be transformed to existing lipase seems necessary to meet production requirement just.And space
Structure can provide a large amount of structural information for the transformation of lipase, preferably instruct the directional transformation of lipase.
Marine source high temperature lipase SL1 has good high temperature and organic solvent tolerance, esterification and hydrolyze lipid
Efficiency it is very high, or even believe that the effect of 435 lactate synthesis triglyceride of lipase is more preferable than commercialization Novi, and with extensive
Fatty acid selectivity can be widely used in the catalytic fields such as daily use chemicals, food processing and medicine, have wide and bright city
Field foreground and application potential.Therefore, it obtains marine source high temperature lipase SL1 crystal and parses its space structure, to determining it
Catalytic pocket composition, all types of substrates and activated centre binding pattern are determined to the contributive structural factor of stability, are deeply ground
There is important theory directive significance and medicine to answer for the design of the molecular mechanism and micromolecular inhibitor of studying carefully its substrate selective
With value.The high temperature lipase of especially currently known structure is simultaneously few.Therefore, in order to more fully understand high temperature tolerance and height
The acquisition of the design feature of catalytic efficiency lipase, marine source high temperature lipase structure just seems necessary.
Invention content
One of the objects of the present invention is to provide a kind of marine source high temperature lipases.
The second object of the present invention is to provide two kinds of marine source high temperature lipase crystal, preparation and structure elucidation side
Method.
The purpose of the present invention is achieved through the following technical solutions：
A kind of marine source high temperature lipase, molecular weight are 30 ± 0.5KD, contain 279 amino acid, amino acid sequence
Such as SEQ ID NO：Shown in 1.
The preparation method of the marine source high temperature lipase, the sequence of marine source high temperature lipase is cloned into
PET22b (+) prokaryotic expression carrier simultaneously converts the carrier to competent escherichia coli cell Rosetta (DE3) expression, passes through
Affinity chromatography and exclusion Gel Chromatography purifying, obtain marine source high temperature lipase aqueous solution.
The preparation method of marine source high temperature lipase crystal, includes the following steps：
(1) preparation of crystallization buffer：The group of crystallization buffer becomes (A) 0.2 ± 0.05M zinc acetates, 0.1 ± 0.02M
Imidazoles, 10 ± 0.5%PEG8000, pH 6.0 ± 0.5 or (B) 1.0 ± 0.1M sodium dihydrogen phosphates-disodium hydrogen phosphate, pH 5.5
±0.5；
(2) the marine source high temperature lipase aqueous solution for preparing claim 2 by volume 1：1 is prepared with step 1)
Crystallization buffer mixing, in 15-25 DEG C of indoor placement, culture obtains the crystal of marine source high temperature lipase in 5~7 days.With inclined
Light stereoscopic microscope observing crystal growth condition, the crystal that two kinds of crystallization conditions are obtained are respectively outside hexagon and corynebacterium
Shape, as shown in Figure 2.
The group of the crystallization buffer becomes：(A) 0.21M zinc acetates, 0.1M imidazoles, 10%PEG8000, pH 6.0 or
(B) 1.05M sodium dihydrogen phosphates-disodium hydrogen phosphate, pH 5.0.
A concentration of 18~25mg/mL of the marine source high temperature lipase aqueous solution.
A concentration of 21mg/mL of the marine source high temperature lipase aqueous solution.
Diffraction Data Collection：Utilize the quick-frozen crystallization buffer for having added 20% glycerine as antifreezing agent that is immersed in of ultra-low temperature liquid nitrogen
Single crystal in liquid is sent to Shanghai synchrotron radiation light source, and the crystal data obtained by x-ray source, diffraction data uses
MOSFLM programs are collected and are arranged.
The parsing of crystal structure：Crystal structure is by anti-in the diffraction data of marine source high temperature lipase SL1 crystal A
Normal scattered signal come determine with the phase of 4 zinc ions of marine source high temperature lipase SL1 non-specific bindings, then use
Buccaneer softwares have built the initial model of marine source high temperature lipase SL1, with Refmac5 and Coot softwares hand repeatedly
It is dynamic to correct and optimize the initial configuration, and with the architecture quality after MolProbity software inspections modification.Point of the correcting principle
Resolution isRworkThe factor is 17.8%, RfreeThe factor is 20.58%.RfreeThe factor and RworkThe difference of the factor is
2.78%, bond distance and bond angle root-mean-square-deviation are respectivelyWithMarine source high temperature lipase SL1 crystal
The structure of B is oriented using molecular replacement technique using marine source high temperature lipase SL1 crystal A as template, with Refmac5 and
Coot softwares repeatedly manual correction and optimize the initial configuration.The resolution ratio of the structure isRworkThe factor is
19.14%, RfreeThe factor is 22.16%.RfreeThe factor and RworkThe difference of the factor is 3.02%, and bond distance and bond angle root mean square are inclined
Difference is respectively With
Structure cell and structure feature：Two kinds of marine sources high temperature lipase SL1 crystal A and B, fat enzyme crystal A and B difference
For hexagon and corynebacterium pattern, space group is respectively P64And P3221, the lattice constant of wherein crystal A is α=β=90 °, γ=120 °；The lattice constant of crystal B isα=β=90 °, γ
=120 °.In an asymmetry unit, crystal A is monomer, and there are three asymmetric molecults by crystal B.The two crystal have symbol
Close the design feature of table 1.The folding center of marine source high temperature lipase SL1 is made of 6 parallel β-pleated sheet pieces, and by 12
A α spiral packagings, wherein the α 5 being made of glycine (G146) to proline (P171) is covered in as lid on active pocket
Side (Fig. 3)；Catalytic triads are by serine (S115), aspartic acid (D206) and histidine (H238) composition (Fig. 3)；Oxygen bear from
Sub- hole is made of (Fig. 4) the main chain imino group of threonine (T44) and glutamine (Q116).
Influence of the temperature to hydrolysis vigor：To emulsify olive oil as substrate, in the phosphate buffer of 0.1M pH 7.0
Under measure marine source high temperature lipase SL1 in 50-90 DEG C of hydrolysis vigor, result such as (Fig. 5).Marine source high temperature fat
Thus the optimal reactive temperature range of enzyme SL1 can determine whether that the lipase is high temperature enzyme at 70 DEG C or so.
The esterification of oleic acid and glycerine：By the high-purity marine source high temperature lipase SL1 immobilised enzymes prepared with have
The commercialization Novozym 435 of height esterification effect is carried out at the same time the catalysis reaction of the esterification of oleic acid and glycerine.Lipase SL1 is 24
Esterification yield and content of triglyceride respectively reach 99.5% and 91.3% after the vacuum reaction of hour；And Novozym 435 can only
Reach 79.3% and 48.7%.Experimental result shows that lipase SL1 has better catalyzing esterification efficiency.
Temperature tolerance：In the high-purity marine source high temperature lipase SL1 aqueous solutions prepared respectively 55,60,
65 and 70 DEG C of incubations, per half an hour sample detection, it remains enzyme activity (with the enzyme enzyme activity that is not incubated for 100%).As a result such as Fig. 6,
It is incubated 3 hours at 55 DEG C, which still retains 95% or more vigor；3 hours enzymes are incubated at 70 DEG C also still to have more than
50% vigor.It can be seen that the enzyme is a kind of thermal stable lipase.
Organic solvent tolerance：It is separately added into the high-purity marine source high temperature lipase SL1 aqueous solutions prepared
Various organic solvents：Methanol, ethyl alcohol, isopropanol, acetone and hexamethylene, a concentration of 90% (v/v), 4 DEG C of placements, the sampling inspection per 4h
It surveys it and remains enzyme activity (be not added with enzyme activity when organic solvent as 100%).As a result such as Fig. 7, the marine source in each organic solvent
High temperature lipase SL1 shows good stability, remain 90.2% after being incubated 24 hours respectively, 94.7%, 97.2%,
92.3% and 96.1% vigor, it is seen that the lipase has good stability in organic solvent.
The gene order of marine source high temperature lipase SL1 is the streptomycete isolated from the sediment of Chinese Jiaozhou Bay
It is found in the genome sequence of (marine Streptomyces).
Compared with prior art, the present invention has the advantages that：
Present invention finds a kind of structures of completely new marine source high temperature lipase SL1.It is determined that it is big by one with one
The unique cap structure of small two α spirals, wherein glycine (G146) are covered to the α 5 that proline (P171) forms as lid
It covers above active pocket；Determine its catalytic triads by serine (S115), aspartic acid (D206) and histidine
(H238) it forms；Negative oxygen ion hole is made of the main chain imino group of threonine (T44) and glutamine (Q116).And the fat
Enzyme has good high temperature and organic solvent tolerance, and esterification is efficient with hydrolyze lipid, or even compares commercial lipases
The effect of 435 (Novi's letter) lactate synthesis triglycerides is more preferable.In the case where vacuumizing environment, the esterification yield of SL1 up to 99.5%, and
Lipase 435 is 79.3%.Therefore, marine source high temperature lipase SL1 can be widely used in daily use chemicals, food processing and
The catalytic fields such as medicine have wide and bright market prospects and application potential.
Description of the drawings
Fig. 1 marine source high temperature lipases SL1 SDS-PAGE electrophoresis after purification.
Fig. 2 marine source high temperature lipase SL1 crystal petrographic microscope photos, A are crystal A, and B is crystal B.
The schematic diagram of Fig. 3 marine source high temperature lipase SL1 monomer structures.
Fig. 4 marine source high temperature lipase SL1 negative oxygen ions hole schematic diagram.
Fig. 5 temperature hydrolyzes marine source high temperature lipase SL1 the influence of vigor.
Tolerances of Fig. 6 marine source high temperature lipase SL1 to temperature.
Tolerances of Fig. 7 marine source high temperature lipase SL1 to organic solvent.
Specific implementation mode
The present invention is further illustrated the present invention with the following example, but protection scope of the present invention is not limited to following reality
Apply example.
The expression and purification of 1 marine source high temperature lipase SL1 of embodiment
It is prepared by the crude enzyme liquid of marine source high temperature lipase SL1：Marine source high temperature lipase SL1 gene orders are cloned
It is converted to competent escherichia coli cell Rosetta (DE3) to pET22b (+) prokaryotic expression carrier and by the carrier.Contain
The low-temperature preservation of above-mentioned transformant is added in the test tube of the LB culture mediums 5mL of 1mM ampicillins (Amp) with 5% inoculum concentration
Glycerine bacterium solution, 37 DEG C of overnight isothermal vibration cultures.Above-mentioned seed liquor is seeded to the LB that 100mL contains 1mM ampicillins again
Culture medium, 37 DEG C, it is 0.8 that 200rpm, which is cultivated to bacteria suspension 600nm absorbances,.Isopropyl-beta D-thio galactopyranose is added
For glycosides (IPTG) derivant to final concentration of 0.2mM, 20 DEG C induce 16h.Thalline is collected with 4500r/min low-temperature centrifugations 20min, with
Appropriate affinity chromatography (Ni chelatings) Buffer A (20mM sodium dihydrogen phosphates-disodium hydrogen phosphate, 20mM imidazoles, 0.5M sodium chloride,
0.5% Bio-Rad-Laboratories, pH 7.4) buffer solution resuspension bacterium.With sonioation method crack thalline, with 12000r/min low temperature from
Heart 10min removals precipitation and other particulate contaminations.
Affinity chromatography (Ni chelatings)：Tomographic system is balanced with the Buffer A of 2 column volumes, by marine source high temperature grease
Fat enzyme SL1 crude enzyme liquid loadings.Continue to be rinsed to baseline with Buffer A after end of the sample and balance.Then affinity chromatography is used again
(Ni chelatings) Buffer B (20mM sodium dihydrogen phosphates-disodium hydrogen phosphate, 0.3M imidazoles, 1M sodium chloride, pH 7.4) are eluted, monitoring
The situation of change of absorption value at UV detector 280nm collects out the protein sample of peak position.
S75 gel exclusion chromatographies：Protein sample is crossed into 0.45 μm of filter membrane except impurity and at 4 DEG C, 12000rpm centrifuges 10min
Removal denaturation protein precipitation.Gel is rinsed with gel exclusion chromatography Buffer (0.1M NaCL, 20mM Tris-HCl pH8.0)
Exclusion chromatography system to baseline balances.5mL protein samples are injected into loading ring loading.Monitor the suction at UV detector 280nm
The situation of change of receipts value collects out the protein sample of peak position, and purity of protein (figure) is detected by SDS-PAGE.
It is prepared by the crystal of 2 marine source high temperature lipase SL1 of embodiment
The crystal growth of marine source high temperature lipase SL1 uses the sitting-drop methods of gas phase diffusion (Vapor Diffusion)
To complete.(no using Crystal Screen2 of HamptonResearch companies, Index (sparse matrix screening), SaltRx
With pH buffer environments and salt) with PEG/Ion (screening different high polymer, salt and pH buffer environments) totally 4 kinds of crystallization of protein
Kit screens crystallization condition.96 orifice plates are added in 50 μ L of crystallization buffer in each crystalline reagents box to correspond in lower slot.Make
So that the albumen of 1 μ L is mixed with the lower slot buffer solution of 1 μ L with robotic arm, 96 hole sitting drop plates is sealed with sealed membrane, in 15-25
DEG C constant temperature and humidity is stood.
Crystallization condition optimizes：After crystal growth comes out, the crystal situation grown under the conditions of observation is each under the microscope is (big
It is small, shape).Crystallization condition to that can grow preferable crystal optimizes that (it is excellent that pH and precipitant concentration carry out two-dimensional gradient
Change), to improve the quality of crystal.Condition optimizing is albumen concentration about 21mg/mL, and pond liquid is 1 with protein solution volume ratio:1, pond
Liquid is (A) 0.21M zinc acetates, 0.1M imidazoles, 10%PEG8000, pH 6.0 and (B) 1.05M sodium dihydrogen phosphates-phosphoric acid hydrogen two
Sodium, pH 5.0.
Diffraction conditions：The crystal of marine source high temperature lipase SL1 is contained into 5%, 10%, 15% He with corresponding successively
The anti-frost protection agent of 25% (v/v) is impregnated, and is placed into the set of fixed crystal, is then submerged in liquid nitrogen and preserves.MAS1 crystal
Diffraction be Shanghai synchrotron radiation light source (BL17U1) carry out, be with wavelength respectively(Zn characteristic absorption wavelengths) andX-ray diffraction.
Structure elucidation：The diffraction data of two kinds of crystal is collected and is arranged using MOSFLM programs, their space group difference
For P64And P3221.Wherein 0.2M Zn (AC)2, 0.1M imidazoles, the space group to grow under the conditions of pH 6.5,10%PEG8000
For crystal, it is to look for phase with the anomalous scattering signal of heavy atom zinc, has then built marine source with Buccaneer softwares
The initial model of high temperature lipase SL1.With Refmac5 and Coot softwares repeatedly manual correction and optimize the initial configuration, and with
Architecture quality after MolProbity software inspections modification.The resolution ratio of final correcting principle is 2.3, RworkIt is 17.8%,
RfreeIt is 20.58%.RfreeAnd RworkDifference be 2.78%, bond distance and bond angle root-mean-square-deviation are respectively 0.0096 He
1.3595。
The marine source high temperature lipase obtained under 1.0M sodium dihydrogen phosphates-disodium hydrogen phosphate crystallization condition of pH 5.0
SL1 crystal is above-mentioned P64Space group marine source high temperature lipase SL1 structures are starting model, are obtained by molecular replacement.
The resolution ratio of the structure is 2.34, RworkFor 19.14%, RfreeIt is 22.16%.RfreeAnd RworkDifference be 3.02%, bond distance
It is respectively 0.0118 and 1.5301 with bond angle root-mean-square-deviation.In the detail parameters of the above crystal characteristic and resolving and table 1
Summarize.
The identification of 3 marine source high temperature lipase SL1 biochemical properties of embodiment and catalytic property
Influence of the temperature to enzyme activity：To emulsify olive oil as substrate, marine source high temperature lipase SL1 is surveyed at 50-90 DEG C
Hydrolysis vigor.Reaction system is that 4g emulsifies olive oil (olive oil：Polyvinyl alcohol=1:And the phosphorus of the pH of 5g 0.1M 7.0 3)
Phthalate buffer mixing and at a certain temperature preincubate 10min.1mL enzyme solutions are added, 15min are reacted, with 95% ethyl alcohol of 15mL
Terminate reaction；Using phenolphthalein as indicator, the aliphatic acid of 0.05M KOH titration hydrolysis releases.The enzyme of high-temperature inactivation does control experiment.
Thus the optimal reactive temperature range of marine source high temperature lipase SL1 can determine whether that the lipase is high temperature enzyme at 70 DEG C or so.
The esterification of oleic acid and glycerine：By the high-purity marine source high temperature lipase SL1 immobilised enzymes prepared with have
The commercialization Novozym 435 of height esterification effect is carried out at the same time the catalysis reaction of the esterification of oleic acid and glycerine.Wherein oleic acid and sweet
The ratio between amount of substance of oil is 1:3, reaction condition is 65 DEG C.The enzyme of 1500U is added in reaction system altogether, in the case where vacuumizing environment into
Row reaction.Sample after reaction carries out constituent analysis through processing using the method for HPLC.Vacuum of the lipase SL1 at 24 hours
Esterification yield and content of triglyceride respectively reach 99.5% and 91.3% after reaction；And Novozym 435 can only achieve 79.3%
With 48.7%.Experimental result shows that lipase SL1 has better catalyzing esterification efficiency.Detailed results see the table below 2.
Table 2
Temperature tolerance：In the high-purity marine source high temperature lipase SL1 aqueous solutions prepared respectively 55,60,
65 and 70 DEG C of incubations, per half an hour sample detection, it remains enzyme activity (with the enzyme enzyme activity that is not incubated for 100%), and detection method is
Olive oil emulsion process.As a result it such as Fig. 6, is incubated 3 hours at 55 DEG C, which still retains 90% or more vigor；3 are incubated at 70 DEG C
Hour, the enzyme also still had more than 50% vigor.It can be seen that the enzyme is a kind of thermal stable lipase.
Organic solvent tolerance：It is separately added into the high-purity marine source high temperature lipase SL1 aqueous solutions prepared
Various organic solvents：Methanol, ethyl alcohol, isopropanol, acetone and hexamethylene, a concentration of 90% (v/v), 4 DEG C of placements, the sampling inspection per 4h
It surveys it and remains enzyme activity (be not added with enzyme activity when organic solvent as 100%), detection method is olive oil emulsion process.As a result such as Fig. 7,
Marine source high temperature lipase SL1 shows good stability in each organic solvent, is all remained after being incubated 24 hours
90% or more vigor, it is seen that the lipase has good stability in organic solvent.
1 marine source high temperature lipase SL1 structural parameters of table
Sequence table
<110>South China Science ＆ Engineering University
<120>A kind of marine source high temperature lipase and its crystal and preparation method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 279
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Met Asp Ile Gly Ile Asn Ser Asp Pro Asn Ser Ala Thr Pro Ala Ala
1 5 10 15
Glu Ala Thr Ser Arg Gly Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala
20 25 30
Ala His Pro Arg Pro Val Val Leu Val His Gly Thr Phe Gly Asn Ser
35 40 45
Ile Asp Asn Trp Leu Val Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr
50 55 60
Cys Val Phe Ser Leu Asp Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe
65 70 75 80
His Gly Leu Gly Pro Ile Asp Lys Ser Ala Glu Gln Leu Asp Val Phe
85 90 95
Val Asp Lys Val Leu Asp Ala Thr Gly Ala Pro Lys Ala Asp Leu Val
100 105 110
Gly His Ser Gln Gly Gly Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu
115 120 125
Gly Gly Ala Asp Lys Val Asn Ala Leu Val Gly Ile Ala Pro Asp Asn
130 135 140
His Gly Thr Thr Leu Leu Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro
145 150 155 160
Gly Val Glu Lys Phe Ile Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln
165 170 175
Val Ala Gly Ser Pro Phe Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr
180 185 190
Val Pro Gly Val Arg Tyr Thr Val Ile Ala Thr Lys Tyr Asp Gln Val
195 200 205
Val Thr Pro Tyr Arg Thr Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn
210 215 220
Val Leu Leu Gln Asp Leu Cys Pro Val Asp Leu Ser Glu His Val Ala
225 230 235 240
Ile Gly Thr Ile Asp Arg Ile Ala Phe His Glu Val Ala Asn Ala Leu
245 250 255
Asp Pro Ala Arg Ala Thr Pro Thr Thr Cys Ala Ser Val Ile Gly Leu
260 265 270
Glu His His His His His His
275
Claims (10)
1. a kind of marine source high temperature lipase, which is characterized in that its amino acid sequence such as SEQ ID NO：Shown in 1.
2. the preparation method of marine source high temperature lipase described in claim 1, which is characterized in that by marine source high temperature fat
The sequence of enzyme is cloned into pET22b (+) prokaryotic expression carrier and converts the carrier to competent escherichia coli cell Rosetta
(DE3) it expresses, is purified by affinity chromatography and exclusion Gel Chromatography, obtain marine source high temperature lipase aqueous solution.
3. the preparation method of marine source high temperature lipase crystal, which is characterized in that include the following steps：
(1) preparation of crystallization buffer：The group of crystallization buffer become (A) 0.2 ± 0.05M zinc acetates, 0.1 ± 0.02M imidazoles,
10 ± 0.5%PEG8000, pH 6.0 ± 0.5 or (B) 1.0 ± 0.1M sodium dihydrogen phosphates-disodium hydrogen phosphate, pH 5.5 ± 0.5；
(2) the marine source high temperature lipase aqueous solution for preparing claim 2 by volume 1：1 with the prepared knot of step 1)
Crystalline substance buffering mixing, in 15-25 DEG C of indoor placement, culture obtains the crystal of marine source high temperature lipase in 5~7 days.
4. preparation method according to claim 3, which is characterized in that the group of the crystallization buffer becomes：(A)0.21M
Zinc acetate, 0.1M imidazoles, 10%PEG8000, pH 6.0 or (B) 1.05M sodium dihydrogen phosphates-disodium hydrogen phosphate, pH 5.0.
5. preparation method according to claim 3, which is characterized in that the marine source high temperature lipase aqueous solution it is dense
Degree is 18~25mg/mL.
6. preparation method according to claim 5, which is characterized in that the marine source high temperature lipase aqueous solution it is dense
Degree is 21mg/mL.
7. marine source high temperature lipase crystal prepared by claim 3~6 any one the method.
8. marine source high temperature lipase crystal according to claim 7, which is characterized in that the crystal is crystal A or crystalline substance
Body B, crystal A, B are respectively hexagon and corynebacterium pattern, and space group is respectively P64And P3221, the wherein lattice constant of crystal A
Forα=β=90 °, γ=120 °；The lattice constant of crystal B is α=β=90 °, γ=120 °；In an asymmetry unit, crystal A is monomer, and there are three asymmetric by crystal B
Molecule.
9. marine source high temperature lipase crystal according to claim 8, which is characterized in that the folding of described crystal A, B
Center is made of 6 parallel β-pleated sheet pieces, and by 12 α spiral packagings.
10. the marine source high temperature lipase crystal according to claim 7 or 8 or 9, which is characterized in that described crystal A, B
Space structure be made of fine three dimensional structure；Wherein glycine (G146) is covered to the α 5 that proline (P171) forms as lid
It covers above active pocket；Catalytic triads are by serine (S115), aspartic acid (D206) and histidine (H238) composition；Oxygen
Anion hole is made of the main chain imino group of threonine (T44) and glutamine (Q116).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110540980A (en) * | 2019-09-07 | 2019-12-06 | 华南理工大学 | Streptomyces marinus lipase mutant and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103642772A (en) * | 2013-12-09 | 2014-03-19 | 中国水产科学研究院黄海水产研究所 | Marine low-temperature alkaline lipase Bohai Sea-9145 crystal and preparation method thereof |
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2018
- 2018-04-08 CN CN201810307147.3A patent/CN108342372A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642772A (en) * | 2013-12-09 | 2014-03-19 | 中国水产科学研究院黄海水产研究所 | Marine low-temperature alkaline lipase Bohai Sea-9145 crystal and preparation method thereof |
Non-Patent Citations (5)
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| DONGJUAN YUAN ET AL.: "Screening and characterization of a thermostable lipase from marine Streptomyces sp. strain W007", 《BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY》 * |
| SLIM CHERIF ET AL.: "A newly high alkaline lipase: an ideal choice for application in detergent formulations", 《LIPIDS IN HEALTH AND DISEASE》 * |
| XIUMEI WANG ET AL.: "Immobilized MAS1 lipase showed high esterification activity in the production of triacylglycerols with n-3 polyunsaturated fatty acids", 《FOOD CHEMISTRY》 * |
| ZEXIN ZHAO ET AL.: "Crystal structure of a lipase from Streptomyces sp. strain W007 – implications for thermostability and regiospecificity", 《THE FEBS JOURNAL》 * |
| 赵泽鑫: "脂肪酶MAS1的结构与耐热及底物选择性关系的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110540980A (en) * | 2019-09-07 | 2019-12-06 | 华南理工大学 | Streptomyces marinus lipase mutant and application thereof |
| CN110540980B (en) * | 2019-09-07 | 2021-06-11 | 华南理工大学 | Streptomyces marinus lipase mutant and application thereof |
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