CN108018273A - A kind of long-chain unsaturated fatty acid specific fat enzyme mutant and its application - Google Patents
A kind of long-chain unsaturated fatty acid specific fat enzyme mutant and its application Download PDFInfo
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- CN108018273A CN108018273A CN201810097101.3A CN201810097101A CN108018273A CN 108018273 A CN108018273 A CN 108018273A CN 201810097101 A CN201810097101 A CN 201810097101A CN 108018273 A CN108018273 A CN 108018273A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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)
Abstract
The invention discloses a kind of long-chain unsaturated fatty acid specific fat enzyme mutant and its application, belong to gene engineering technology field.The present invention carries out rite-directed mutagenesis to it, obtains the mutant enzyme that aliphatic acid specifically sexually revises according to rhizopus chinensis lipase (RCL) structural analysis.Compare wild type lipase and be mutated the ability of enzymatic hydrolysis soybean oil, obtained the mutant enzyme HQL that percent hydrolysis is far above wild type lipase, percent hydrolysis reaches 98% or so.
Description
Technical field
The present invention relates to a kind of long-chain unsaturated fatty acid specific fat enzyme mutant and its application, belong to genetic engineering
Technical field.
Background technology
Aliphatic acid is most basic in oil and fat chemical, one of the most widely used raw material.Natural oil hydrolysis is to be swum
One of important channel from aliphatic acid.The production technology of aliphatic acid mainly has saponification method, Hydrolyze method, steam splitting process and enzyme water
Solution, wherein enzyme hydrolysis method due to reaction condition is gentle, unrighted acid is not easy the advantage that is aoxidized, accessory substance is few,
As the research hotspot of aliphatic acid preparation method.The aliphatic acid of different type, different carbon chain lengths and different proportion has difference
Function.Short chain fatty acids can be used as feed addictive, promote growth of animal.Medium chain fatty acid is widely used in medicine, health care
Edible oil and aquaculture.Chain saturated fatty acids such as palmitic acid and stearic acid, mainly for the production of the aliphatic acid as emulsifying agent
Salt, is widely present in the animal oil such as pig, ox and sheep, and the sum of all kinds of saturated fatty acid mass fractions are usually more than 60%.It is long
Chain unrighted acid has extensive physiological activity in metabolism, for health care.Unrighted acid in vegetable fat
Content is higher, as unsaturated fatty acid content is respectively 84.34%, 77.70% and in soybean oil, rapeseed oil and oil tea oil
81.43%.
Lipase (Triacylglycerol hydrolase, Lipase, EC 3.1.1.3) is a kind of triglyceride hydrolysis
Enzyme, is the essential industry enzyme that " green " fats and oils processing manufactures in the hydrolysis or synthesis of oil-water interfaces catalysis ester bond.Vegetable oil is
The important sources of long chain fatty acids are prepared, but most lipase are low to the catalytic efficiency of unrighted acid, or tend to water
The utilization of its raw material is reduced when solving medium chain fatty acid, therefore being hydrolyzed applied to the vegetable oil of long-chain unsaturated fatty acid content higher
Rate.For example, Alves et al. studies have shown thats utilize single commercial enzyme (Novozym435, Lipozyme TL-IM and Lipozyme
RM-IM) percent hydrolysis of catalyzing hydrolysis soybean oil is less than 50%, compounds lipase (the Lipozyme RM- of different substrate specificities
IM and Novozym435) percent hydrolysis brought up to more than 80% (RSCAdvances, 2014,4:6863-6868).It is although compound
The catalytic efficiency of enzyme significantly improves, but current research fails inherently to solve the low problem of vegetable oil percent hydrolysis, such as carries
High enzyme improves long chain fatty acids specificity etc., so as to further improve plant grease to the substrate specificity of unrighted acid
Solution rate.
The content of the invention
To solve the above problems, the present invention passes through design and rational rhizopus chinensis (Rhizopus chinensis) lipase
(RCL) mutational site, obtains the mutant enzyme that long-chain unsaturated fatty acid specificity improves, and improves the hydrolysis of soybean oil
Rate.
First purpose of the present invention is to provide a kind of lipase mutant, the lipase mutant relative to
SEQ ID NO:The lipase of amino acid sequence shown in 1, includes the one, two or three amino of lipase substrate binding pocket
The change of sour residue；The change increase pocket hydrophily, the change is with lipase to the special of long-chain unsaturated fatty acid
Property it is related.
In one embodiment of the invention, the lipase mutant is in amino acid sequence such as SEQ ID NO.1
Glutamine is inserted between the histidine of the 284th of shown sequence and the leucine of the 285th.
In one embodiment of the invention, the amino acid sequence of the lipase mutant such as SEQ ID NO.2 institutes
Show.
In one embodiment of the invention, paddy ammonia is inserted between the histidine of the 284th and the leucine of the 285th
Acid amides obtains mutant HQL.
Second object of the present invention is to provide the gene order for encoding the lipase mutant.
Third object of the present invention is to provide the plasmid or cell for carrying the gene order.
In one embodiment of the invention, the cell is bacterium, fungi or ancient bacterium.
Fourth object of the present invention is to provide application of the lipase mutant in food, health products, field of medicaments.
In one embodiment of the invention, the application is to utilize the lipase mutant hydrolyzing plant grease.
In one embodiment of the invention, the application include hydrolyzed soy oil, corn oil, peanut oil, rapeseed oil,
Olive oil, cottonseed oil, walnut oil.
Beneficial effects of the present invention：
The present invention carries out rite-directed mutagenesis to it, it is special to obtain aliphatic acid according to rhizopus chinensis lipase (RCL) structural analysis
The mutant enzyme sexually revised.Compare wild type lipase with being mutated the ability of enzymatic hydrolysis soybean oil, it is far high to have obtained percent hydrolysis
In the mutant enzyme HQL of wild type lipase, percent hydrolysis reaches 98% or so.
Brief description of the drawings：
Fig. 1 is the fatty acid chain length specificity (C2 of lipase:pNPC2、C4:pNPC4、C5:pNPC5、C8:pNPC8、
C12:pNPC12、C14:pNPC14、C16:pNPC16)；
Fig. 2 is hydrolysis properties of the lipase to saturated fatty acid in soybean oil and unrighted acid；
Fig. 3 is influence of the temperature to lipase activity and stability, and wherein A is influence of the temperature to activity, and B is temperature pair
The influence of stability；
Fig. 4 is influences of the pH to lipase activity and stability, and ApH is to active influence, influences of the B pH to stability；
Fig. 5 is lipase-catalyzed soybean oil percent hydrolysis.
Embodiment
The measure of raw soybeans oil saponification number is with reference to GB/T 5534-2008.
Percent hydrolysis=(AV0- AV)/(SV-AV) × 100%
In formula：AV0For the acid number of sample after hydrolysis, mgKOH/g；AV, SV are respectively raw soybeans oleic acid value and saponification number,
mgKOH/g。
Lipase hydrolysis soybean oil test method：Every part of sample weighs 5g soybean oils, and a certain amount of 50mM potassium phosphates are delayed
Fliud flushing is added in 50mL triangular flasks, and ultrasound makes substrate fully emulsified；The reaction condition of soybean oil hydrolysis is set as the reaction time
24h, water oil quality is than 1:1, enzyme concentration 500U/g (oil weight), pH8.0,40 DEG C of temperature, it is whole to add 10mL95% ethanol after reaction
Only react, measure the acid number of hydrolysate.Acid value measuring method is with reference to GB/T 5009.37-2003.
Aliphatic acid component analysis：Rich mixture after being reacted with n-hexane extraction, is separated by thin-layer chromatography TLC
Obtain free fatty to be placed in 10ml color-comparison tubes, add 1ml 2%H2SO4- methanol solutions, 80 DEG C of water-bath 30min, take
Go out colorimetric cylinder, be cooled to room temperature, add 2ml n-hexanes, fully mix, add saturation NaCl solution to bottleneck, acutely shaking, quiet
Centrifugation layering is put, collects upper strata n-hexane phase, adds suitable anhydrous sodium sulfate, nitrogen blows concentrated sample introduction analysis.
Chromatographic column：DB-Wax(30m×250μm×0.25μm)；Sample size：1μL；Split ratio：50:1；Injector temperature：
225℃；Carrier gas：Nitrogen, 30mLmin-1；Hydrogen, 45mLmin-1；Air, 450mLmin-1；Temperature programming：180℃
1.5min is kept, 210 DEG C is raised to 10 DEG C of min-1, keeps 2min, is then raised to 220 DEG C of holding 5min with 5 DEG C of min-1；
Detector：Flame ionic detector (FID), temperature are 250 DEG C.Each sample detection 13.5min.
Even if area normalization method soya fatty acid group is used on Agilent GC 6890N gas chromatograph work stations
The percentage composition divided.
Lipase hydrolysis vigour-testing method：Lipase hydrolysis p-nitrophenyl phenolic ester produces p-nitrophenol and aliphatic acid,
P-nitrophenol displaing yellow in aqueous, has the light absorbs of maximum, by measuring p-nitrophenol at 410nm in 410nm
Light absorbs can measure the vigor of lipase.The definition of enzyme activity is：1 μm of ol p-nitrophenyl of generation per minute under certain reaction condition
The enzyme amount of phenol is a lipase hydrolysis enzyme activity international unit.
Embodiment 1：The rite-directed mutagenesis of rhizopus chinensis lipase
Compared according to the crystal structure analysis of rhizopus chinensis lipase and similar structures lipase crystal structure, utilize full plasmid
Round pcr carries out rite-directed mutagenesis and combinatorial mutagenesis.Pinpointed by rizolipase substrate binding pocket to China and carry out rite-directed mutagenesis.
Primer needed for 1 mutational site of table
The factor such as the hydrophilic and hydrophobic of amino acid and side-chain radical size may influence fat in zymolyte binding pocket
The substrate specificity of enzyme.Therefore, from increase hydrophilic three mutant of angle design of pocket, including L285Q, T286Q and
A hydrophilic amino acid Q is inserted among sequence H284 and L285, mutant is named as HQL；From the angle of increase steric hindrance
Design three mutational sites, including A116W, I281F, A116W/I281F.
Embodiment 2：The special Journal of Sex Research of aliphatic acid of mutant enzyme
Using the p-nitrophenol fatty acid ester of different alkyl carbon chain lengths as substrate (pNPC2, pNPC4, pNPC5, pNPC8,
PNPC12, pNPC14, pNPC16), the fatty acid chain length that 1 mutant enzyme of embodiment is detected at being 40 DEG C in pH 8.0 and temperature is special
Property.
As shown in Figure 1, for compared to wild type lipase, mutant enzyme HQL strengthens the specificity of long chain fatty acids, its
In the specific highest of pNPC16, hydrolysis vigor are improved to 2.72 times of wild type.Mutant enzyme A116W and L285Q is right
The specific highest of pNPC16, hydrolysing activity are 1.23 times and 1.50 times of wild type respectively.Mutant enzyme I281F has widened hydrolysis
Fatty acid chain length scope, adds the hydrolysing activity to pNPC2, and to the specific highest of pNPC12, hydrolysing activity is wild type
2 times.In addition, mutant enzyme L285Q, T286Q, HQL, A116W, A116W/I281F can not hydrolyze pNPC4, and it is all prominent
Becoming enzyme reduces the hydrolysing activity of pNPC8.By rizolipase substrate binding pocket rite-directed mutagenesis to China, we obtain
A series of mutant enzyme that aliphatic acid specifically sexually revise, is especially that of obtaining the mutant enzyme to long chain fatty acids specificity enhancing.
Embodiment 3：Mutant enzyme studies the hydrolysis properties of aliphatic acid in soybean oil
In order to investigate substrate specificity of the lipase to saturation and unsaturated long chain fatty acids, with rich in the quasi-glycerol three-ester
Soybean oil be measured as substrate.The aliphatic acid composition of soybean oil is determined first, contains 13.77% saturated fatty acid
(palmitic acid 11.19%, stearic acid 2.58%), and 86.23% unrighted acid (oleic acid 25.21%, linoleic acid
54.67%, leukotrienes 6.34%).From table 2 and Fig. 2, mutant enzyme HQL enhances the specificity to unrighted acid, water
The vigor of solution unrighted acid is 1.45 times of saturated fatty acid, and wild type hydrolysis unrighted acid is only saturated fat
1.10 times of acid.Mutant enzyme HQL is stronger to unrighted acid specificity, therefore, it is expected to percent hydrolysis of the HQL to soybean oil
Wild-type enzyme will be higher than.In addition, mutant enzyme T286Q, I281F and A116W/I281F are enhanced to the special of saturated fatty acid
Property, the vigor for hydrolyzing saturated fatty acid is the 1.20 of unrighted acid, 1.83 and 1.34 times respectively, these types fat enzyme mutant
Body would be more suitable for the high animal fat of saturated fatty acid content.
Table 2 is mutated the hydrolysis of enzymatic soybean oil
Embodiment 4：The THERMAL STABILITY of mutant enzyme
The vigor of lipase is detected using p-nitrophenol palmitate as substrate (pNPC16).Different temperature (20 DEG C-
60 DEG C) under by standard method measure enzyme liquid lipase activity, be opposite enzyme activity 100% using highest enzyme activity, research temperature is to dashing forward
Become the influence of enzyme activity.The lipase activity of enzyme liquid is measured after keeping the temperature 1h under different temperatures (20 DEG C -60 DEG C), it is uninsulated
Enzyme activity calculates residual enzyme activity as control, investigates influence of the temperature to mutation enzyme stability.
The optimum temperature that all mutation enzymic catalytic reactions are understood by Fig. 3 A is 40 DEG C, the optimum temperature one with wild type
Cause.In the range of 20-40 DEG C, the vigor of mutant enzyme T286Q can be maintained at more than 84%, other mutant enzymes equal energy at 20 DEG C
Retain more than 65% vigor.But after temperature is higher than 40 DEG C, zymoprotein is gradually denatured, and the vigor of all mutant enzymes is notable
Decline, when temperature rises to 60 DEG C, the vigor of mutant enzyme HQL remains in 20% or so, and the vigor of other mutant enzymes declines
To less than 10%.
Fig. 3 B show the temperature stability of lipase, and at 45 DEG C, the vigor of mutant enzyme HQL and I281F can be maintained at
The vigor of 80% or so, only mutant enzyme A116W drop to 50% or so, and other mutation enzyme activities are then retained in 69%-
Between 75%.When temperature is more than 55 DEG C, enzyme activity rapid decrease is mutated.
Embodiment 5：The pH stability studies of mutant enzyme
PH influences the conformation of enzyme, also influences and dissociation situation of the catalysis in relation to group and the dissociated state of substrate molecule.Enzyme
Vigor influenced by environment pH, under certain pH, the maximum vigor of enzyme performance, higher or lower than this pH, enzyme activity reduces.Respectively
Prepare the phosphate buffer (pH6.5-pH8) of 0.05mol/L, 0.05mol/L Tris-HCl buffer solutions (pH8-pH9),
The carbonate buffer (pH9-pH10) of 0.05mol/L, enzyme liquid is added in respectively in the buffer solution of above-mentioned difference pH, in standard conditions
Lower measure lipase activity, using highest enzyme activity as opposite enzyme activity 100%, influences of the research pH to mutation enzyme activity.Enzyme liquid is upper
The lipase activity of measure enzyme liquid after keeping the temperature 1h in 25 DEG C in the buffer solution of different pH is stated, uninsulated enzyme activity is counted as control
Residual enzyme activity is calculated, investigates influences of the pH to mutation enzyme stability.
The optimum pH that all mutation enzymic catalytic reactions are understood by Fig. 4 A is 8.0, consistent with the optimal pH of wild type.pH
Scope is between pH 7.5-9.0, and only mutant enzyme HQLT and I28F vigor can be maintained at more than 50%, other mutant enzymes exist
Vigor is below 50% during pH9.0.pH<7.5 and pH>In the case of 9, enzyme activity declines substantially, illustrates that reacting pH significantly affects
The vigor of mutant enzyme.
As shown in Figure 4 B, all mutant enzymes are best in 8.0 stability inferior of optimal pH.Mutant enzyme T286Q is relative to wild
Type stability is poor, and enzyme activity can just be maintained at more than 60% between pH 7.5-8.5, and other mutant enzymes in pH7.0 and
Also more than 50% vigor can be retained during pH9.0.In pH<7 and pH>In the case of 9, the very fast decrease of stability, only mutant enzyme
I281F can also keep 22% vigor in pH10.0.
Embodiment 6：Difference mutation enzymatic soybean oil hydrolysis ability compares
The reaction condition of soybean oil hydrolysis is set as reaction time 24h, and water oil quality is than 1:1, enzyme concentration 500U/g (oil
Weight), pH8.0,40 DEG C of temperature, investigates the ability that mutation enzymatic soybean oil hydrolyzes.As shown in Figure 5, mutant enzyme L285Q with it is wild
The percent hydrolysis of type catalysis soybean oil approaches, percent hydrolysis 82%.The catalysis soybean of mutant enzyme A116W, I281F, A116W/I281F
The ability of grease solution is weaker than wild type, percent hydrolysis as low as 70% or so.Mutant enzyme L285Q hydrolysis abilities are slightly above wild type, water
Solution rate reaches 88%.The ability highest of mutant enzyme HQL catalysis soybean oil hydrolysis, reaches 98% or so.Due to insatiable hunger in soybean oil
It is high with content of fatty acid, more than 80% is accounted for, and mutant enzyme HQL compares long-chain saturated fat to long-chain unsaturated fatty acid specificity
Sour specificity is stronger, therefore the ability of catalyzing hydrolysis soybean oil significantly improves.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Sequence table
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<120>A kind of long-chain unsaturated fatty acid specific fat enzyme mutant and its application
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Ser Pro Lys Asn Leu Ser Ile Tyr Thr Val Gly Cys Pro Arg Val Gly
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Asn Asn Ala Phe Ala Tyr Tyr Val Asp Ser Thr Gly Ile Pro Phe His
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245 250 255
Ala Asp Val Gln Ile Cys Thr Ser Asn Ile Glu Thr Lys Gln Cys Ser
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Claims (10)
1. a kind of lipase mutant, it is characterised in that the lipase mutant is relative to SEQ ID NO:Ammonia shown in 1
The lipase of base acid sequence, includes the change of the one, two or three amino acid residue of lipase substrate binding pocket；It is described
Change increase pocket hydrophily, the change is to lipase to the specific related of long-chain unsaturated fatty acid.
2. lipase mutant according to claim 1, it is characterised in that the lipase mutant is in amino acid sequence
Glutamine is inserted between the histidine of the 284th and the leucine of the 285th of sequence of the row as shown in SEQ ID NO.1.
3. lipase mutant according to claim 2, it is characterised in that the amino acid sequence of the lipase mutant
As shown in SEQ ID NO.2.
4. lipase mutant according to claim 2, it is characterised in that the histidine of the 284th and the 285th 's is bright
Glutamine is inserted between propylhomoserin and obtains mutant HQL.
5. encode the gene order of any lipase mutant of Claims 1 to 4.
6. carry the plasmid or cell of gene order described in claim 5.
7. cell according to claim 6, it is characterised in that the cell is bacterium, fungi or ancient bacterium.
8. any lipase mutant of Claims 1 to 4 is in the application of food, health products, field of medicaments.
9. apply according to claim 8, it is characterised in that the application is to utilize any fat of Claims 1 to 44
Enzyme mutant hydrolyzing plant grease.
10. application according to claim 9, it is characterised in that the application includes hydrolyzed soy oil, corn oil, peanut
Oil, rapeseed oil, olive oil, cottonseed oil, walnut oil.
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| CN109750012A (en) * | 2019-03-27 | 2019-05-14 | 云南师范大学 | A kind of lipase mutant and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899427A (en) * | 2009-11-11 | 2010-12-01 | 江南大学 | Lipase mutant with improved activity and built by orthogenesis |
| CN104080908A (en) * | 2012-02-03 | 2014-10-01 | 诺维信公司 | Lipase variants and polynucleotides encoding same |
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2018
- 2018-01-31 CN CN201810097101.3A patent/CN108018273B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101899427A (en) * | 2009-11-11 | 2010-12-01 | 江南大学 | Lipase mutant with improved activity and built by orthogenesis |
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| CN109750012A (en) * | 2019-03-27 | 2019-05-14 | 云南师范大学 | A kind of lipase mutant and its application |
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