CN114015673B - Lipase Sv-lip5 and application thereof in hydrolysis of astaxanthin ester - Google Patents
Lipase Sv-lip5 and application thereof in hydrolysis of astaxanthin ester Download PDFInfo
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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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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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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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- C12N2800/00—Nucleic acids vectors
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- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention discloses lipase Sv-lip5 and application thereof in hydrolyzing astaxanthin ester, belonging to the technical field of functional enzyme screening, wherein the amino acid sequence of the lipase Sv-lip5 is shown as SEQ ID NO. 1. The nucleotide sequence of the gene for coding the lipase Sv-lip5 is shown as SEQ ID NO. 2. The lipase Sv-lip5 is used for hydrolyzing astaxanthin ester or preparing free astaxanthin. The invention also provides a method for hydrolyzing astaxanthin ester or preparing free astaxanthin. The invention also provides a recombinant expression vector and a recombinant engineering bacterium containing the gene of the coded lipase Sv-lip 5. The lipase Sv-lip5 has astaxanthin ester hydrolysis activity, can break ester bonds in astaxanthin ester, realizes efficient preparation of free astaxanthin, solves the technical problem of low enzyme catalysis efficiency, and has important significance for industrial application of astaxanthin ester.
Description
Technical Field
The invention relates to lipase Sv-lip5 and application thereof in hydrolyzing astaxanthin ester, belonging to the technical field of functional enzyme screening.
Background
Free astaxanthin is fat-soluble ketone type carotenoid, has strong effects of resisting oxidation, resisting inflammation, regulating immunity and the like, and has great application potential in the aspects of treating cardiovascular and cerebrovascular diseases, diabetes, cancers, immune system diseases and the like.
The main existing form of astaxanthin in nature is fatty acid ester combined with fatty acid, the diversity of fatty acid causes the astaxanthin ester to be more complex in structure, and the low water solubility and the low bioavailability of the astaxanthin ester limit the application of the astaxanthin ester in the food industry. Astaxanthin cannot be synthesized in most animals, and can only be obtained from other ways such as food. Thus, there is a need for hydrolysis of astaxanthin esters to astaxanthin for a variety of applications. The method commonly used for hydrolyzing astaxanthin ester comprises saponification method and enzymolysis method, wherein the saponification method needs strong alkali reagent such as sodium hydroxide, etc., and free astaxanthin can be prepared efficiently by utilizing the catalytic activity of lipase, and the reaction process is mild.
The enzymolysis method can directly treat astaxanthin-containing ester substances with wide sources, the hydrolysis reaction can be carried out in an aqueous phase system, and the reaction process does not involve toxic reagents except the extracted products, so the method can be used for qualitative and quantitative determination of astaxanthin. Most studies in the current reports use commercial enzymes or lipases obtained by different expression systems to hydrolyze astaxanthin ester, but the enzymes have low catalytic efficiency and the catalytic efficiency is the highest by using extracellular crude extracts, but the biological safety of pseudomonas aeruginosa is unknown and the enzymes are difficult to be used in industrial production.
Disclosure of Invention
Aiming at the prior art, the invention provides a new lipase, namely the lipase Sv-lip5, which utilizes the hydrolysis activity of the lipase to catalyze the hydrolysis of astaxanthin ester, can realize the high-efficiency preparation of free astaxanthin, provides more references for the research field and provides more choices for industrial application.
The invention is realized by the following technical scheme:
the amino acid sequence of the lipase Sv-lip5 is shown in SEQ ID NO. 1.
The amino acid sequence of the lipase Sv-lip5 is shown as follows (shown as SEQ ID NO: 1):
VHGRAGPRRARGRAGPDQGRQGRAKVEQGPFQLDQGPCTAEKRQRVEPRRTTVGAARSAAEESSLFGHPEAAPDTTAAYGPDQDQVVDFYAPRGPGAAPGTPLVVVVHGGAWRARYDRRHISPFAAFLAGRGFAVASVEYRRGDGGRGAGRWPDTFDDVAAAFDALPELAAGAVPEADPGRTVLTGHSAGGHLALWAAARHVLPAGSPWRLPEAPAVRGVVALAPIADFATSAELNVCDGAFTQLLGEEHEERLAHADLTALLPTGIATTLVQGTDDAEVPPAVAEAYADAATAAGETPGFTLVDGVGHFPLIDPASAACALVAEEIAQLAW。
the nucleotide sequence of the gene for coding the lipase Sv-lip5 is shown as SEQ ID NO. 2.
The lipase Sv-lip5 is used for hydrolyzing astaxanthin ester or preparing free astaxanthin.
A method for hydrolyzing astaxanthin ester or preparing free astaxanthin comprises: the astaxanthin ester was hydrolyzed by the lipase Sv-lip5 to obtain free astaxanthin.
Further, the specific conditions of the hydrolysis are as follows: adding lipase Sv-lip5 into the astaxanthin ester solution, and hydrolyzing at 25-50 ℃ and pH value of 8.0-10.0.
Further, ethanol is added to the astaxanthin ester solution during hydrolysis, and the volume ratio of the ethanol to the astaxanthin ester solution is preferably 1: 12.
Further, Ca is added to the astaxanthin ester solution during hydrolysis2+、Co2+And/or Ba2+(soluble salts of corresponding metal ions, such as calcium chloride, cobalt chloride and barium chloride, can be added); the Ca2+Is preferably 1 mM; the Co2+Is preferably 10 mM; said Ba2+The concentration of (B) is preferably 10 mM.
Furthermore, the concentration of astaxanthin ester in the astaxanthin ester solution is 40 mg/ml, the addition amount of lipase Sv-lip5 is 11U, the hydrolysis temperature is 40 ℃, the pH value is 9.0, and the hydrolysis time is 12 h.
A recombinant expression vector comprising the gene encoding the lipase Sv-lip 5.
Further, the vector used by the recombinant expression vector is pP43NMK (+).
A recombinant engineering bacterium comprises the recombinant expression vector or a gene coding lipase Sv-lip5 and can express lipase Sv-lip 5.
The recombinant expression vector and the recombinant engineering bacteria are applied to preparation of lipase Sv-lip 5.
The lipase Sv-lip5 has astaxanthin ester hydrolysis activity, can break ester bonds in astaxanthin ester to obtain a product of free astaxanthin, has high hydrolysis rate which can reach 98.27 percent, has high astaxanthin yield, and has higher utilization rate of the product of free astaxanthin obtained by reaction in the fields of medicine preparation and the like. The invention has important significance for industrial application of astaxanthin ester.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: optimum temperature diagram of lipase Sv-lip 5.
FIG. 2: schematic representation of the temperature stability of lipase Sv-lip 5.
FIG. 3: schematic pH optimum of lipase Sv-lip 5.
FIG. 4: pH stability of Lipase Sv-lip 5.
FIG. 5: schematic representation of the effect of metal ions on the activity of lipase Sv-lip 5.
FIG. 6: schematic representation of the effect of surfactant on lipase Sv-lip5 activity.
FIG. 7: the lipase catalyzes the astaxanthin ester hydrolysis reaction, and the substances of reaction liquid before and after the reaction are compared and schematically shown.
FIG. 8: and (3) a schematic diagram of the optimized result of the reaction pH.
FIG. 9: the results of the optimization of the ethanol to buffer volume ratio are shown schematically.
FIG. 10: and (3) a schematic diagram of the optimization results of enzyme adding amount and time.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
EXAMPLE 1 obtaining of Lipase Sv-lip5
In order to find out high-activity lipase, the invention obtains Streptomyces lividans purchased from the research center of marine microorganism culture preservation and application engineering technology in Shandong province (Streptomyces violascensAnd the code is SDUM 420004), the culture solution of the strain is found to be capable of detecting higher lipase and esterase activities through earlier experimental research, in order to discover high-activity lipase or esterase in the strain, whole genome sequencing is carried out on the strain, a potential target gene segment for expressing lipase is screened according to gene annotation information obtained by sequencing, the nucleotide sequence of the gene segment is shown as SEQ ID NO. 2, the amino acid sequence of the expressed protein is shown as SEQ ID NO. 1, and the strain possibly has lipase activity and is named as lipase Sv-lip 5.
The gene sequence of lipase Sv-lip5 was analyzed using methods reported in the literature, the MEGA 9.0 software was used to construct a phylogenetic tree of lipase Sv-lip5 and other families of lipid hydrolases, the Clustal X software was used for multiple sequence alignment of lipid hydrolases, and ESPrip 3.0 (http:// ESPript. ibcp. fr/ESPrip /) was used for export of aligned sequences. The results show that the lipase Sv-lip5 belongs to the IV family of lipid hydrolases.
Choose to usep43NMK is used as a vector, and the gene of lipase Sv-lip5 is heterogeneously expressed in the bacillus subtilis WB800 by utilizing the principle of homologous recombinationThe recombinant engineering bacteria are inoculated in LB culture medium with the inoculation amount of 1% after successful expression, and are fermented for 12 h in a shaking table at 37 ℃, then fermentation liquor is centrifuged for 20 min at the rotating speed of 6000 rpm, supernatant is taken and placed in a plate, the plate is pre-frozen for 4 h in a refrigerator at minus 80 ℃, crude enzyme powder is harvested after 3 days of freeze-drying, and the subsequent experiments use the crude enzyme powder for experiments in order to reduce the experiment cost and improve the experiment efficiency.
EXAMPLE 2 study of the enzymatic Properties of the Lipase Sv-lip5
(1) Optimum temperature of lipase Sv-lip5
The optimum temperature of lipase Sv-lip5 was determined by measuring the activity of the enzyme at various temperatures (25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃). The activity at the optimum temperature is defined as 100%, and the activities at other temperatures are expressed as a percentage of the maximum activity. From the results (FIG. 1), it can be seen that the enzyme activity gradually increased with increasing temperature at a temperature of less than 45 ℃ and reached a maximum at 45 ℃, that the enzyme activity gradually decreased with increasing temperature at a temperature of more than 45 ℃ and that the lipase Sv-lip5 was substantially inactive at a temperature of more than 60 ℃. Therefore, the optimum temperature of the lipase Sv-lip5 was 45 ℃.
(2) Temperature stability of Lipase Sv-lip5
The temperature stability was measured at 35 ℃, 40 ℃, 45 ℃ and 50 ℃ based on the optimum temperature of 45 ℃ for lipase Sv-lip 5. Incubating the enzyme solutions at 35 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C, sampling at different time intervals to determine activity, and detecting A405Lower absorbance values. The enzyme activity at 0 h was defined as 100% and the activity at other times is expressed as a percentage of the highest activity. As can be seen from the results (FIG. 2), the enzyme activity gradually decreased with the increase of time at each temperature, and the residual enzyme activity was above 36.8% in all experimental groups after 42 h incubation.
(3) Optimum pH of lipase Sv-lip5
The optimum pH of the lipase Sv-lip5 was determined by measuring the activity of the enzyme at various pH conditions. Buffers used in the detection process included: 100 mM citric acid-sodium citrate buffer (pH 4.0-6.0), 100 mM phosphate buffer (pH 6.0-8.0), 100 mM Tris-HCl buffer (pH 8.0-9.0) and 100 mM glycine-sodium hydroxide buffer (pH 9.0-10.6). The enzyme activity at the optimum pH is defined as 100%, and the enzyme activities at other pH conditions are expressed as relative percentages. The results (FIG. 3) show that the lipase Sv-lip5 is essentially inactive under acidic conditions (citrate-sodium citrate buffer, pH 4.0-6.0). Under alkaline conditions (Tris-HCl buffer, glycine-sodium hydroxide buffer, pH 8.0-10.6), the activity of the lipase Sv-lip5 is all higher than 50.36%, and the enzyme activity reaches the maximum value at the pH of 10.0, so that the optimum pH of the lipase Sv-lip5 is 10.0.
(4) pH stability of Lipase Sv-lip5
The pH stability was measured by selecting pH 7.0, pH 8.0, pH 9.0 and pH10.0 based on the optimum pH10.0 of lipase Sv-lip 5. Placing equal amount of enzyme solution in buffer solution of pH 7.0, pH 8.0, pH 9.0, and pH10.0 respectively, incubating at 4 deg.C, sampling at different time intervals to determine activity, and detecting A405Lower absorbance values. The enzyme activity at 0 h was defined as 100% and the activity at other times is expressed as a percentage of the highest activity. As can be seen from the results (FIG. 4), the lipase Sv-lip5 has poor stability at the optimum pH of 10.0, and the residual enzyme activity after 96 h is 54.6%. The stability in glycine-sodium hydroxide buffer solution of pH 9.0 is the best, and 78.23% of enzyme activity can be still remained after 96 hours.
(5) Influence of metal ions on lipase Sv-lip5 enzyme activity
Effect of Metal ions on the Activity of Lipase Sv-lip5 by adding Metal ions (Zn) to the reaction System at final concentrations of 1 mM and 10 mM2+，Ca2+，Fe3+，K+，Mg2+，Mn2+，Ba2+，Co2+，Cu2+，Ni2+) And Na2EDTA. Without addition of metal ions and Na2EDTA as a control, the activity of which is defined as 100%, the results being shown (FIG. 5), Ca at a concentration of 1 mM2+And 10 mM Co2+And Ba2+Enhanced enzyme activity, and Mg2+An inhibitory effect on lipase activity is exhibited. K removal at 10 mM concentration+And Ni2+In addition to significantly reducing the enzyme activity, the remaining metal ions all showed slight inhibition.
(6) Effect of surfactants on the Activity of Lipase Sv-lip5
The effect of the surfactant on the activity of lipase Sv-lip5 was determined by adding different surfactants (final concentration 0.5%) to the enzyme solution. The surfactants used include: triton X-100, Span 20, Span 80, Tween 20, Tween 60 and Tween 80. The enzyme activity was defined as 100% for the control group without added surfactant, and the enzyme activity for the remaining experimental groups was expressed as a relative percentage. The results (fig. 6) show that, compared with the sample without the surfactant, the activity of lipase Sv-lip5 is significantly affected by the addition of the surfactant, and span 20, span 80, tween 60, tween 80 and triton X-100 all have a certain degree of inhibition effect on the activity thereof, and tween 20 and tween 60 reduce the activity thereof by 25.7% and 55.3%, respectively.
Example 3 high efficiency preparation of free astaxanthin by hydrolysis of astaxanthin ester catalyzed by Lipase Sv-lip5
The lipase Sv-lip5 enzyme powder prepared in example 1 is used for astaxanthin ester hydrolysis, and the reaction system is as follows: 5 mL Tris-HCl buffer (100 mM, pH 9.0), 200 mg astaxanthin ester, 11U Lipase Sv-lip5 (topThe hydrolytic activity of NPP is defined), placing the reaction mixture in a water bath shaker at 40 ℃ for shaking reaction for 12 hours, and detecting the yield of free astaxanthin by using liquid chromatography.
The liquid chromatography test results are shown in fig. 7, 8, 9 and 10, and fig. 7 shows the comparison of the reaction solution substances before and after the reaction, respectively, and the results show that the lipase Sv-lip5 has a powerful hydrolysis effect on astaxanthin ester, and can hydrolyze most of astaxanthin ester into astaxanthin. FIGS. 8, 9 and 10 show the optimization results of pH, ethanol/buffer volume ratio (ethanol is added to the reaction system to increase the solubility of astaxanthin ester in the system and thus the reaction efficiency), enzyme amount and time, respectively, when the enzyme powder is sufficiently contacted with the substrate, the reaction can be carried out within 1 hourThe lipase Sv-lip5 is alkaline lipase, so the lipase has the best activity and good stability under alkaline conditions, and can effectively inhibit the growth of mixed bacteria. The method can obtain higher astaxanthin yield and simultaneously produce less byproducts, such as astaxanthin, semiastaxanthin and the like. The optimization results show that when the ratio of ethanol to buffer is 1:12, under the condition that the reaction pH is 9.0, after 11U of enzyme powder is added for reaction for 12 hours, the conversion rate of the astaxanthin ester can reach 98.27 percent, and the yield of free astaxanthin in 200 mu g of astaxanthin ester is 147.48 mu g. OUC-Sb-lip12 expressed by Gao et al clone can also be used for hydrolysis of astaxanthin ester, and the hydrolysis rate is 96.29% in 12 h (GAO K P, et al Identification of a GDSL lipase fromStreptomyces bacillarisand its application in the preparation of free activity, Journal of Biotechnology, 2021, 325: 280-; HUANG et al used lipases heterologously expressed in yeast to perform hydrolysis of astaxanthin esters with a maximum hydrolysis rate of 96% (HUANG J J J, et al, effective serology expression of an alkaline lipase and an ate application in a hydrolytic process of free activity. Biotechnology Biofuels, 2018, 11: 181); the yield and hydrolysis rate of the free astaxanthin in the invention are higher than those reported in related documents, probably because the side products such as astaxanthin and the like are generated less in the reaction process. The conversion rate of astaxanthin ester and the yield of free astaxanthin of lipase Sv-lip5 are both high, and the method has the application potential of industrial popularization of the preparation of free astaxanthin by enzymolysis.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Sequence listing
<110> China oceanic university
<120> lipase Sv-lip5 and application thereof in hydrolysis of astaxanthin ester
<141> 2021-12-27
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 332
<212> PRT
<213> Streptomyces violascens
<400> 1
Val His Gly Arg Ala Gly Pro Arg Arg Ala Arg Gly Arg Ala Gly Pro
1 5 10 15
Asp Gln Gly Arg Gln Gly Arg Ala Lys Val Glu Gln Gly Pro Phe Gln
20 25 30
Leu Asp Gln Gly Pro Cys Thr Ala Glu Lys Arg Gln Arg Val Glu Pro
35 40 45
Arg Arg Thr Thr Val Gly Ala Ala Arg Ser Ala Ala Glu Glu Ser Ser
50 55 60
Leu Phe Gly His Pro Glu Ala Ala Pro Asp Thr Thr Ala Ala Tyr Gly
65 70 75 80
Pro Asp Gln Asp Gln Val Val Asp Phe Tyr Ala Pro Arg Gly Pro Gly
85 90 95
Ala Ala Pro Gly Thr Pro Leu Val Val Val Val His Gly Gly Ala Trp
100 105 110
Arg Ala Arg Tyr Asp Arg Arg His Ile Ser Pro Phe Ala Ala Phe Leu
115 120 125
Ala Gly Arg Gly Phe Ala Val Ala Ser Val Glu Tyr Arg Arg Gly Asp
130 135 140
Gly Gly Arg Gly Ala Gly Arg Trp Pro Asp Thr Phe Asp Asp Val Ala
145 150 155 160
Ala Ala Phe Asp Ala Leu Pro Glu Leu Ala Ala Gly Ala Val Pro Glu
165 170 175
Ala Asp Pro Gly Arg Thr Val Leu Thr Gly His Ser Ala Gly Gly His
180 185 190
Leu Ala Leu Trp Ala Ala Ala Arg His Val Leu Pro Ala Gly Ser Pro
195 200 205
Trp Arg Leu Pro Glu Ala Pro Ala Val Arg Gly Val Val Ala Leu Ala
210 215 220
Pro Ile Ala Asp Phe Ala Thr Ser Ala Glu Leu Asn Val Cys Asp Gly
225 230 235 240
Ala Phe Thr Gln Leu Leu Gly Glu Glu His Glu Glu Arg Leu Ala His
245 250 255
Ala Asp Leu Thr Ala Leu Leu Pro Thr Gly Ile Ala Thr Thr Leu Val
260 265 270
Gln Gly Thr Asp Asp Ala Glu Val Pro Pro Ala Val Ala Glu Ala Tyr
275 280 285
Ala Asp Ala Ala Thr Ala Ala Gly Glu Thr Pro Gly Phe Thr Leu Val
290 295 300
Asp Gly Val Gly His Phe Pro Leu Ile Asp Pro Ala Ser Ala Ala Cys
305 310 315 320
Ala Leu Val Ala Glu Glu Ile Ala Gln Leu Ala Trp
325 330
<210> 2
<211> 996
<212> DNA
<213> Streptomyces violascens
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gtgcacggcc gggcagggcc ccgcagggcc cgcggtcgag ctgggccgga ccagggtcgg 60
cagggccggg ccaaggtcga gcagggtccg ttccagctcg accagggccc ctgcacggcc 120
gagaagaggc agcgagtcga gccgaggagg acgacggtgg gcgcagcgcg gagcgcggcg 180
gaggagagct ccctgttcgg gcacccggag gcggcgccgg acaccacggc ggcgtacggc 240
ccggaccagg accaggtggt ggacttctac gccccgcgcg gcccgggcgc ggcgccgggc 300
acgccgctgg tcgtggtggt gcacggcggg gcgtggcggg ccaggtacga ccggcgccac 360
atctccccgt tcgcggcgtt cctggcgggg cgcgggttcg cggtggcgag cgtggagtac 420
cggcggggcg acggtgggcg gggcgccggg cgctggcccg acaccttcga cgacgtcgcc 480
gccgccttcg acgcgctgcc ggagctggcg gccggggcgg tgccggaggc ggatccgggc 540
cgtaccgtcc tcaccgggca ctcggccggc ggccacctcg cgctctgggc cgccgcccgg 600
cacgttttgc ccgccggttc cccgtggcgc ctgcccgagg cgccggcggt gcgcggggtg 660
gtggcgctcg cccccatcgc ggacttcgcc acctcggccg aactgaacgt ctgcgacggc 720
gcgttcaccc agctcctcgg ggaggagcac gaggagcggc tggcgcacgc cgacctcacc 780
gccctgctgc cgaccgggat cgccaccacc ctggtccagg gcaccgacga cgcggaggtg 840
ccgcccgcgg tggcggaggc gtacgccgac gccgcgacgg ccgccgggga aaccccgggc 900
ttcaccctgg tcgacggcgt ggggcacttc ccgctgatcg acccggcgtc ggccgcctgc 960
gccctggtcg cggaggagat agcccaactg gcctgg 996
Claims (10)
1. A lipase Sv-lip5, the amino acid sequence of which is shown in SEQ ID NO. 1.
2. The gene of the lipase Sv-lip5 as claimed in claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 2.
3. Use of the lipase Sv-lip5 according to claim 1 for hydrolyzing astaxanthin esters or for preparing free astaxanthin.
4. A method of hydrolyzing an astaxanthin ester or preparing free astaxanthin, characterized in that: hydrolysis of astaxanthin ester by the lipase Sv-lip5 according to claim 1 to obtain free astaxanthin.
5. The method according to claim 4, characterized in that the specific conditions of the hydrolysis are: adding lipase Sv-lip5 into the astaxanthin ester solution, and hydrolyzing at 25-50 ℃ and pH value of 8.0-10.0.
6. The method of claim 5, wherein: adding ethanol into astaxanthin ester solution during hydrolysis; the volume ratio of the ethanol to the astaxanthin ester solution is 1: 12;
adding Ca to astaxanthin ester solution during hydrolysis2+、Co2+Or Ba2+(ii) a The Ca2+Is 1 mM; the Co2+Is 10 mM; said Ba2+Is 10 mM;
the astaxanthin ester concentration in the astaxanthin ester solution is 40 mg/mL, the addition amount of lipase Sv-lip5 is 5 mL, 100 mM, pH 9.0, Tris-HCl buffer solution is added with 11U, the hydrolysis temperature is 40 ℃, the pH value is 9.0, and the hydrolysis time is 12 h.
7. A recombinant expression vector comprising the gene encoding lipase Sv-lip5 according to claim 2.
8. Use of the recombinant expression vector of claim 7 for the preparation of the lipase Sv-lip5 of claim 1.
9. A recombinant engineered bacterium comprising the recombinant expression vector of claim 7 or the gene encoding the lipase Sv-lip5 of claim 2, which is capable of expressing the lipase Sv-lip5 of claim 1.
10. Use of the recombinant engineered bacterium of claim 9 in the preparation of the lipase Sv-lip5 of claim 1.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008143679A2 (en) * | 2006-06-01 | 2008-11-27 | Verenium Corporation | Nucleic acids and proteins and methods for making and using them |
| CN104293874A (en) * | 2014-09-23 | 2015-01-21 | 中国海洋大学 | Method for preparing free astaxanthin |
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