CN113528498B - Preparation method of S-cyanohydrin lyase and product thereof - Google Patents
Preparation method of S-cyanohydrin lyase and product thereof Download PDFInfo
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- CN113528498B CN113528498B CN202110926742.7A CN202110926742A CN113528498B CN 113528498 B CN113528498 B CN 113528498B CN 202110926742 A CN202110926742 A CN 202110926742A CN 113528498 B CN113528498 B CN 113528498B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/02—Aldehyde-lyases (4.1.2)
- C12Y401/02047—(S)-Hydroxynitrile lyase (4.1.2.47)
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Abstract
The invention provides a preparation method and application of a plant-derived novel S-cyanohydrin lyase mutant. Specifically, various mutant cyanohydrin lyase are obtained by constructing random mutation libraries and point saturation mutation libraries and high-throughput screening, and the catalytic activity of the mutant cyanohydrin lyase is 200-500% of that of wild-type cyanohydrin lyase.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of S-cyanohydrin lyase.
Background
Cyanohydrin lyase is an industrial enzyme which is very useful in chemical production, the natural activity of which is to catalyze the cleavage of cyanohydrin with the release of hydrocyanic acid. The cyanohydrin lyase can catalyze the reverse reaction, namely the addition of HCN and aldehyde ketone, and an alpha-cyanohydrin product with optical activity is obtained.
The natural S-cyanohydrin lyase exists in a few plant tissues such as rubber, cassava, sorghum and the like, and has low abundance and high purification difficulty. In 1995, Wajant isolated cassava cyanohydrin lyase MeHNL from cassava using a five-step purification method (Plant Sci, 1995, 108, 1); white et al extracted MeHNL from cassava leaves in a three-step process and obtained the enzyme solution by salting out and dialysis, but the stereoselectivity for application in chemical catalysis was not high (Plant Physiol 1998, 116, 1219). The cyanohydrin lyase (MeHNL) derived from cassava (Manihot esculenta) is an S-cyanohydrin lyase, and the MeHNL is reported to be used for catalyzing the chemical synthesis of S-type chiral cyanohydrin in documents, the ee value is more than 99 percent, and the enzyme has important application value, for example, the S-m-phenoxy benzaldehyde cyanohydrin is a general intermediate of novel pyrethroid pesticides. In 1993, Wajant et al reported the cDNA sequence and protein sequence encoding MeHNL.
The adoption of microorganisms as host bacteria is an effective method for rapidly obtaining a large amount of cyanohydrin lyase. Effenberger et al reported in 1996 recombinant expression of MeHNL in E.coli, but most of the proteins were inclusion bodies and the soluble protein content was low (Angew 1996, 35, 437). In 2001, Chengshouhua and the like, the MeHNL gene is cloned into a plasmid PPIC9K, so that the expression in yeast is realized (in the report of bioengineering, 2001, 17(1), 78), but the enzyme activity is still not high enough, and the requirement of practical application is difficult to achieve.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art methods for preparing S-cyanohydrin lyase.
Therefore, one of the objects of the present invention is to provide a method for preparing S-cyanohydrin lyase and the use thereof, which overcomes the defect of the prior art that the activity of the prepared S-cyanohydrin lyase is not high enough.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a preparation method of S-cyanohydrin lyase comprises the following steps,
carrying out mutation treatment on a wild type gene of the cyanohydrin lyase: carrying out mutation treatment on a cyanohydrin lyase gene and a wild type gene sequence of the cyanohydrin lyase as shown in Seq ID No.1 to obtain a cyanohydrin lyase mutant gene, wherein the sequence of the cyanohydrin lyase mutant gene is shown in Seq ID No. 2.
Adding enzyme cutting sites: inserting double enzyme cutting sites into a mutation gene of the cyanohydrin lyase;
preparing a recombinant plasmid: inserting the cyanohydrin lyase mutator gene into an expression vector to obtain a recombinant plasmid;
introducing a strain: introducing the recombinant plasmid with the cyanohydrin lyase gene into a strain to obtain a recombinant expression strain;
secretion and expression of the strains: inducing the recombinant expression strain in culture solution to express and collecting enzyme solution.
As a preferable embodiment of the method for producing S-cyanohydrin lyase of the present invention, there is provided a method wherein: the mutation treatment, wherein the mutation treatment mode is error-prone PCR.
As a preferable embodiment of the method for producing S-cyanohydrin lyase of the present invention, there is provided a method wherein: in the added enzyme cutting sites, the double enzyme cutting sites are NdeI/HindIII.
As a preferable embodiment of the method for producing S-cyanohydrin lyase of the present invention, there is provided a method wherein: in the recombinant plasmid preparation, the expression vector was pET26b (+).
As a preferable embodiment of the method for producing S-cyanohydrin lyase of the present invention, there is provided a method wherein: the introduced strain is E.coli BL21(DE 3).
As a preferable embodiment of the process for producing S-cyanohydrin lyase of the present invention, there is provided a process wherein: in the secretion and expression of the strain, induction culture is also included, when OD is in the culture medium600After 1.0, 0.2mM IPTG was added and the temperature was maintained at 30 ℃ to induce expression for 4-5 h.
The product prepared by the preparation method of the S-cyanohydrin lyase comprises the following steps: the catalytic activity of the product is 200-500% times of that of wild-type cyanohydrin lyase.
The invention has the beneficial effects that:
the invention provides a preparation method and application of a plant-derived novel S-cyanohydrin lyase mutant. Specifically, various mutant cyanohydrin lyase are obtained by constructing random mutation libraries and point saturation mutation libraries and high-throughput screening, and the catalytic activity of the mutant cyanohydrin lyase is 200-500% of that of wild-type cyanohydrin lyase.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
In the examples of the present invention, the cyanohydrin lyase used is cassava cyanohydrin lyase.
The raw materials and reagents used in the present invention are commercially available unless otherwise specified.
Example 1
The specific principle of the design of the invention is to obtain various mutant cyanohydrin lyase by constructing random mutation libraries and point saturation mutation libraries and high-throughput screening.
The source of the S-cyanohydrin lyase mutator is wild-type cyanohydrin lyase, and the specific synthetic method comprises the following steps: the method comprises the steps of adding enzyme cutting sites NdeI and HindIII at two ends of a cyanohydrin lyase wild gene shown in Seq ID No.1 or a cyanohydrin lyase mutant gene shown in Seq ID No.2, performing DNA enzyme cutting purification, inserting the purified product into an expression vector pET26b (+) to obtain a recombinant plasmid, and transforming the recombinant plasmid into E.coliBL21(DE3) to construct a recombinant expression strain. Culturing the recombinant strain in LB culture medium to OD600After 1.0, 0.2mM IPTG is cultured for 4-5 hours at 30 ℃, and the wild type or mutant cells are obtained by centrifugal collection, and crude enzyme liquid is obtained after the cells are subjected to ultrasonic lysis.
Example 2
The preparation method of the wild-type cyanhydrin lyase mutant comprises the following steps:
the method comprises the steps of introducing random mutation and (or) introduced point saturation mutation based on enzyme protein structure/simulated substrate docking into a wild gene sequence of cyanohydrin lyase of cassava (Manihot esculenta) as shown in Seq ID No.1 in an error-prone PCR mode, and then carrying out high-throughput screening to obtain a wild type mutant of the cyanohydrin lyase in the invention as shown in Seq ID No. 2.
The protein sequence of cassava (Manihot esculenta) after transcription and translation by a cyanohydrin lyase wild gene is shown as Seq ID No.3, the protein sequence of cassava (Manihot esculenta) translated by a wild-type cyanohydrin lyase mutant is shown as Seq ID No.4, the wild-type cyanohydrin lyase mutant has the difference that 50-bit glutamic acid is mutated into glycine, 128-bit tryptophan is mutated into alanine, 226-bit lysine is mutated into proline compared with the wild-type cyanohydrin lyase, and Seq ID No.4 shows the possibility of one of the protein sequences, but the possibility of mutation of other various protein sequences has the same performance.
Except for Seq ID No.2, the mutant gene of the wild-type cyanohydrin lyase is changed into GGG at the 148 th to 150 th sites, GCG at the 382 th to 384 th sites and CCG at the 676 th to 678 th sites, and any situation that any site is changed as described above is included.
Example 3
The high-throughput screening of cyanohydrin enzyme comprises the following steps: at 25 ℃, the following reagents, 130 μ L of 100mM potassium phosphate-citric acid buffer (pH 5.0), 20 μ L of diluted lyase solution, and finally 50 μ L of phenethyl cyanol substrate solution are sequentially added into a 96-well plate, and the change of the absorbance at 280nm is read for 5 minutes by using an enzyme-linked immunosorbent assay, wherein the change of the absorbance represents the enzyme activity.
Phenethyl cyanide alcohol substrate solution: 100mM potassium phosphate-citric acid buffer (pH 3.5) was used to prepare a buffer solution at a concentration of 8. mu.l/ml.
Example 4
Determination of enzyme Activity
The wild-type cyanohydrin lyase gene was inserted into an expression vector pET26b (+) by the same procedure as in the example to obtain a recombinant plasmid, and the plasmid was transformed into e.coli BL21(DE3) to construct an expression strain. Bacteria causingCulturing the strain in LB culture medium to OD600After changing to 1.0, 0.2mM IPTG was cultured at 30 ℃ for 4-5 hours under induction, and the wild-type cells were collected by centrifugation, and the cells were subjected to ultrasonic lysis to obtain a crude enzyme solution.
Simultaneously calculating the activity of the wild-type cyanohydrin lyase and the enzyme of the mutant of the wild-type cyanohydrin lyase
To a 1.0 ml cuvette were added the following reagents: 0.70 ml of 100mM potassium phosphate-citric acid buffer (pH 5.0) and 0.10 ml of diluted enzyme solution.
Put into an ultraviolet spectrophotometer, mixed evenly, adjusted to zero at 280nm, added with 0.2 ml of phenethyl cyanol substrate solution (same as example 3), mixed evenly, and read the change of the light absorption value at 280nm for 5 minutes.
The formula for calculating the activity of the enzyme is 7.267 Xthe dilution factor of the enzyme solution is multiplied by delta A280
ΔA280: change in absorbance per minute at 280nm
TABLE 1 Activity of wild-type cyanohydrin lyase mutant (cell lysate)
The invention provides a preparation method and application of a plant-derived novel S-cyanohydrin lyase mutant. Specifically, various mutant cyanohydrin lyase are obtained by constructing random mutation libraries and point saturation mutation libraries and high-throughput screening, and the catalytic activity of the S-cyanohydrin lyase mutant is 200-500% times that of wild-type cyanohydrin lyase.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<120> preparation method of S-cyanohydrin lyase and product thereof
<141> 2021-08-09
<160> 4
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atggcacttg cccattttgt tctcattcat accatatgcc atggtgcttg gatttggtac 60
aagctcgtac cattgctgga agctgctggc cacaacgtaa ctgcactgga ccttgcagcc 120
agtggcattg accaaagaca aataaatcag cttggctcat ttgatgacta ttctgaacct 180
ctgttgaagt atatggcttc agtcccaaaa ggaaaaaagg taatacttgt tggagagagc 240
tgtggaggaa tcaatattgc cattgctgct gataaatacc ctgaaaagat tgctgctgct 300
gtcttccaca attccctaat gccagatact gtgcacaacc ctgcttatgt tgtggacaag 360
ctattggagg tgtttcctga ctggaaagat acagtgtttt ctaactacac tttcaaaggg 420
gagaatataa cagcattgag tttgggcttc aaacttatga aggagaatat atatactaac 480
tgcatatata aggattatga actggcaaag cgattggtaa ggaagggatc attattccaa 540
aatgttttgg caaagagaaa attcttcact aaagaaggat atggatcaat taagagaata 600
tatgtgtatg gagatgaaga taaaatattt ctaccaaaat ttcatcgctg gcaaatagca 660
aactacaaac cacacaaggt gtatttggtc ccaggtggtg atcacaagct gatgctttca 720
aagcgtattg agctctttca cattctgcaa gaggtggctc aaacatatgc ttga 774
<210> 2
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<212> DNA
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atggcacttg cccattttgt tctcattcat accatatgcc atggtgcttg gatttggtac 60
aagctcgtac cattgctgga agctgctggc cacaacgtaa ctgcactgga ccttgcagcc 120
agtggcattg accaaagaca aataaatggg cttggctcat ttgatgacta ttctgaacct 180
ctgttgaagt atatggcttc agtcccaaaa ggaaaaaagg taatacttgt tggagagagc 240
tgtggaggaa tcaatattgc cattgctgct gataaatacc ctgaaaagat tgctgctgct 300
gtcttccaca attccctaat gccagatact gtgcacaacc ctgcttatgt tgtggacaag 360
ctattggagg tgtttcctga cgcgaaagat acagtgtttt ctaactacac tttcaaaggg 420
gagaatataa cagcattgag tttgggcttc aaacttatga aggagaatat atatactaac 480
tgcatatata aggattatga actggcaaag cgattggtaa ggaagggatc attattccaa 540
aatgttttgg caaagagaaa attcttcact aaagaaggat atggatcaat taagagaata 600
tatgtgtatg gagatgaaga taaaatattt ctaccaaaat ttcatcgctg gcaaatagca 660
aactacaaac cacacccggt gtatttggtc ccaggtggtg atcacaagct gatgctttca 720
aagcgtattg agctctttca cattctgcaa gaggtggctc aaacatatgc ttga 774
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<212> PRT
<213> Artificial Sequence
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Met Ala Leu Ala His Phe Val Leu Ile His Thr Ile Cys His Gly Ala
1 5 10 15
Trp Ile Trp Tyr Lys Leu Val Pro Leu Leu Glu Ala Ala Gly His Asn
20 25 30
Val Thr Ala Leu Asp Leu Ala Ala Ser Gly Ile Asp Gln Arg Gln Ile
35 40 45
Asn Gln Leu Gly Ser Phe Asp Asp Tyr Ser Glu Pro Leu Leu Lys Tyr
50 55 60
Met Ala Ser Val Pro Lys Gly Lys Lys Val Ile Leu Val Gly Glu Ser
65 70 75 80
Cys Gly Gly Ile Asn Ile Ala Ile Ala Ala Asp Lys Tyr Pro Glu Lys
85 90 95
Ile Ala Ala Ala Val Phe His Asn Ser Leu Met Pro Asp Thr Val His
100 105 110
Asn Pro Ala Tyr Val Val Asp Lys Leu Leu Glu Val Phe Pro Asp Trp
115 120 125
Lys Asp Thr Val Phe Ser Asn Tyr Thr Phe Lys Gly Glu Asn Ile Thr
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Ala Leu Ser Leu Gly Phe Lys Leu Met Lys Glu Asn Ile Tyr Thr Asn
145 150 155 160
Cys Ile Tyr Lys Asp Tyr Glu Leu Ala Lys Arg Leu Val Arg Lys Gly
165 170 175
Ser Leu Phe Gln Asn Val Leu Ala Lys Arg Lys Phe Phe Thr Lys Glu
180 185 190
Gly Tyr Gly Ser Ile Lys Arg Ile Tyr Val Tyr Gly Asp Glu Asp Lys
195 200 205
Ile Phe Leu Pro Lys Phe His Arg Trp Gln Ile Ala Asn Tyr Lys Pro
210 215 220
His Lys Val Tyr Leu Val Pro Gly Gly Asp His Lys Leu Met Leu Ser
225 230 235 240
Lys Arg Ile Glu Leu Phe His Ile Leu Gln Glu Val Ala Gln Thr Tyr
245 250 255
Ala
<210> 4
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<213> Artificial Sequence
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Met Ala Leu Ala His Phe Val Leu Ile His Thr Ile Cys His Gly Ala
1 5 10 15
Trp Ile Trp Tyr Lys Leu Val Pro Leu Leu Glu Ala Ala Gly His Asn
20 25 30
Val Thr Ala Leu Asp Leu Ala Ala Ser Gly Ile Asp Gln Arg Gln Ile
35 40 45
Asn Gly Leu Gly Ser Phe Asp Asp Tyr Ser Glu Pro Leu Leu Lys Tyr
50 55 60
Met Ala Ser Val Pro Lys Gly Lys Lys Val Ile Leu Val Gly Glu Ser
65 70 75 80
Cys Gly Gly Ile Asn Ile Ala Ile Ala Ala Asp Lys Tyr Pro Glu Lys
85 90 95
Ile Ala Ala Ala Val Phe His Asn Ser Leu Met Pro Asp Thr Val His
100 105 110
Asn Pro Ala Tyr Val Val Asp Lys Leu Leu Glu Val Phe Pro Asp Ala
115 120 125
Lys Asp Thr Val Phe Ser Asn Tyr Thr Phe Lys Gly Glu Asn Ile Thr
130 135 140
Ala Leu Ser Leu Gly Phe Lys Leu Met Lys Glu Asn Ile Tyr Thr Asn
145 150 155 160
Cys Ile Tyr Lys Asp Tyr Glu Leu Ala Lys Arg Leu Val Arg Lys Gly
165 170 175
Ser Leu Phe Gln Asn Val Leu Ala Lys Arg Lys Phe Phe Thr Lys Glu
180 185 190
Gly Tyr Gly Ser Ile Lys Arg Ile Tyr Val Tyr Gly Asp Glu Asp Lys
195 200 205
Ile Phe Leu Pro Lys Phe His Arg Trp Gln Ile Ala Asn Tyr Lys Pro
210 215 220
His Pro Val Tyr Leu Val Pro Gly Gly Asp His Lys Leu Met Leu Ser
225 230 235 240
Lys Arg Ile Glu Leu Phe His Ile Leu Gln Glu Val Ala Gln Thr Tyr
245 250 255
Ala
Claims (7)
1. A preparation method of S-cyanohydrin lyase is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
carrying out mutation treatment on a wild type gene of the cyanohydrin lyase: carrying out mutation treatment on a cyanohydrin lyase gene and a wild type gene sequence of the cyanohydrin lyase as shown in Seq ID No.1 to obtain a cyanohydrin lyase mutant gene, wherein the sequence of the cyanohydrin lyase mutant gene is shown in Seq ID No. 2;
adding enzyme cutting sites: inserting double enzyme cutting sites into two ends of a mutation gene of the cyanohydrin lyase;
preparing a recombinant plasmid: inserting the cyanohydrin lyase mutator gene into an expression vector to obtain a recombinant plasmid;
introducing a strain: introducing the recombinant plasmid with the cyanohydrin lyase gene into a strain to obtain a recombinant expression strain;
secretion and expression of the strains: inducing the recombinant expression strain in culture solution to express and collecting enzyme solution.
2. The process for producing S-cyanohydrin lyase as claimed in claim 1, wherein: the mutation treatment, wherein the mutation treatment mode is error-prone PCR.
3. The process for producing S-cyanohydrin lyase as claimed in claim 1, wherein: in the added enzyme cutting sites, the double enzyme cutting sites are NdeI/HindIII.
4. The process for producing S-cyanohydrin lyase as claimed in claim 1, wherein: in the recombinant plasmid preparation, the expression vector was pET26b (+).
5. The process for producing S-cyanohydrin lyase as claimed in claim 1, wherein: the introduced strain is E.coli BL21(DE 3).
6. The process for producing S-cyanohydrin lyase as claimed in claim 1, wherein: in the secretion and expression of the strain, induction culture is also included, when OD is in the culture medium600After =1.0, 0.2mM IPTG was added and the temperature was kept at 30 ℃, inducing expression for 4-5 h.
7. The product obtained by the method for producing S-cyanohydrin lyase according to claim 1 to 6, wherein the method comprises: the catalytic activity of the product is 200-500% times of that of wild-type cyanohydrin lyase.
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| JP2005312431A (en) * | 2004-03-31 | 2005-11-10 | Nippon Shokubai Co Ltd | New modified s-hydroxynitrile lyase |
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| WO2018127143A1 (en) * | 2017-01-06 | 2018-07-12 | 上海弈柯莱生物医药科技有限公司 | Highly active s-cyanohydrin lyase and application thereof |
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| EP2092060B1 (en) * | 2006-12-14 | 2014-01-22 | DSM IP Assets B.V. | R-hnl random variants and their use for preparing optically pure, sterically hindered cyanohydrins |
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| JP2005312431A (en) * | 2004-03-31 | 2005-11-10 | Nippon Shokubai Co Ltd | New modified s-hydroxynitrile lyase |
| CN106032531A (en) * | 2015-08-07 | 2016-10-19 | 南京博优康远生物医药科技有限公司 | Cyanohydrins lyase and preparing method and application thereof |
| WO2018127143A1 (en) * | 2017-01-06 | 2018-07-12 | 上海弈柯莱生物医药科技有限公司 | Highly active s-cyanohydrin lyase and application thereof |
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| CN113046337A (en) * | 2021-03-18 | 2021-06-29 | 赤峰制药股份有限公司 | Pantolactone hydrolase mutant strain and application thereof |
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