CN106011092A - Engineered ketoreductase polypeptide and method for preparing montelukast midbody by means of same - Google Patents

Engineered ketoreductase polypeptide and method for preparing montelukast midbody by means of same Download PDF

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CN106011092A
CN106011092A CN201610440251.0A CN201610440251A CN106011092A CN 106011092 A CN106011092 A CN 106011092A CN 201610440251 A CN201610440251 A CN 201610440251A CN 106011092 A CN106011092 A CN 106011092A
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sequence
host cell
compound
ketoreductase
coenzyme
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原海亮
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SUZHOU ENZYMEWORKS Inc
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SUZHOU ENZYMEWORKS Inc
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

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Abstract

The invention discloses an engineered ketoreductase polypeptide and a method for preparing a montelukast midbody by means of the same. Compared with the prior art, the engineered ketoreductase polypeptide has the advantages that enzyme consumption is low, reaction time is short, substrate concentration is high, and the engineered ketoreductase polypeptide is more suitable for industrial application when used for preparation of the montelukast midbody.

Description

A kind ofThrough engineering approaches Ketoreductase polypeptides and the method being used for preparing Montelukast intermediate thereof
Technical field
The present invention relates to bio-pharmaceuticals and technical field of biotransformation, be specifically related to a kind of through engineering approaches Ketoreductase polypeptides and the method preparing Montelukast intermediate thereof.
Background technology
Montelukast (Montelukast, CAS 158966-92-8) is a kind of selectivity LTRA (LTRA), is used for preventing and treat asthma, bronchoconstriction and allergic rhinitis, has important market value.The molecular structure patent of Montelukast expired on August 3rd, 2012, therefore, it is possible to the method effectively synthesizing its effective ingredient 2-(3-(3-((E)-2-(7-chloroquinoline base) vinyl) phenyl)-(3S)-3-hydroxypropyl) essence of Niobe has broad application prospects.Article one, common synthetic route is such asFollowing formula instituteShow:
Chemical process utilizes (-)-diisopinocampheylchloroborane base chloroborane class catalyst ((-)-DIP-Cl) carry out asymmetric hydrogenation catalysis (J.Am.Chem.Soc.1996,118,2521 and WO 2008/009970).It is poor to there is Atom economy in (-)-DIP-Cl, complex operation step, separates the problems such as complicated, seriously polluted.Bioconversion method such as Org.Process Res.Dev.2010,14,193 and WO 2009/042984A1 etc. report, there is enzyme amount higher (3%) in the method, the problems such as response time length (24h), concentration of substrate low (12%) and consumption of organic solvent (60%) are higher.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of through engineering approaches Ketoreductase polypeptides and the method preparing Montelukast intermediate thereof.
For reaching above-mentioned purpose, the invention provides one and substrate 2-(3-(3-((E)-2-(7-chloroquinoline base) vinyl) phenyl)-3-ketone propyl group) essence of Niobe can be converted into the through engineering approaches Ketoreductase polypeptides of product 2-(3-(3-((E)-2-(7-chloroquinoline base) vinyl) phenyl)-(3S)-3-hydroxypropyl) essence of Niobe, it is characterized in that, the aminoacid sequence of described Ketoreductase polypeptides and SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, any sequence in 42 and 44 has the homology of at least 90%.Wherein, sequence 2 is the wild type ketoreductase from Candida magnoliae, other sequence is obtained by directed mutagenesis method such as random mutation and avtive spot cassette mutagenesis (CASTing) such as fallibility PCR, mutant obtains the activity and stability strengthened, can be found out by methods such as high flux screenings (HTS), above-mentioned change aminoacid sequence and screening mutant library method, the routine techniques being in this area.
The invention provides the polynucleotide of a kind of coded polypeptide.Preferably, described polynucleotide any sequence in SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43, its albumen expressed is as indicated above.
The invention provides a kind of expression vector, described expression vector comprises and is suitable to the polynucleotide that the control sequence of the expression that guidance is coded of polypeptide is operably connected in host cell.Preferably, the sequence of these polynucleotide is SEQ ID NO:43.
Present invention also offers a kind of host cell, described host cell comprises above-mentioned expression vector.Preferably, this host cell is escherichia coli.
It is a further object to provide a kind of method preparing Montelukast intermediate, described method, with compound 1 as substrate, contacts with described Ketoreductase polypeptides, wherein under being suitable to the reaction condition being reduced to compound 2:
The structural formula of described compound 1 is:
The structural formula of described compound 2 is:
Preferably, described reduction reaction is carried out in the presence of coenzyme and regenerating coenzyme system, and wherein, described coenzyme is NADP, and described regenerating coenzyme system is glucose and glucose dehydrogenase.
It is further preferred that described glucose dehydrogenase is the glucose dehydrogenase that the trade mark is EW002 produced purchased from Suzhou Chinese biotechnology of enzymes company limited.
It is further preferred that described reduction reaction is carried out under the conditions of cosolvent, described cosolvent is toluene.
Utilization due to technique scheme, the present invention compared with prior art has the advantage that during preparing Montelukast intermediate, have employed the through engineering approaches ketoreductase of the present invention, overcome the problem that reactivity worth in prior art is not good enough, its enzyme dosage low (2%), in the response time short (12h), concentration of substrate is high, up to 20%, it is more suitable for commercial application.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following example.The implementation condition used in embodiment can do further adjustment according to specifically used different requirement, and not marked implementation condition is the condition in normal experiment.
Embodiment 1 (preparation of ketoreductase):
Conventional method is used to prepare ketoreductase catalyst: sequenceIn table1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 genetic fragments are synthesized by Jin Weizhi bio tech ltd, Suzhou, it is connected with the digestion products of pET30a (Novagen) plasmid, proceed to competence E.coli BL21 (DE3) bacterial strain, screening obtains positive colony, it is inoculated in the LB liquid medium containing resistance, cultivate to OD600 to 0.8 in 37 DEG C, add derivant IPTG, continue to cultivate 16 hours, centrifugal collecting precipitation, add phosphate buffer to suspend, ultrasonic disruption 10 minutes in ice-water bath, centrifuging and taking supernatant, freezing obtains ketoreductase enzyme powder.
Embodiment 2 (screening of ketoreductase)
nullBy sequence 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、The 43 ketoreductase 2mg obtained at expression in escherichia coli respectively，(buying is from Suzhou Chinese biotechnology of enzymes company limited for glucose dehydrogenase，Trade mark EW002) 2mg，NADP 1mg，Substrate 2-(3-(3-((E)-2-(7-chloroquinoline base) vinyl) phenyl)-3-ketone propyl group) essence of Niobe 50mg、Glucose 50mg adds in the 5mL reactor equipped with 2mL 0.05M Triethanolamine buffer，30 DEG C of 1000rpm stirrings，HPLC detection is sampled after 1h，Conversion ratio and the ee of sequence 43 are > 99%，Therefore use sequence 43 as further object of study.
Embodiment 3 (enzymic catalytic reaction):
100mg ketoreductase (being obtained by sequence 43) it is sequentially added at expression in escherichia coli in 50mL reaction there-necked flask, (buying is from Suzhou Chinese biotechnology of enzymes company limited for 20mg glucose dehydrogenase, trade mark EW002) and the Triethanolamine buffer of 5mg NADP, 5g substrate 2-(3-(3-((E)-2-(7-chloroquinoline base) vinyl) phenyl)-3-ketone propyl group) essence of Niobe, 3.5g glucose, 20mL 0.05M pH 7.0 Triethanolamine buffer, 5mL toluene, temperature regulates to 30 DEG C, 900r/min stirs, 15%Na2CO3Solution maintains pH to 7.0, reaction 12h, HPLC detects conversion ratio 99%, adds methanol 30ml, 60 DEG C of backflow 2h, filter and backward filtrate drips deionized water to no longer there being solid to separate out, crossing and add methanol at leaching filter cake 60 DEG C to after just dissolving, dropping deionized water, to no longer there being solid to separate out, is filtrated to get product 4.2g, purity 99%, content 99%.
Above-described embodiment is only for technology design and the feature of the explanation present invention; its object is to allow person skilled in the art will appreciate that present disclosure and to be carried out; can not limit the scope of the invention with this; all equivalence changes made according to spirit of the invention or modification, all should contain within the scope of the present invention.

Claims (10)

1. one kind can be by substrate 2-(3-(3-((E)-2-(7-chloroquinoline base) vinyl) phenyl)-3-ketone propyl group) Essence of Niobe is converted into product 2-(3-(3-((E)-2-(7-chloroquinoline base) vinyl) phenyl)-(3S)-3- Hydroxypropyl) the through engineering approaches Ketoreductase polypeptides of essence of Niobe, it is characterised in that described Ketoreductase polypeptides Aminoacid sequence and SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26, 28, any sequence in 30,32,34,36,38,40,42 and 44 has the homology of at least 90%.
2. the polynucleotide encoding polypeptide according to claim 1.
Polynucleotide the most according to claim 2, its selected from SEQ ID NO:1,3,5,7,9, 11, in 13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 Any sequence.
4. an expression vector, described expression vector comprise be suitable in host cell instruct be coded of many The polynucleotide described in claim 3 that the control sequence of the expression of peptide is operably connected.
5. a host cell, described host cell comprises the expression vector described in claim 4.
Host cell the most according to claim 5, it is escherichia coli.
7. the method preparing Montelukast intermediate, it is characterised in that described method with compound 1 is Substrate, with the ketoreductase described in claim 1 under being suitable to the reaction condition being reduced to compound 2 Polypeptide contacts, wherein:
The structural formula of described compound 1 is:
The structural formula of described compound 2 is:
Method the most according to claim 7, it is characterised in that described reduction reaction is at coenzyme and auxiliary Carrying out in the presence of enzyme regenerative system, wherein, described coenzyme is NADP, described regenerating coenzyme system For glucose and glucose dehydrogenase.
Method the most according to claim 8, it is characterised in that described glucose dehydrogenase is for being purchased from The glucose dehydrogenase that the trade mark is EW002 that Suzhou Chinese biotechnology of enzymes company limited produces.
Method the most according to claim 7, it is characterised in that described reduction reaction is at cosolvent Under the conditions of carry out, described cosolvent is toluene.
CN201610440251.0A 2016-06-20 2016-06-20 Engineered ketoreductase polypeptide and method for preparing montelukast midbody by means of same Pending CN106011092A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106011096A (en) * 2016-07-27 2016-10-12 苏州汉酶生物技术有限公司 Engineered ketoreductase polypeptide and method for preparing (S)-3-(dimethylamino)-1-(thiophene -2-yl)-1-propanol by using same
CN106011094A (en) * 2016-07-27 2016-10-12 苏州汉酶生物技术有限公司 Engineered ketoreductase polypeptide and method for preparing (3R, 5S)-6-chloro-3, 5-dihydroxy caproic acid tert-butyl ester by using same

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1152054A1 (en) * 1999-12-03 2001-11-07 Kaneka Corporation Novel carbonyl reductase, gene thereof and method of using the same
US8455223B2 (en) * 2005-09-19 2013-06-04 Basf Se Dehydrogenases, the derivatives thereof, and method for the production of optically active alkanols
CN103710405A (en) * 2013-12-31 2014-04-09 苏州汉酶生物技术有限公司 Method for preparing Montelukast intermediate by using biological method
CN104326976A (en) * 2014-10-20 2015-02-04 张家港市信谊化工有限公司 Preparation method of montelukast sodium intermediate
CN105039361A (en) * 2015-06-30 2015-11-11 浙江工业大学 Carbonyl reductase gene, codase, vector, strain and application of gene

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1152054A1 (en) * 1999-12-03 2001-11-07 Kaneka Corporation Novel carbonyl reductase, gene thereof and method of using the same
US8455223B2 (en) * 2005-09-19 2013-06-04 Basf Se Dehydrogenases, the derivatives thereof, and method for the production of optically active alkanols
CN103710405A (en) * 2013-12-31 2014-04-09 苏州汉酶生物技术有限公司 Method for preparing Montelukast intermediate by using biological method
CN104326976A (en) * 2014-10-20 2015-02-04 张家港市信谊化工有限公司 Preparation method of montelukast sodium intermediate
CN105039361A (en) * 2015-06-30 2015-11-11 浙江工业大学 Carbonyl reductase gene, codase, vector, strain and application of gene

Non-Patent Citations (1)

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JACK LIANG ET AL.: "Development of a Biocatalytic Process as an Alternative to the （-）-DIP-Cl-Mediated Asymmetric Reduction of a Key Intermediate of Montelukast", 《ORGANIC PROCESS RESEARCH & DEVELOPMENT》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106011096A (en) * 2016-07-27 2016-10-12 苏州汉酶生物技术有限公司 Engineered ketoreductase polypeptide and method for preparing (S)-3-(dimethylamino)-1-(thiophene -2-yl)-1-propanol by using same
CN106011094A (en) * 2016-07-27 2016-10-12 苏州汉酶生物技术有限公司 Engineered ketoreductase polypeptide and method for preparing (3R, 5S)-6-chloro-3, 5-dihydroxy caproic acid tert-butyl ester by using same
CN106011094B (en) * 2016-07-27 2020-11-03 苏州汉酶生物技术有限公司 Engineered ketoreductase polypeptide and method for preparing (3R,5S) -6-chloro-3, 5-dyhydroxyl hexanoic acid tert-butyl ester by using same
CN106011096B (en) * 2016-07-27 2020-11-03 苏州汉酶生物技术有限公司 Engineered ketoreductase polypeptide and method for preparing (S) -3- (dimethylamino) -1- (thiophene-2-yl) -1-propanol by using same

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