CN109913429A - A kind of novel keto reductase and its it is used for chiral alcohol synthetic method - Google Patents
A kind of novel keto reductase and its it is used for chiral alcohol synthetic method Download PDFInfo
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- CN109913429A CN109913429A CN201910284143.2A CN201910284143A CN109913429A CN 109913429 A CN109913429 A CN 109913429A CN 201910284143 A CN201910284143 A CN 201910284143A CN 109913429 A CN109913429 A CN 109913429A
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Abstract
The invention discloses a kind of novel keto reductase and its it is used for chiral alcohol synthetic method, more particularly to field of biotechnology, the 49th amino acids residue, the 127th amino acids residue, the 196th amino acids residue, the 324th amino acids residue and the 828th amino acids residue of the novel keto reductase mutate；The 49th amino acids residue sports C by G, and the 127th amino acids residue sports A by T, and the 196th amino acids residue sports E by A, and the 324th amino acids residue sports K by A.Invention required time when actually preparing chiral alcohol is greatly shortened compared with prior art, effectively shorten the time required for preparing chiral alcohol, the preparation efficiency of chiral alcohol is improved, is suitable for industrialized production and is prepared by laboratory, the scope of application is wider.
Description
Technical field
The present invention relates to technical field of biotechnology, it is more particularly related to a kind of novel keto reductase
And its it is used for chiral alcohol synthetic method.
Background technique
Because chiral, secondary alcohols are the key that many important chiral drugs of synthesis, agricultural chemicals and natural prodcuts chirality are cut
Block, it is cheap using other from the angle of environment and sustainable development, it is easy to get industrial chemicals and carrys out synthesis of chiral secondary alcohol
Obviously have great importance.
The patent of invention of patent application publication CN 106032347A discloses a kind of method of synthesis of chiral alcohol.Including
Following steps: in the reaction vessel, alkynes, golden chloride [Au (L) Cl], solvent methanol and water is added；Reaction mixture is 110
Room temperature is arrived after reacting 6-12 hours at DEG C；Add sodium formate, water, transition metal rhodium catalyst Cp*RhCl [(R, R)-
TsDPEN], after reaction mixture reacts 5 hours at 30-40 DEG C again, it is cooled to room temperature, rotary evaporation is gone out solvent, is then led to
Column separation obtains target compound.The invention is from the alkynes for being commercialized or being readily synthesized, the chiral alcohol directly obtained, instead
Significant advantage should be shown: commodity in use is easy the alkynes of preparation as starting material, directly obtains hand by tandem reaction
Property alcohol, does not need separation of intermediates, avoids solvent, waste of time etc., and therefore, which meets the requirement of Green Chemistry,
With vast potential for future development.
But it is in actual use, still there are some disadvantages, as transition metal rhodium catalyst Cp*RhCl is being added in it
When [(R, R)-TsDPEN] is reacted, the reaction time is too long, and then affects the preparation efficiency of chiral alcohol.
Summary of the invention
The present invention overcomes the shortcomings of the prior art, technical problem to be solved are as follows: provides a kind of novel ketone group
Reductase and its be used for chiral alcohol synthetic method, when actually preparing chiral alcohol, the required time is big compared with prior art
Amplitude shortens, and effectively shortens the time required for preparing chiral alcohol, improves the preparation efficiency of chiral alcohol, is suitable for industrialization
Production and laboratory preparation, the scope of application are wider.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows: a kind of novel keto reductase, it is described
The 49th amino acids residue, the 127th amino acids residue, the 196th amino acids residue, the 324th of novel keto reductase
Amino acids residue and the 828th amino acids residue mutate；
The 49th amino acids residue sports C by G, and the 127th amino acids residue sports A by T, and described
196 amino acids residues sport E by A, and the 324th amino acids residue sports K by A, and the 828th amino acids are residual
Base, which mutates, sports A by C.
The specific preparation method of novel keto reductase the following steps are included:
S1: culture gives expression to the genetic engineering bacterium of the novel keto reductase；
S2: by keto reductase by being separated in said gene engineering bacteria.
The G is set as glycine, and the C is set as cysteine, and the T is set as threonine, and the A is set as third
Propylhomoserin, the E are set as glutamic acid, and the K is set as lysine.
The 49th amino acids residue is cysteine by glycine mutation, and the 127th amino acids residue is by reviving
Histidine mutations are alanine, and the 196th amino acids residue is glutamic acid, the 324th amino acids by alanine mutation
Residue is lysine by alanine mutation, and it is alanine that the 828th amino acids residue, which mutates by cysteine mutation,.
The host cell of the genetic engineering bacterium is set as escherichia coli MC1061.
The present invention also provides a kind of novel keto reductases to be used for chiral alcohol synthetic method, specifically includes following step
It is rapid:
S1, keto reductase preparation: novel keto reductase described in claim 1 is prepared；
S2, substrate preparation: 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester is added to containing phosphate buffer
Container in heated, heating temperature is set as 45-85 DEG C, and heating time is set as 20-25min, be made mixed liquor；
S3, mixed liquor centrifugation: by novel keto reductase obtained in the mixed liquor and S1 in container be added to from
It being centrifuged in heart equipment, centrifugal rotational speed is set as 8000-12000RPM, and centrifugation time is set as 3-5min,
S4, semi-finished product preparation: the mixed liquor after centrifugation is stood into 40-60min and is reacted, in novel keto reductase
Under the action of, semi-finished product light is made by asymmetric reduction in the 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester in mixed liquor
Learn chiral alcohol mixed liquor；
S5, finished product preparation: double of completed optical chiral alcohol mixed liquor carries out lyophilization, then by the production after lyophilization
Object carries out the processing of column chromatographic purifying, obtains corresponding chiral alcohol finished product.
The phosphate buffer pH value is set as 7.0.
Also contain NADP+, glucose dehydrogenase and glucose in the container of the S2.
Technical effect and advantage of the invention: the present invention required time and prior art when actually preparing chiral alcohol
Compared to being greatly shortened, the time required for preparing chiral alcohol is effectively shortened, the preparation efficiency of chiral alcohol is improved, is suitable for
Industrialized production and laboratory preparation, the scope of application are wider.
Detailed description of the invention
The present invention will be further described in detail with reference to the accompanying drawing.
Fig. 1 is chiral alcohol synthesis flow schematic diagram of the invention.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiments of the present invention, instead of all the embodiments；Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without creative efforts, shall fall within the protection scope of the present invention.
Embodiment 1
A kind of novel keto reductase, the 49th amino acids residue of the novel keto reductase, the 127th ammonia
Base acid residue, the 196th amino acids residue, the 324th amino acids residue and the 828th amino acids residue mutate；
The 49th amino acids residue sports C by G, and the 127th amino acids residue sports A by T, and described
196 amino acids residues sport E by A, and the 324th amino acids residue sports K by A, and the 828th amino acids are residual
Base, which mutates, sports A by C.
The specific preparation method of novel keto reductase the following steps are included:
S1: culture gives expression to the genetic engineering bacterium of the novel keto reductase；
S2: by keto reductase by being separated in said gene engineering bacteria.
The G is set as glycine, and the C is set as cysteine, and the T is set as threonine, and the A is set as third
Propylhomoserin, the E are set as glutamic acid, and the K is set as lysine；
The 49th amino acids residue is cysteine by glycine mutation, and the 127th amino acids residue is by reviving
Histidine mutations are alanine, and the 196th amino acids residue is glutamic acid, the 324th amino acids by alanine mutation
Residue is lysine by alanine mutation, and it is alanine that the 828th amino acids residue, which mutates by cysteine mutation,；
The host cell of the genetic engineering bacterium is set as escherichia coli MC1061；
The present invention also provides a kind of novel keto reductases to be used for chiral alcohol synthetic method, specifically includes following step
It is rapid:
S1, keto reductase preparation: novel keto reductase described in claim 1 is prepared；
S2, substrate preparation: 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester is added to containing phosphate-buffered
It is heated in liquid, the container containing NADP+, glucose dehydrogenase and glucose, heating temperature is set as 45 DEG C, heating time
It is set as 20min, mixed liquor is made, the phosphate buffer pH value is set as 7.0；
S3, mixed liquor centrifugation: by novel keto reductase obtained in the mixed liquor and S1 in container be added to from
It being centrifuged in heart equipment, centrifugal rotational speed is set as 8000RPM, and centrifugation time is set as 3min,
S4, semi-finished product preparation: the mixed liquor after centrifugation is stood into 40min and is reacted, in novel keto reductase
Under effect, semi-finished product optics is made by asymmetric reduction in the 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester in mixed liquor
Chiral alcohol mixed liquor；
S5, finished product preparation: double of completed optical chiral alcohol mixed liquor carries out lyophilization, then by the production after lyophilization
Object carries out the processing of column chromatographic purifying, obtains corresponding chiral alcohol finished product.
As can be seen from the above embodiments: heating temperature is set as 45 DEG C in the present embodiment, and heating time is set as 20min, from
Heart revolving speed is set as 8000RPM, and centrifugation time is set as 3min, and time of repose is set as 40min, 63min the time required to experiment,
Chiral alcohol conversion 92.4%.
Embodiment 2
A kind of novel keto reductase, the 49th amino acids residue of the novel keto reductase, the 127th ammonia
Base acid residue, the 196th amino acids residue, the 324th amino acids residue and the 828th amino acids residue mutate；
The 49th amino acids residue sports C by G, and the 127th amino acids residue sports A by T, and described
196 amino acids residues sport E by A, and the 324th amino acids residue sports K by A, and the 828th amino acids are residual
Base, which mutates, sports A by C.
The specific preparation method of novel keto reductase the following steps are included:
S1: culture gives expression to the genetic engineering bacterium of the novel keto reductase；
S2: by keto reductase by being separated in said gene engineering bacteria.
The G is set as glycine, and the C is set as cysteine, and the T is set as threonine, and the A is set as third
Propylhomoserin, the E are set as glutamic acid, and the K is set as lysine；
The 49th amino acids residue is cysteine by glycine mutation, and the 127th amino acids residue is by reviving
Histidine mutations are alanine, and the 196th amino acids residue is glutamic acid, the 324th amino acids by alanine mutation
Residue is lysine by alanine mutation, and it is alanine that the 828th amino acids residue, which mutates by cysteine mutation,；
The host cell of the genetic engineering bacterium is set as escherichia coli MC1061；
The present invention also provides a kind of novel keto reductases to be used for chiral alcohol synthetic method, specifically includes following step
It is rapid:
S1, keto reductase preparation: novel keto reductase described in claim 1 is prepared；
S2, substrate preparation: 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester is added to containing phosphate-buffered
It is heated in liquid, the container containing NADP+, glucose dehydrogenase and glucose, heating temperature is set as 65 DEG C, heating time
It is set as 23min, mixed liquor is made, the phosphate buffer pH value is set as 7.0；
S3, mixed liquor centrifugation: by novel keto reductase obtained in the mixed liquor and S1 in container be added to from
It being centrifuged in heart equipment, centrifugal rotational speed is set as 10000RPM, and centrifugation time is set as 4min,
S4, semi-finished product preparation: the mixed liquor after centrifugation is stood into 50min and is reacted, in novel keto reductase
Under effect, semi-finished product optics is made by asymmetric reduction in the 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester in mixed liquor
Chiral alcohol mixed liquor；
S5, finished product preparation: double of completed optical chiral alcohol mixed liquor carries out lyophilization, then by the production after lyophilization
Object carries out the processing of column chromatographic purifying, obtains corresponding chiral alcohol finished product.
As can be seen from the above embodiments: heating temperature is set as 65 DEG C in the present embodiment, and heating time is set as 23min, from
Heart revolving speed is set as 10000RPM, and centrifugation time is set as 4min, and time of repose is set as 50min, the time required to experiment
77min, chiral alcohol conversion 98.2%.
Embodiment 3
A kind of novel keto reductase, the 49th amino acids residue of the novel keto reductase, the 127th ammonia
Base acid residue, the 196th amino acids residue, the 324th amino acids residue and the 828th amino acids residue mutate；
The 49th amino acids residue sports C by G, and the 127th amino acids residue sports A by T, and described
196 amino acids residues sport E by A, and the 324th amino acids residue sports K by A, and the 828th amino acids are residual
Base, which mutates, sports A by C.
The specific preparation method of novel keto reductase the following steps are included:
S1: culture gives expression to the genetic engineering bacterium of the novel keto reductase；
S2: by keto reductase by being separated in said gene engineering bacteria.
The G is set as glycine, and the C is set as cysteine, and the T is set as threonine, and the A is set as third
Propylhomoserin, the E are set as glutamic acid, and the K is set as lysine；
The 49th amino acids residue is cysteine by glycine mutation, and the 127th amino acids residue is by reviving
Histidine mutations are alanine, and the 196th amino acids residue is glutamic acid, the 324th amino acids by alanine mutation
Residue is lysine by alanine mutation, and it is alanine that the 828th amino acids residue, which mutates by cysteine mutation,；
The host cell of the genetic engineering bacterium is set as escherichia coli MC1061；
The present invention also provides a kind of novel keto reductases to be used for chiral alcohol synthetic method, specifically includes following step
It is rapid:
S1, keto reductase preparation: novel keto reductase described in claim 1 is prepared；
S2, substrate preparation: 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester is added to containing phosphate-buffered
It is heated in liquid, the container containing NADP+, glucose dehydrogenase and glucose, heating temperature is set as 85 DEG C, heating time
It is set as 25min, mixed liquor is made, the phosphate buffer pH value is set as 7.0；
S3, mixed liquor centrifugation: by novel keto reductase obtained in the mixed liquor and S1 in container be added to from
It being centrifuged in heart equipment, centrifugal rotational speed is set as 12000RPM, and centrifugation time is set as 5min,
S4, semi-finished product preparation: the mixed liquor after centrifugation is stood into 60min and is reacted, in novel keto reductase
Under effect, semi-finished product optics is made by asymmetric reduction in the 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester in mixed liquor
Chiral alcohol mixed liquor；
S5, finished product preparation: double of completed optical chiral alcohol mixed liquor carries out lyophilization, then by the production after lyophilization
Object carries out the processing of column chromatographic purifying, obtains corresponding chiral alcohol finished product.
As can be seen from the above embodiments: heating temperature is set as 85 DEG C in the present embodiment, and heating time is set as 25min, from
Heart revolving speed is set as 12000RPM, and centrifugation time is set as 5min, and time of repose is set as 60min, the time required to experiment
90min, chiral alcohol conversion 93.5%.
Following table can be obtained by above-described embodiment 1-3:
As seen from the above table: the time required for embodiment 1-3 is greatly shortened compared with prior art, in practice
When, effectively shorten the time required for preparing chiral alcohol, improve the preparation efficiency of chiral alcohol, be suitable for industrialized production with
And laboratory preparation, the scope of application is wider, while every numerical value is when implementing in embodiment 2, chiral alcohol conversion highest.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (9)
1. a kind of novel keto reductase, it is characterised in that: the 49th amino acids residue of the novel keto reductase,
127th amino acids residue, the 196th amino acids residue, the 324th amino acids residue and the 828th amino acids residue occur
Mutation；
The 49th amino acids residue sports C by G, and the 127th amino acids residue sports A by T, and the described 196th
Amino acids residue sports E by A, and the 324th amino acids residue sports K, the 828th amino acids residue by A
It mutates and A is sported by C.
2. the novel keto reductase of one kind according to claim 1, it is characterised in that: the novel keto reductase
Specific preparation method the following steps are included:
S1: culture gives expression to the genetic engineering bacterium of the novel keto reductase；
S2: by keto reductase by being separated in said gene engineering bacteria.
3. the novel keto reductase of one kind according to claim 1, it is characterised in that: the G is set as glycine, institute
It states C and is set as cysteine, the T is set as threonine, and the A is set as alanine, and the E is set as glutamic acid, the K
It is set as lysine.
4. the novel keto reductase of one kind according to claim 3, it is characterised in that: the 49th amino acids residue
It is cysteine by glycine mutation, the 127th amino acids residue sports alanine by threonine, and described 196th
Amino acid residue is glutamic acid by alanine mutation, and the 324th amino acids residue is lysine by alanine mutation, described
It is alanine that 828th amino acids residue, which mutates by cysteine mutation,.
5. the novel keto reductase of one kind according to claim 1, it is characterised in that: the host of the genetic engineering bacterium
Cell is set as escherichia coli MC1061.
6. a kind of novel keto reductase is used for chiral alcohol synthetic method, which is characterized in that specifically includes the following steps:
S1, keto reductase preparation: novel keto reductase described in claim 1 is prepared；
S2, substrate preparation: 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester is added to the appearance containing phosphate buffer
It is heated in device, heating temperature is set as 45-85 DEG C, and heating time is set as 20- 25min, and mixed liquor is made；
S3, mixed liquor centrifugation: novel keto reductase obtained in the mixed liquor and S1 in container is added to centrifugation and is set
It being centrifuged in standby, centrifugal rotational speed is set as 8000-12000RPM, and centrifugation time is set as 3-5min,
S4, semi-finished product preparation: the mixed liquor after centrifugation is stood into 40-60min and is reacted, in the work of novel keto reductase
Under, semi-finished product optics hand is made by asymmetric reduction in the 5R-6- cyano -5- hydroxyl -3- oxo hecanoic acid t-butyl ester in mixed liquor
Property alcohol mixed liquor；
S5, finished product preparation: double completed optical chiral alcohol mixed liquor carries out lyophilization, then by the product after lyophilization into
The processing of row column chromatographic purifying, obtains corresponding chiral alcohol finished product.
7. the novel keto reductase of one kind according to claim 6 is used for chiral alcohol synthetic method, it is characterised in that:
The phosphate buffer pH value is set as 6.0-8.0.
8. the novel keto reductase of one kind according to claim 7 is used for chiral alcohol synthetic method, it is characterised in that:
The phosphate buffer pH value is set as 7.0.
9. the novel keto reductase of one kind according to claim 6 is used for chiral alcohol synthetic method, it is characterised in that:
Also contain NADP+, glucose dehydrogenase and glucose in the container of the S2.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671010A (en) * | 2016-03-04 | 2016-06-15 | 浙江工业大学 | Aldehyde ketone reductase mutant, gene, engineering bacterium and application of mutant |
| CN106459952A (en) * | 2014-05-23 | 2017-02-22 | 国立大学法人大阪大学 | Mutein, method for producing said mutein, and gene encoding said mutein |
| CN108048417A (en) * | 2018-01-22 | 2018-05-18 | 吉林凯莱英医药化学有限公司 | Ketoreductase mutant and its application |
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- 2019-04-10 CN CN201910284143.2A patent/CN109913429A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106459952A (en) * | 2014-05-23 | 2017-02-22 | 国立大学法人大阪大学 | Mutein, method for producing said mutein, and gene encoding said mutein |
| CN105671010A (en) * | 2016-03-04 | 2016-06-15 | 浙江工业大学 | Aldehyde ketone reductase mutant, gene, engineering bacterium and application of mutant |
| CN108048417A (en) * | 2018-01-22 | 2018-05-18 | 吉林凯莱英医药化学有限公司 | Ketoreductase mutant and its application |
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Application publication date: 20190621 |