CN101418269B - Streptomycete and application thereof in reduction of aromatic ketone - Google Patents
Streptomycete and application thereof in reduction of aromatic ketone Download PDFInfo
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- CN101418269B CN101418269B CN2008100135126A CN200810013512A CN101418269B CN 101418269 B CN101418269 B CN 101418269B CN 2008100135126 A CN2008100135126 A CN 2008100135126A CN 200810013512 A CN200810013512 A CN 200810013512A CN 101418269 B CN101418269 B CN 101418269B
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- streptomycete
- fluorenone
- aromatic ketone
- reduction
- streptomyces
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- 241000187180 Streptomyces sp. Species 0.000 claims abstract description 8
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the field of biocatalysis, and in particular relates to a streptomyces (Streptomyces sp.DUT003 with the preserving number of CGMCC2518) and application thereof in the reduction of rigid macrocyclic aromatic ketone such as substituted fluorenone. With the streptomyces, aromatic ketone, in particular the rigid macrocyclic aromatic ketone such as the substituted fluorenone can be reduced with high efficiency; products are aromatic alcohol and the aromatic alcohol with optical activity, the problems of synthesizing the aromatic alcohol and the aromatic alcohol with optical activity chemically and industrially are solved, the conversion rate can reach between 89 and 98 percent, the products have high stereoselectivity, and the ee value is between 83 and 100 percent. The streptomyces has the advantages of easy culture, mild reduction and reaction condition, simple operation steps, less environmental pollution, and good prospect of industrial application and development.
Description
Technical field
The present invention relates to biocatalysis field, particularly relate to a kind of streptomycete and encircle the rigidity aromatic ketone greatly, as replacing the application in the Fluorenone in reduction.
Technical background
Aromatic alcohol or optical activity aromatic alcohol are the important structure unit of many pharmaceutical intermediates, catalyzer, analytical reagent foodstuff additive and advanced light-guide material.At present, chemically mainly be that reduction by aromatic ketone obtains.But there are problems such as complex steps, productive rate is low, cost is high, environmental pollution is serious in its synthetic method chemically.It is high sterically hindered that reason is that the rigid structure of aromatic ketone produces, and stoped the approaching of catalyzer.Especially reduction or the Stereoselective reduction for some rigid macrocyclic aromatic ketones is still a challenging problem as replacing Fluorenone in organic synthesis.
For example treat antimalarial drug--in the building-up process of benzfluorenol, though synthetic route is through improving (clock scape magnitude, Chinese patent: 1999,1029680), but from 2,7-dichloro fluorenes-4-chloracetyl fluorenes (IV) needs through two steps in the reduction process of alcohol, wherein explosive catalyzer KBH
4Hydrogenating reduction, reaction conditions acutely are difficult to control, and serious three wastes and productive rate are low.For another example, in the building-up process of important organic synthesis intermediate--fluorenol, need to obtain fluorenol, severe reaction conditions, product separation difficulty (Chen Zhongxiu, chemical reagent, 2003,25 (3), 177～178) by the alkaline boiled condition hydrolyzable bromine fluorenes that gets off.
The optical activity aromatic alcohol is synthetic just difficult more for another example.Though can obtain the chirality aromatic alcohol by methods such as asymmetric synthesis or reduction back chiral separation, these two kinds of method stepss are loaded down with trivial details, split apparatus expensive, realize the large-scale industrial production difficulty.For example in the liquid crystal material preparation process, remove and adopt explosive catalyst n aBH
4Behind the reduction intermediate product aromatic ketone, the method that need split with half preparative high-performance liquid chromatographic also, obtain containing the midbody compound of optical activity aromatic alcohol structure, still face problems such as productive rate and stereoselectivity are low (Robert P.Lemieux etc., J.AM.CHEM.SOC.2004 (126), 11611167).
In view of the importance of aromatic alcohol compound and the difficulty that chemical synthesis process faced, adopting biocatalysis technology to prepare aromatic alcohol compound should be an important channel that addresses the above problem.Biological catalyst is biological enzyme or biological viable cell, chemistry, zone and stereoselectivity with height, and can under the condition of gentleness, carry out, as room temperature, neutrality or near neutral pH, avoid or reduce decomposition, isomerization, racemization and the rearrangement reaction of product, and multiple advantage (John M.Woodley, Trends in Biotechnology such as productive rate height, energy consumption are low, environmental friendliness, 2008 (26), 321-327; Permitted to build and etc., biological processing, 2008 (6), 1-9).
Summary of the invention
The objective of the invention is at aromatic ketone especially rigid macrocyclic aromatic ketone difficult reductive characteristics and chemical process condition harshness, be difficult to control, productive rate and stereoselectivity low, have a problem such as environmental pollution, by scientific approach screening, the efficient reduction of aromatic ketone bacterial strain of domestication, to solve the difficult problem of chemosynthesis and industrial preparation aromatic alcohol and optical activity aromatic alcohol, solve problems such as environmental pollution simultaneously.
Technical scheme of the present invention is: the concrete screening of streptomycete of the present invention domestication step is as follows:
The soil sample that takes a morsel from suburb of Shanghai soil joins and carries out shaking table in the big screening culture medium of liquid and cultivate 28 ℃ of temperature, rotating speed 170rpm, the big screening culture medium prescription of liquid is: Zulkovsky starch 10g, glucose 20g, analysis for soybean powder 25g, yeast powder 4g, extractum carnis 1g, NaCl2g, K
2HPO
40.05g, water 1L, pH7.0-7.2; When substratum becomes muddy, the branch bottle of transferring dilution is cultivated, and the applying solid screening is dull and stereotyped carrying out, solid plate is put into illumination box cultivate, 30 ℃, cultivated 4-5 days, choose single bacterium colony, the dull and stereotyped prescription of solid screening is: Zulkovsky starch 5g, glucose 5g, peptone 1g, yeast powder 1g, extractum carnis 1g, agar 15g, water 1L, pH7.0-7.2; The single thalline of each that will select carries out the liquid nutrient medium enlarged culturing then, and centrifugal (5000rpm 10min) collects somatic cells; Add aromatic ketone-replacement Fluorenone respectively, screening obtains the bacterial strain that a strain has the aromatic ketone reducing activity, again this bacterial strain is tamed, add different concns (0.05mol, 0.1mol, 0.2mol, 0.4mol, 0.8mol) the cultivation of going down to posterity of replacement Fluorenone, make it can tolerate the replacement Fluorenone substrate of high density, obtain screening the bacterial strain of domestication at last.
Bacterial strain through the screening domestication has following microbial characteristic:
The bacterium colony cocoon is white or greyish white on the big screening culture medium of actinomycetes, have an even surface, the mycelia prosperity, multi-branched, do not have every, be shaft-like.Can be on most of substratum well-grown, aerial hyphae and substrate mycelium are all based on white or lime.Gelatine liquefication, milk are solidified and starch hydrolysis, tyrosine hydrolysis are positive, and do not grow on the Mierocrystalline cellulose, do not produce hydrogen sulfide, can utilize the most carbon source of being tried, and full cell amino acid analysis shows and contain aspartic acid, and full cell glycan analysis shows and contains semi-lactosi.Reach phylogenetic analysis and morphological feature according to document " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) based on 16S rDNA (the GeneBank accession number is EU216596), a novel species that can tentatively be decided to be streptomycete is therefore with its called after Streptomycessp.DUT003.In on May 23rd, 2008 in the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of State Intellectual Property Office, depositary institution is called for short CGMCC, deposit number is 2518, classification called after Streptomyces sp.DUT003, Datun Road, Chaoyang District, Beijing City, depositary institution address, Institute of Microorganism, Academia Sinica, postcode 100101.
The application concrete operations of streptomycete of the present invention in reduction of aromatic ketone-replacement Fluorenone are:
With weight in wet base is that the streptomycete of 10-100g/L is suspended among the 20mM Tris-HCl or 20mM HEPES damping fluid of pH7.0-8.0, adding concentration is aromatic ketone compound-replacement Fluorenone (fluorine Fluorenone, chlorine Fluorenone, bromine Fluorenone, iodine Fluorenone or methyl Fluorenone) of 0.1-1mmol/L, place 30 ℃, the shaking table of rotating speed 170rpm then, reaction 2-12h, obtain corresponding aromatic alcohol or optical activity aromatic alcohol, reaction conversion ratio is 89-98%, and stereoselectivity is 83-100%;
Or be that the streptomycete of 10-100g/L is suspended in pH7.0-8.0 with weight in wet base, contain among the 20mMTris-HCl or 20mM HEPES damping fluid of volume ratio 5% solubility promoter, adding concentration is aromatic ketone compound-replacement Fluorenone (fluorine Fluorenone of 0.1-10mmol/L, the chlorine Fluorenone, the bromine Fluorenone, iodine Fluorenone or methyl Fluorenone), place 30 ℃ then, in the shaking table of rotating speed 170rpm, reaction 2-12h, obtain corresponding aromatic alcohol or optical activity aromatic alcohol, reaction conversion ratio is 89-98%, stereoselectivity is 83-100%, the solubility promoter of selecting for use is DMSO, methyl alcohol, ethanol, acetonitrile, DMF, SDS, Triton X-100 or Tween-20.
The reaction expression of using streptomycete reduction substituted fluorene ketogenesis corresponding aromatic alcohol or optical activity aromatic alcohol is:
The invention has the beneficial effects as follows: streptomycete provided by the invention is easy to cultivate, efficient reduction of aromatic ketone, especially big ring rigidity aromatic ketone is as replacing Fluorenone, and product is aromatic alcohol and optical activity aromatic alcohol, has solved the difficult problem of chemosynthesis and the pure and mild optical activity aromatic alcohol of industrial synthetic aroma, and while the reaction conditions gentleness, operation steps is simple, has very high stereoselectivity, does not have other by product and produces, environmental pollution is little, has good industrial application DEVELOPMENT PROSPECT.
Description of drawings
Accompanying drawing 1 is microscope and the electromicroscopic photograph of streptomycete of the present invention.
Embodiment
Embodiment 1
The step of screening, domestication streptomycete is as follows:
From suburb of Shanghai soil, take a sample, screen, tame cultivation as the bacterium source.With a small amount of soil sample, join and carry out shaking table cultivation, 28 ℃ of temperature, rotating speed 170rpm in the big screening culture medium of liquid; The big screening culture medium prescription of liquid is: Zulkovsky starch 10g, glucose 20g, analysis for soybean powder 25g, yeast powder 4g, extractum carnis 1g, NaCl2g, K
2HPO
40.05g, water 1L, pH7.0-7.2.When substratum becomes muddy, the branch bottle of transferring dilution is cultivated, carrying out the applying solid screening dull and stereotyped, solid plate is put into illumination box cultivates, 30 ℃, cultivated 4-5 days, choose single bacterium colony.The dull and stereotyped prescription of solid screening is: Zulkovsky starch 5g, glucose 5g, peptone 1g, yeast powder 1g, extractum carnis 1g, agar 15g, water 1L, pH7.0-7.2.The single thalline of each that will select carries out the liquid nutrient medium enlarged culturing again, and centrifugal (5000rpm 10min) collects somatic cells, add aromatic ketone-replacement Fluorenone respectively, screening obtains the bacterial strain that a strain has the aromatic ketone reducing activity, this bacterial strain is tamed again, and adds the replacement Fluorenone (0.05mol of different concns, 0.1mol, 0.2mol, 0.4mol, 0.8mol) cultivation of going down to posterity, be its replacement Fluorenone substrate that can tolerate high density, obtain screening the bacterial strain of domestication at last.And this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 23rd, 2008, and depositary institution is called for short CGMCC, and deposit number is 2518.
Embodiment 2
The streptomycete bacterial strain optimum growth temperature is 20-37 ℃, and the suitableeest growth pH is 6-8.The bacterium colony cocoon is white or greyish white on big screening solid medium, have an even surface, the mycelia prosperity, multi-branched, do not have every, be shaft-like.Can be on most of substratum well-grown, aerial hyphae and substrate mycelium are all based on white or lime.
The amplification of the 16S rDNA of streptomycete:
By the 16S rDNA of this bacterial strain that increases, having obtained length is the 16S rDNA sequence of 1424bp.The PCR primer adopts 16SrDNA amplification universal primer, 27f (5 '-GAGTTTGATC (AC) TGGCTCAG-3 ') and 1492r (5 '-TACGG (CT) TACCTTGTTACGACTT-3 ').Carry out amplified reaction with the PCR instrument.Reaction system is: TaKaRa PCR10 * Buffer, 5 μ l; MgCl
2(25mM), 4 μ l; DNTP Mixture (each 2.5mM), 4 μ l; Primer 8f (20 μ m), 0.25 μ l; Primer 1492r (20 μ m), 0.25 μ l; Template DNA, 1 μ l; TaKaRa Taq (5U/ μ l), 0.25 μ l; Ultrapure water, Up to50 μ l.The PCR reaction conditions is: 94 ℃ 5 minutes, 30 circulations afterwards comprise: 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 2 minutes, extended 5 minutes down at 72 ℃ at last.1% agarose gel electrophoresis, EB dyeing back ultraviolet detection.Carry out sequence alignment by Blast program among the NCBI, show that this bacterial strain and Streptomyces recifensis homology reach 99%.
The cultivation of streptomycete is as follows with the collection step:
The solid culture based formulas is: Zulkovsky starch 5g, glucose 5g, peptone 1g, yeast powder 1g, extractum carnis 1g, agar 15g, water 1L, pH7.0-7.2.The liquid culture based formulas is: Zulkovsky starch 10g, glucose 20g, analysis for soybean powder 25g, yeast powder 4g, extractum carnis 1g, NaCl2g, K
2HPO
40.05g, water 1L, pH7.0-7.2.Based on the volume of liquid nutrient medium, colony inoculation is in liquid nutrient medium in the picking solid medium, and inoculum size is 2%, and culture temperature is 30 ℃, and shaking speed is 200rpm, and incubation time is 4 days.It is centrifugal that (5000rpm 10min) collects thalline, and-20 ℃ of preservations are standby.
The physio-biochemical characteristics of streptomycete see Table 1.
The cultural characters of streptomycete sees Table 2.
The physio-biochemical characteristics of table 1 streptomycete Streptomyces sp.DUT003
The cultural characters of table 2 streptomycete Streptomyces sp.DUT003
Embodiment 3
The applying step of streptomycete in the biocatalysis reduction of aromatic ketone is as follows:
(1) streptomycete being placed pH is the Tris-HCl damping fluid of 7.0-8.0, making its cell concn is 50g (weight in wet base)/L, add aromatic ketone 2-and replace (fluorine, chlorine, bromine, iodine, methyl) Fluorenone, concentration is 0.5mmol/l, and placing rotating speed is that 200rpm, temperature are that 30 ℃ shaking table reacts 12h;
(2) process of usefulness HPLC monitoring reaction;
(3) after question response is finished, add equal volume of ethyl acetate and go out organic compound, cross the anhydrous magnesium sulfate post and remove moisture content and partial impurities, 40 ℃ of concentrating under reduced pressure separate obtaining SOLID ORGANIC compound-aromatic alcohol replacement fluorenol.
(4) the silica gel column chromatography separation obtains pure aromatic alcohol compounds--and replace fluorenol, carry out nuclear-magnetism and mass spectrum and identify, and measure optical value and corresponding body excessive value.
Embodiment 4
Aromatic ketone compound-replacement Fluorenone that the streptomycete reduction is different, step is as follows:
(1) streptomycete being placed pH is the Tris-HCl damping fluid of 7.0-8.0, making its cell concn is 50g (weight in wet base)/L, add aromatic ketone 2-and replace (fluorine, chlorine, bromine, iodine, methyl) Fluorenone, concentration is 0.5mmol/l, adds solubility promoter＜5% (v/v) (organic solvent DMSO or methyl alcohol or ethanol or acetonitrile or DMF; Surfactant SDS or Triton X-100 or Tween-20), placing rotating speed is that 200rpm, temperature are that 30 ℃ shaking table reacts, the reaction times is 12h.
(2) process of usefulness HPLC monitoring reaction: chromatographic instrument Agilent1100, chromatographic column: Cromasil C18 (250mm * 4.6mm, 5 μ m); Moving phase: 70% methyl alcohol 50min; Flow velocity: 0.8ml/min; Detect wavelength: 254nm; Column temperature: room temperature; Sampling volume: 10 μ l.
(3) after question response is finished, add equal volume of ethyl acetate and go out organic compound, cross the anhydrous magnesium sulfate post and remove moisture content and partial impurities, 40 ℃ of concentrating under reduced pressure separate obtaining SOLID ORGANIC compound-aromatic alcohol replacement fluorenol.(data see attached list 3)
(4) the silica gel column chromatography separation obtains pure aromatic alcohol compounds--and replace fluorenol, carry out nuclear-magnetism and mass spectrum and identify, and measure optical value and corresponding body excessive value, data see Table 3.
The different Fluorenone situations that replace of table 3 streptomycete Streptomyces sp.DUT003 reduction
Claims (6)
1. streptomycete, this streptomycete are preserved on May 23rd, 2008 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number are CGMCC2518, classification called after Streptomyces sp.DUT003.
2. a kind of streptomycete according to claim 1 is characterized in that, this streptomycete was cultivated 4-5 days under on the big screening culture medium of actinomycetes 28 ℃, the bacterium colony cocoon is white or greyish white, has an even surface mycelia prosperity, multi-branched, nothing every, be shaft-like, aerial hyphae and substrate mycelium are based on white or lime.
3. use a kind of to replace the method that Fluorenone is the streptomycete reduction of aromatic ketone of substrate screening, it is characterized in that, after streptomycete Streptomyces sp.DUT003 cultivated domestication, centrifugal collection thalline, with weight in wet base is that the streptomycete Streptomyces sp.DUT003 of 10-100g/L is suspended in pH 7.0-8.0, contains in the damping fluid of volume ratio 5% solubility promoter, adding concentration is aromatic ketone compound-replacement Fluorenone of 0.1-1mmol/L, place 30 ℃, the shaking table of rotating speed 170rpm then, reaction 2-12h.
4. application according to claim 3 is a kind of to replace the method that Fluorenone is the streptomycete reduction of aromatic ketone of substrate screening, it is characterized in that described damping fluid is 20mM Tris-HCl or 20mM HEPES.
5. application according to claim 3 is a kind of to replace the method that Fluorenone is the streptomycete reduction of aromatic ketone of substrate screening, it is characterized in that described aromatic ketone is fluorine Fluorenone, chlorine Fluorenone, bromine Fluorenone, iodine Fluorenone or methyl Fluorenone.
6. application according to claim 3 is a kind of to replace the method that Fluorenone is the streptomycete reduction of aromatic ketone of substrate screening, it is characterized in that described solubility promoter is DMSO, methyl alcohol, ethanol, acetonitrile, DMF, SDS, TritonX-100 or Tween-20.
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