CN103289922B - Yokenella and the application in preparation α, β-unsaturated enol and aromatic alcohol thereof - Google Patents

Abstract

The invention provides a strain new strains---Yokenella (Yokenella sp.) WZY002, and prepare α at regioselective reduction α, β-unsaturated olefine aldehydr (ketone), the application in aromatic alcohol is prepared in β-unsaturated enol and reduction aromatic aldehyde (ketone).This bacterial strain is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number: CCTCC No:M2013099, preservation date: on March 22nd, 2013.Beneficial effect of the present invention is mainly reflected in: provide the new strains that a strain has high regioselectivity, highly-solid selectively and high enzymatic activity, this bacterial strain catalysis α, the regioselective reduction of β-unsaturated olefine aldehydr (ketone) can obtain multiple α, β-unsaturated enol, this bacterial strain also the reduction of catalysis aromatic aldehyde (ketone) can obtain aromatic alcohol simultaneously.Bacterial strain of the present invention as biological catalyst, regioselectivity and stereoselectivity high, catalytic activity is strong, and the reaction of institute's catalysis does not need to add coenzyme, and reaction conditions is gentle, and suitability for industrialized production has higher using value.

Description

Yorkella sp. and its application in the preparation of α, β-unsaturated enols and aromatic alcohols

(1) Technical field

The present invention relates to a new bacterial strain——Yokenella sp. WZY002, and its regioselective reduction of α, β-unsaturated alkenal or α, β-unsaturated enone to prepare α, β-unsaturated Applications in enols, asymmetric reduction of aromatic ketones to prepare chiral aromatic alcohols, and reduction of aromatic aldehydes to prepare aromatic alcohols.

(2) Background technology

α,β-unsaturated enols are very important intermediates in organic synthesis (including drug synthesis), such as cinnamyl alcohol, limonyl alcohol, crotyl alcohol, etc., which are widely used in the production of spices, drugs and other fine chemicals, and are often used as It is used as food aroma additive, aroma blending agent and pharmaceutical intermediate, etc., with high economic value.

The selective hydrogenation of α,β-unsaturated alkenals (ketones) is a key step in the synthesis of fragrances and pharmaceutical intermediates. Since the activation energy of the C=C bond is lower than that of the C=O bond in thermodynamics, and the C=C bond is more active than the C=O bond in kinetics, and under the action of general chemical catalysts, α, β-unsaturated enaldehyde The main products of (ketones) are mostly saturated aldehydes (ketones), and the yield of more valuable products α, β-unsaturated enols is low. Different from chemical catalysts, biocatalysts have more excellent regioselectivity, and can only selectively hydrogenate the C=O bond of α, β-unsaturated alkenes (ketones) to obtain corresponding α, β-unsaturated Saturated enol. The advantages of biocatalysts such as mild reaction conditions, high stereoselectivity, environmental friendliness, easy separation and recovery, and low production cost also make up for the shortcomings of chemical catalysts. At present, the preparation of α,β-unsaturated enols by biocatalysis is mainly achieved by selective reduction of α,β-unsaturated alkenes (ketones). However, there is no report on the preparation of α,β-unsaturated enols catalyzed by Yorkella microorganisms.

(3) Contents of the invention

The present invention provides a bacterial strain with high regioselectivity, high stereoselectivity and high vigor - Yorkella WZY002 and its application in biological preparation of α, β-unsaturated enol and aromatic alcohol. The strain can reduce α, β-unsaturated enaldehyde (ketone) to obtain α, β-unsaturated enol, can also obtain chiral aromatic alcohol through asymmetric reduction of aromatic ketone, and can also reduce aromatic aldehyde to obtain aromatic alcohol. The reaction has strong specificity, good selectivity and high activity, and the reaction does not require additional coenzymes.

The technical scheme adopted in the present invention is:

Yorkella sp. WZY002, preserved in China Center for Type Culture Collection, address: China, Wuhan, Wuhan University, 430072, preservation number: CCTCC No: M2013099, preservation date: March 22, 2013.

The 16S rDNA sequence of Yorkella WZY002 of the present invention is as follows:

ACATGCAAGTCGAACGGTAGCACAGAGGAGCTTGCTCCTTGGGTGACGAGTGG CGGACGGGTGAGTAATGTCTGGGAAACTGCCCGATGGAGGGGGATAACTACTG GAAACGGTAGCTAATACCGCATAATGTCGCAAGACCAAAGAGGGGGACCTTCG GGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACG GCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGG AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCAC AATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGT TGTAAAGTACTTTCAGCGGGGAGGAAGGCGATACGGTTAATAACCGTGTCGATT GACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAA TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGC GGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCGA AACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTG AAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAA AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG GTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGC TTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTA  AAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCACGGAATTTAGCAGA GATGCTTTAGTGCCTTCGG GAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAG CTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTT GTTGCCAGCGGTTCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGA GGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACAC GTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT CATAAAGTATGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCG GAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTT GTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTT AACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGA 

The bacterial strain screening process of the present invention is as follows:

Sample collection: The soil samples used for screening were collected from Shandong, Hubei, Inner Mongolia and other places. Yorkia WZY002 was screened from vegetable garden soil in Jianli County, Jingzhou City, Hubei Province, China.

Plate initial screening: Weigh 0.5g soil sample and add 1mL sterile water, shake well, let it stand, dilute the supernatant 1000 times with sterile water, then take 100μL of it and apply it on a solid plate, place it for 3-5 minutes, absorb 100 μL of 2-butenol (crotyl alcohol) was applied to the plate again, and incubated overnight at 30°C. Pick the grown single colonies and transfer them to the slant of solid test tubes, and culture them overnight at 30°C. Components of solid medium: 1% peptone, 0.5% yeast extract, 0.5% NaCl, 2% agar, pH 7.0-7.2, sterilized at 121°C for 20 minutes. The composition of the medium in the present invention is expressed in mass volume percentage (W/V), for example, if the concentration of a certain component is 1%, it means that 100mL of the medium contains 1g of the component.

The slant strain was inoculated into the fermentation medium, and its components were as follows: 1% peptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0-7.2, and sterilized at 121°C for 20 minutes. Cultivate for 24-48 hours on a shaker at 30°C with a rotating speed of 200 rpm, and after centrifugation, the bacteria are suspended in the buffer system.

The wet cells obtained according to the above method are suspended in the buffer system, and α, β-unsaturated aldehydes (ketones) or aromatic aldehydes (ketones) are added for reaction. After 1 to 72 hours of reaction, chiral gas chromatography or gas phase The substrates and their conversion products were analyzed by mass spectrometry.

The present invention also relates to the application of the Yorkella WZY002 in the preparation of α, β-unsaturated enols from regioselectively reduced α, β-unsaturated enaldehydes (ketones).

Preferably, the α,β-unsaturated enal (ketone) is one of the following: 2-butenal, 2-hexenal, 2-methyl-2-pentenal, 2-octenal , 2-decenal, citral, cinnamaldehyde, mesityl oxide, ionone, 3-octen-2-one and 4-methoxy-3-buten-2-one.

The present invention also relates to the application of the Yorkella WZY002 in the preparation of chiral aromatic alcohols through the asymmetric reduction of aromatic ketones.

Preferably, the aromatic ketone is one of the following: acetophenone, 2-bromoacetophenone, 4-bromoacetophenone, 2-hydroxyacetophenone, benzylideneacetone.

The present invention also relates to the application of the Yorkella WZY002 in reducing aromatic aldehydes to prepare aromatic alcohols.

Preferably, the aromatic aldehyde is benzaldehyde or vanillin.

The regioselective reduction of α,β-unsaturated alkenals (ketones) and the reduction of aromatic aldehydes (ketones) have catalytic activity at pH 6-9.5, preferably at pH 6.0-8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or pH8.0~9.0 Tris-HCl buffer.

The regioselective reduction of α,β-unsaturated enaldehydes (ketones) and the reduction of aromatic aldehydes (ketones) are carried out in the presence of glucose, ethanol, glycerol or isopropanol.

Specifically, the reaction is as follows: using α, β-unsaturated alkenal (ketone) or aromatic aldehyde (ketone) as the substrate, using the wet cell of Yorkia WZY002 as the catalyst, in a buffer solution with a pH of 6-9 , the substrate concentration is 5-200mM, react at 4-60°C and shaker speed 0-200rpm for 2-72 hours, after the reaction is completed, it is extracted with ethyl acetate, centrifuged to obtain the organic phase, dried over anhydrous sodium sulfate, and The substrates and their conversion products in the organic phase were analyzed by gas chromatography-mass spectrometry and chiral gas chromatography. The added amount of the wet bacteria relative to the buffer solution is 45-450 g/L.

Specifically, the wet bacterium of Yorkia WZY002 can be obtained as follows: fermentation medium composition: peptone 10g/L, yeast extract 5g/L, NaCl 5g/L, solvent is water, pH7.0～7.2, Inoculate Yorkella WZY002 in the fermentation medium, culture at 30°C and shaker speed 200rpm for 12-48 hours, centrifuge the obtained fermentation broth at 10000rpm for 10min, discard the supernatant, and use pH6-9 buffer for the bacteria Wash once with liquid, and the obtained wet cells are biocatalysts.

The beneficial effects of the present invention are mainly reflected in: providing a strain with high regioselectivity, high stereoselectivity and high activity - Yorkella WZY002, through which the α, β-unsaturated alkenal (ketone) is catalyzed Reduction with aromatic aldehydes (ketones) can give α, β-unsaturated enols and aromatic alcohols. As a biocatalyst, the bacterial strain has high regioselectivity and stereoselectivity, strong catalytic activity, the catalyzed reaction does not need to add coenzymes, and the reaction conditions are mild, so it has high application value in industrial production.

(4) Description of drawings

Figure 1 is a phylogenetic tree of Yorkia WZY002.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1:

Using the Blast database to compare the 16S rDNA of Yorkella WZY002 (CCTCC No: M2013099), find out several strains of the same and adjacent genera and species, and use Clastal W software to analyze and draw the phylogenetic tree of Yorkia WZY002, As shown in Figure 1. Yorkella WZY002 is the closest relative to Yorkella regensburgi strain CIP105435.

Example 2:

The composition of the fermentation medium of Yorkella WZY002 (CCTCC No: M2013099): peptone 10g/L, yeast extract 5g/L, NaCl 5g/L, solvent is water, pH7.0-7.2, sterilized at 121℃ for 20min.

Yorkella WZY002 was inoculated in 150 ml of fermentation medium, and cultured at 30° C. and a shaker speed of 200 rpm for 12 to 48 hours. After the fermentation broth was centrifuged at 10000rpm for 10min, the supernatant was discarded, and the cells were washed once with a reaction buffer, and the obtained wet cells were biocatalysts.

Example 3:

Yorkella WZY002 regioselective reduction of 2-butenal (crotonaldehyde): In a 2mL reaction system, contain 100mM phosphate buffer at pH7.2, 0.25g Yorkella wet cells, 50mM crotonaldehyde, 500 mM of various cosubstrates. The control was no addition of co-substrate. React at 30 °C and 200 rpm for 12 hours.

After the reaction was completed, 2 mL of ethyl acetate was added to the reaction liquid, and placed in a shaker for extraction at 30° C. and 200 rpm for 1 hour. Centrifuge the extract at 10,000rpm for 10min, take 400-1000μL of the organic phase, add excess anhydrous Na ₂ SO ₄ to dry, and perform gas chromatography analysis on the substrate and its conversion product. Isopropanol, glycerol, ethanol and Glucose can increase the yield by 2 to 22 times. Among them, the effect of glucose is the most significant, adding 10 times the substrate concentration of glucose, the reaction product yield reaches 85.6%, and the conversion rate reaches 98%.

Example 4:

Yorkella WZY002 regioselective reduction of crotonaldehyde: In a 2mL reaction system, contain 100mM pH7.2 phosphate buffer, 0.25g Yorkella wet bacteria, 50mM 2-butenal (crotonaldehyde) , the glucose concentration was 2 mol/L, and the reaction was carried out at 30° C. and 200 rpm for 12 hours.

After the reaction was completed, 2 mL of ethyl acetate was added to the reaction liquid, and placed in a shaker for extraction at 30° C. and 200 rpm for 1 hour. Centrifuge the extract at 10000rpm for 10min, take 400-1000μL of organic phase, add excess anhydrous Na ₂ SO ₄ to dry, conduct gas chromatographic analysis on the substrate and its conversion product, the product yield is significantly improved when glucose is added than when glucose is not added , and the effect is best when the amount of glucose added is 10 times that of the substrate, the product yield reaches 83%, and the conversion rate also increases to 98%.

Example 5:

In the 2mL reaction system, respectively containing 100mM phosphate buffer solution with pH7.2, 0.25g Yorkella wet bacteria, 50mM crotonaldehyde, and 250mM glucose, the reaction was allowed to stand at different temperatures for 12 hours.

After the reaction was completed, 2 mL of ethyl acetate was added to the reaction liquid, and placed in a shaker for extraction at 30° C. and 200 rpm for 1 hour. Centrifuge the extract at 10000rpm for 10min, take 400-1000μL of the organic phase, add excess anhydrous Na ₂ SO ₄ to dry, and conduct gas chromatography analysis on the substrate and its transformation product. Catalytic activity, but the activity is the highest at 30°C, the product yield reaches 83%, and the conversion rate also increases to 98%.

Embodiment 6:

The 2mL reaction system contains 200mM buffer solution of different pH (phosphate buffer, pH6.0~8.9; glycine-NaOH buffer, pH8.9~9.6), 0.25g Yorkella wet bacteria, 50mM croton Aldehydes, 250 mM glucose. React at 30°C and 200 rpm for 4 hours.

After the reaction was completed, 2 mL of ethyl acetate was added to the reaction liquid, and placed in a shaker for extraction at 30° C. and 200 rpm for 1 hour. Centrifuge the extract at 10,000rpm for 10min, take 400-1000μL of the organic phase, add excess anhydrous Na ₂ SO ₄ to dry, and conduct gas chromatographic analysis on the substrate and its conversion products. Yorkia WZY002 can catalyze crotonaldehyde at pH 6-9.5 Vitality, the activity is the highest in the phosphate buffer solution of pH 8.0, the product yield reaches 83%, and the conversion rate also increases to 98%.

Embodiment 7:

Yorkella WZY002 regioselective reduction of α, β-unsaturated alkenals (ketones): in a 2mL reaction system, containing 200mM pH8.0 phosphate buffer, 0.25g Yorkella wet cells, 50mM each One kind of α,β-unsaturated alkenals or 10mM of various enones, 250mM glucose, reacted at 30°C and 200rpm.

After the reaction was completed, 2 mL of ethyl acetate was added to the reaction liquid, and placed in a shaker for extraction at 30° C. and 200 rpm for 1 to 2 hours. The extract was centrifuged at 10,000 rpm for 10 min, and 400-1,000 μL of the organic phase was taken, dried by adding excess anhydrous Na ₂ SO ₄ , and the substrate and its conversion product were analyzed by gas chromatography and mass spectrometry. The results are shown in Table 1.

Table 1: Catalytic reduction of α,β-unsaturated alkenals (ketones) by Yorkia sp. WZY002

a, Selectivity refers to the percentage of the target product α, β-unsaturated alcohol in the total reduction products after the reaction.

As can be seen from Table 1, Yorkella WZY002 showed high catalytic activity to most enals, and the corresponding regioselectivity was also very high. When the substrates were crotonaldehyde, 2-hexenal, 2- When methyl-2-pentenal is used, the product conversion rate is above 94%, and the regioselectivity based on α,β-unsaturated enol formation is also as high as 99%. Compared with enals, Yorkella WZY002 has lower catalytic activity to all enones, but still has excellent regioselectivity.

Embodiment 8:

Yorkella WZY002 catalytic reduction of aromatic aldehydes (ketones): in a 2mL reaction system, respectively containing 200mM pH8.0 phosphate buffer, 0.25g York Aromatic ketones, 250 mM glucose, reacted at 30°C and 200 rpm.

After the reaction was completed, 2 mL of ethyl acetate was added to the reaction liquid, and placed in a shaker for extraction at 30° C. and 200 rpm for 1 hour. The extract was centrifuged at 10,000 rpm for 10 min, and 400-1,000 μL of the organic phase was taken and dried by adding excess anhydrous Na ₂ SO ₄ . The substrate and its conversion product were analyzed by gas chromatography and mass spectrometry. The results are shown in Table 2.

Table 2: Catalytic reduction of aromatic aldehydes (ketones) by Yorkia sp. WZY002

a, Enantioselectivity for (S)-form.

It can be seen from Table 2 that Yorkella WZY002 has high catalytic activity for both benzaldehyde and vanillin, especially the catalytic conversion rate of benzaldehyde reaches 99.6%. Compared with aromatic aldehydes, Yorkella WZY002 has lower catalytic activity for aromatic ketones, but the products have high enantioselectivity. When the substrate is 2-hydroxyacetophenone, the product e.e. value reaches 99% .

Claims (10)

1. a Yokenella WZY002 is at regioselective reduction α, β-unsaturated olefine aldehydr or α, β-Unsaturated Alkenone prepare α, the application in β-unsaturated enol, described Yokenella (Yokenella sp.) WZY002, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number: CCTCC No:M 2013099, preservation date: on March 22nd, 2013.

2. apply as claimed in claim 1, it is characterized in that described α, β-unsaturated olefine aldehydr or α, β-Unsaturated Alkenone are one of following: crotonic aldehyde, 2-hexenoic aldehyde, 2-methyl-2-pentenal serving, 2-octenal, 2-decenal, citral, phenylacrolein, mesityl oxide, jononeionone, 3-octene-2-ketone and 4-methoxyl group-3-butene-2-one.

3. apply as claimed in claim 1, it is characterized in that described application method is as follows: with α, β-unsaturated olefine aldehydr or α, β-Unsaturated Alkenone is substrate, with Yokenella WZY002 wet thallus for biological catalyst, in the damping fluid of pH6 ~ 9, concentration of substrate is 5 ~ 200mM, react 2 ~ 72 hours under 4 ~ 60 DEG C and shaking speed 0 ~ 200rpm, through extraction into ethyl acetate after reaction terminates, the centrifugal organic phase that obtains is reaction product.

4. apply as claimed in claim 3, its specific features is that described Yokenella WZY002 wet thallus obtains as follows: fermention medium forms: peptone 10g/L, yeast extract 5g/L, NaCl 5g/L, solvent is water, pH7.0 ~ 7.2, Yokenella WZY002 is inoculated in fermention medium, in 30 DEG C, cultivate 12 ~ 48 hours under the condition of shaking speed 200rpm, gained fermented liquid is at the centrifugal 10min of 10000rpm, abandon supernatant liquor, once, gained wet thallus is biological catalyst to the buffer solution of thalline pH6 ~ 9.

5. a Yokenella WZY002 prepares the application in chiral aromatic alcohols at asymmetric reduction aromatic ketone, described Yokenella WZY002, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number: CCTCC No:M 2013099, preservation date: on March 22nd, 2013.

6. apply as claimed in claim 5, it is characterized in that described aromatic ketone is one of following: methyl phenyl ketone, 2-bromoacetophenone, 4-bromoacetophenone, 2-hydroxy acetophenone, benzylideneacetone.

7. apply as claimed in claim 5, it is characterized in that described application method is as follows: take aromatic ketone as substrate, with Yokenella WZY002 wet thallus for biological catalyst, in the damping fluid of pH6 ~ 9, concentration of substrate is 5 ~ 200mM, react 2 ~ 72 hours under 4 ~ 60 DEG C and shaking speed 0 ~ 200rpm, through extraction into ethyl acetate after reaction terminates, the centrifugal organic phase that obtains is reaction product.

8. a Yokenella WZY002 prepares the application in aromatic alcohol at reduction aromatic aldehyde, described Yokenella WZY002, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number: CCTCC No:M 2013099, preservation date: on March 22nd, 2013.

9. apply as claimed in claim 8, it is characterized in that described aromatic aldehyde is phenyl aldehyde or Vanillin.

10. apply as claimed in claim 8, it is characterized in that described application method is as follows: take aromatic aldehyde as substrate, with Yokenella WZY002 wet thallus for biological catalyst, in the damping fluid of pH6 ~ 9, concentration of substrate is 5 ~ 200mM, react 2 ~ 72 hours under 4 ~ 60 DEG C and shaking speed 0 ~ 200rpm, through extraction into ethyl acetate after reaction terminates, the centrifugal organic phase that obtains is reaction product.