WO2019144920A1 - 一种促进氧化葡糖杆菌合成山梨醇脱氢酶及辅酶吡咯喹啉醌的方法 - Google Patents

一种促进氧化葡糖杆菌合成山梨醇脱氢酶及辅酶吡咯喹啉醌的方法 Download PDF

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WO2019144920A1
WO2019144920A1 PCT/CN2019/073044 CN2019073044W WO2019144920A1 WO 2019144920 A1 WO2019144920 A1 WO 2019144920A1 CN 2019073044 W CN2019073044 W CN 2019073044W WO 2019144920 A1 WO2019144920 A1 WO 2019144920A1
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fermentation
gluconobacter oxydans
sorbitol
medium
yeast extract
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French (fr)
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郑裕国
柯霞
鲁阳辉
胡忠策
吴洋
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浙江工业大学
浙江医药股份有限公司新昌制药厂
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Priority to US16/964,999 priority Critical patent/US20210047609A1/en
Priority to JP2020562819A priority patent/JP2021514200A/ja
Priority to EP19744369.0A priority patent/EP3744837A4/en
Publication of WO2019144920A1 publication Critical patent/WO2019144920A1/zh

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  • the invention relates to the field of fermentation engineering and biotransformation, in particular to the fermentation culture of Gluconobacter oxydans and the preparation of cell membrane D-sorbitol dehydrogenase (SLDH) and its coenzyme pQQ, and the biotransformation of the strain into the middle of miglitol Application in the form of 6-deoxy-6-amino(N-hydroxyethyl)- ⁇ -L-furan sorbose (6NSL).
  • SLDH cell membrane D-sorbitol dehydrogenase
  • Miglitol [(2R,3R,4R,5S)-2-hydroxymethyl-1-(2-hydroxyethyl)-3,4,5-piperidinetriol, miglitol] is a product developed by Bayer
  • the hypoglycemic agent reduces the absorption of sugar by inhibiting the ⁇ -glucosidase activity of the epithelial cells of the small intestinal mucosa, and has the advantages of safety, effectiveness, good tolerance and small side effects.
  • the current common method for synthesizing miglitol is chemical biocombination.
  • the key catalytic step of the process requires the use of D-sorbitol dehydrogenase (EC 1.1.) on the cell membrane of Gluconobacter oxydans. 99.22)
  • D-sorbitol dehydrogenase EC 1.1.
  • the obtained intermediate product is directly coupled to the chemical hydrogenation ring.
  • Synthesis of miglitol, and finally chemical hydrogenation to produce miglitol ( Figure 1).
  • the method simplifies the production process, increases the yield of the product, and reduces the synthesis cost.
  • biochemical catalytic synthesis of intermediate 6NSL is a key step in the production of miglitol, which is mainly divided into two steps: the first step, the fermentation of Gluconobacter oxydans produces high-activity sorbitol dehydrogenase; the second step is to utilize Resting cells catalyze the synthesis of the key synthetic precursor 6NSL of miglitol by N-hydroxyethylglucosamine.
  • the second step is to utilize Resting cells catalyze the synthesis of the key synthetic precursor 6NSL of miglitol by N-hydroxyethylglucosamine.
  • SLDH membrane sorbitol dehydrogenase
  • Gluconobacter oxydans has been widely used in the catalytic oxidation synthesis of other polyols such as dihydroxyacetone and vitamin C. .
  • Patent CN 101302549A discloses a process for preparing a corresponding resting cell for producing miglitol by culturing a glucoamyces oxidizing bacterium from a medium consisting of sorbitol, yeast extract, etc., but the mass percentage of the yeast extract is as high as 2.4%, fermentation The cost is significant.
  • U.S. Patent No. 4,806,650, U.S. Patent No. 5,401, 645 issued to U.S. Pat. Key parameters, such as the determination of membrane sorbitol dehydrogenase activity and its coenzyme pQQ content and monitoring.
  • the incomplete oxidation of the highly active Gluconobacter oxysporum sorbitol dehydrogenase obtained by applying this fermentation medium formulation is not limited to the synthesis of miglitol, but also includes other polyols such as dihydroxyacetone, vitamin C and the like. Catalytic synthesis of chiral compounds.
  • the present invention is based on the fermentation metabolism characteristics of Gluconobacter oxydans and membrane sorbitol dehydrogenase and The anabolic pathway of its coenzyme pQQ proposes an inexpensive and efficient fermentation medium.
  • a small amount of sodium glutamate is added to replace the yeast extract, which can further increase the fermentation volume of sorbitol dehydrogenase while reducing the fermentation cost.
  • the enzyme activity provides a good basis for the subsequent resting cell catalyzed synthesis of miglitol intermediate 6NSL.
  • the invention provides a method for promoting D-sorbitol dehydrogenase (SLDH) and pyrroloquinoline quinone (pQQ) by fermentation of Gluconobacter oxydans, and inoculating Gluconobacter oxydans into a fermentation medium.
  • SLDH D-sorbitol dehydrogenase
  • pQQ pyrroloquinoline quinone
  • Fermentation culture was carried out at 28 to 32 ° C and 150 to 180 rpm for 6 to 24 hours, and the fermentation broth was centrifuged to collect wet cells to obtain bacterial cells containing D-sorbitol dehydrogenase and pyrroloquinoline quinone; the fermentation medium
  • the composition is: D-sorbitol 50-80g / L, yeast extract 0.5 ⁇ 30g / L, KH 2 PO 4 5g / L, K 2 HPO 4 5g / L, amino acid 0.2 ⁇ 2g / L, solvent deionization Water, pH 6.5.
  • the amino acid in the fermentation medium is sodium glutamate, leucine, tyrosine or valine.
  • the fermentation medium has an amino acid concentration of 0.5 to 1 g/L, more preferably 1 g/L.
  • the amino acid is sodium glutamate
  • sodium glutamate is added to the fermentation medium during the fermentation and partially replaces the yeast extract.
  • the yeast extract has a concentration of 1 to 7 g/L, more preferably 5 g/L.
  • the fermentation medium composition is: D-sorbitol 50 g / L, yeast extract 5 g / L, KH 2 PO 4 5 g / L, K 2 HPO 4 5 g / L, sodium glutamate 1 g / L,
  • the solvent was deionized water, pH 6.5.
  • the Gluconobacter oxydans is subjected to bevel activation and seed culture, and then the seed liquid is introduced into the fermentation medium at a volume concentration of 5-15% to be fermented.
  • the bevel activation culture is to inoculate Gluconobacter oxydans to the slant culture medium, and culture in a constant temperature incubator at 28 ° C for 3 to 5 days, and the CaCO 3 in the slant culture medium becomes transparent and bright, and the naked eye can be inoculated by visual inspection.
  • Oblique sputum cells Oblique sputum cells; slant medium composition: yeast extract 2 ⁇ 10g / L, calcium carbonate 5 ⁇ 20g / L, glucose 10 ⁇ 60g / L, agar 24g / L, solvent deionized water, pH
  • the composition of the slant culture medium is preferably: yeast extract 5 g/L, calcium carbonate 5 g/L, glucose 50 g/L, agar 24 g/L, the solvent is deionized water, and the pH is natural.
  • the seed culture is to inoculate the slanted surface bacteria to the seed culture medium, and culture at 28 ° C, 225 rpm shaker for 24 to 48 h, OD 600 ⁇ 8 to reach the transfer standard, and the microscopic shape is short rod-shaped, and the coloration is deep.
  • the seed medium composition is: D-sorbitol 30-60 g/L, yeast extract 10-30 g/L, KH 2 PO 4 1-5 g/L, K 2 HPO 4 0.1 ⁇ 1g/L, the solvent is deionized water, pH pH 4.0 ⁇ 7.0, preferably seed medium composition: D-sorbitol 50g/L, yeast extract 20g/L, KH 2 PO 4 5g/L, K 2 HPO 4 5g/L, the solvent is deionized water, pH 6.5.
  • Gluconobacter oxydans is preferably deposited by the China Center for Type Culture Collection (CCTCC), Gluconobacter oxydans, and the accession number is No. M 208069, and the patent publication number is CN101591681A.
  • the relative saturated dissolved oxygen needs to be controlled at 5% to 30%, and the stirring speed is decreased, and when the sorbitol is substantially depleted in the medium, the fermentation is terminated, and the fermentation cycle is controlled to 14 ⁇ 16h; end-fermentation cell microscopic examination: the shape of the fungus is short rod-shaped, dark coloring, no bacteria.
  • the collection and washing of the cells are also carried out: the Gluconobacter oxydans obtained by fermentation is collected by a ceramic microfiltration membrane, and the cells are washed by adding ultrapure water during the filtration process.
  • the filtrate is colorless and transparent.
  • the present invention uses the fermented Gluconobacter oxydans resting cells to catalyze the production of miglitol precursor 6NSL.
  • the preferred procedure is as follows: using N-hydroxyethyl glucosamine as a substrate, adding MgSO 4 ⁇ 7H 2 O Then, adding resting cells (based on the weight of the wet cells), using deionized water as the reaction medium, adjusting the pH to 4.5-5.5, forming a reaction system, and converting the reaction at a temperature of 5 to 30 ° C and 400 to 600 rpm for 36 hours.
  • the reaction solution was taken to obtain 6NSL; in the reaction system, the final concentration of the substrate was 40-100 g/L, the final concentration of MgSO 4 ⁇ 7H 2 O was 1-5 g/L, and the final concentration of resting cells was 20-100 g/L.
  • the present invention is directed to the fermentation characteristics of Gluconobacter oxydans and the catalytic characteristics of the membrane-localized D-sorbitol de-enzyme, and the addition of a small amount of sodium glutamate in the fermentation medium and partial replacement of the components of the yeast extract significantly reduces The production cost of the enzyme produced by the fermentation of Gluconobacter oxydans. On this basis, the synthesis of coenzyme pQQ and the enzyme activity per unit volume of D-sorbitol dehydrogenase were further promoted.
  • the biotransformation of Gluconobacter oxydans obtained by this method was used to synthesize MiGlip alcohol precursor 6NSL, and the product 6NSL
  • the conversion process increased by 21-35%, shortening the biotransformation step cycle, and the resting cell transformation process was shortened from 48h to 36h.
  • the cumulative concentration of the product 6NSL was increased by 10g. /L or above.
  • the proposal of the invention effectively reduces the production cost of the biocatalytic synthesis of 6NSL in the key section of the chemical biocombination process for the production of miglitol, and effectively improves the space-time yield of the 6NSL of the miglitol precursor, and industrially produces the rice in large scale. Glycerol laid the foundation.
  • the present invention provides a fermentation medium cultured with Gluconobacter oxydans resting cells with high activity D-sorbitol dehydrogenase. Its coenzyme pQQ is not only applied to the synthesis of miglitol precursor 6NSL, but also can be applied to the selective oxidation catalytic process of other polyols.
  • Gluconobacter oxydans (CCTCC No. M 208069, Patent Publication No. CN101591681A) was inoculated to a slant medium, and cultured in a constant temperature incubator at 28 ° C for 3 to 5 days to obtain a slanted surface;
  • the slant medium consisted of: yeast Extract 5g / L, calcium carbonate 5g / L, glucose 50g / L, agar 24g / L, the solvent is deionized water, the pH is natural.
  • the slanted surface cells were inoculated into the seed culture medium, and cultured at 28 ° C, 225 rpm shaker for 48 hours to obtain a seed liquid of Gluconobacter oxydans CCTCC No. M 208069; the seed medium composition was: D-sorbitol 50 g / L, yeast The extract was 20 g/L, KH 2 PO 4 5 g/L, K 2 HPO 4 5 g/L, and the solvent was deionized water, and the pH was 6.5.
  • Example 2 Effect of amino acid species in fermentation medium on enzyme activity of Gluconobacter oxydans SLDH
  • the fermentation medium of different components was prepared, and the effects of different amino acids on the synthesis of sorbitol dehydrogenase by fermentation of Gluconobacter oxydans were investigated.
  • the fermentation medium formula of different amino acids is: D-sorbitol 50g/L, yeast extract 20g/L, KH 2 PO 4 5g/L, K 2 HPO 4 5g/L, 1g/L (sodium glutamate, Leucine, tyrosine and valine), the solvent was deionized water, pH 6.5; under the same conditions, no amino acid was added as a control.
  • the seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 was inoculated to the fermentation medium at a volume concentration of 2%, fermented at 28 ° C, 150 rpm for 24 h, and the fermentation broth was centrifuged, and the obtained cells were used for detecting sorbitol dehydrogenation.
  • the enzyme SLDH is relatively active.
  • the enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the formation of 1.0 ⁇ mol L-sorbose per minute of D-sorbitol in a phosphate buffer system at 30 ° C, pH 6.0 at 20 g/L sorbitol concentration. D-sorbitol and L-sorbose concentrations were determined by high performance liquid chromatography (HPLC).
  • SLDH enzyme activity detection procedure Take 20 mL of fermentation broth and centrifuge at 10000 rpm for 10 min, discard the supernatant, wash once, centrifuge to discard the supernatant, prepare buffer 100 mM PBS, and dissolve 20 g/L sorbitol and 5 g/L magnesium sulfate heptahydrate.
  • Buffer re-adjust the pH to 6.0, establish 10mL reaction system, react at 28 °C for 30min, sample 1mL centrifugation for liquid phase analysis and analyze the conversion of sorbitol and the yield of sorbose product;
  • the liquid phase detection condition is: mobile phase is 5mM H 2 SO 4 solution; flow rate 0.6 mL/min; injection volume 20 ⁇ L; column temperature 60 ° C, time 12 min.
  • Example 3 Effect of sodium glutamate addition on synthesis of coenzyme pQQ and SLDH by fermentation of Gluconobacter oxydans
  • the fermentation medium of different components was prepared, and the effect of partial replacement of yeast extract by sodium glutamate on the synthesis of sorbitol dehydrogenase and coenzyme pQQ by fermentation of Gluconobacter oxydans was investigated.
  • the fermentation medium formula of different nitrogen sources is: D-sorbitol 50g/L, yeast extract (30g/L, 20g/L, 15g/L, 10g/L, 5g/L), KH 2 PO 4 5g/ L, K 2 HPO 4 5g / L, 1g / L sodium glutamate, the solvent is deionized water, pH 6.5; under the same conditions, without adding 1g / L sodium glutamate as a control.
  • the seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 was inoculated to the fermentation medium at a volume concentration of 2%, fermented at 28 ° C, 150 rpm for 24 h, and the fermentation broth was centrifuged, and the supernatant was used to detect the relative accumulation of pQQ. Concentration, the cells were used to detect the relative viability of SLDH, the former was determined by glucose dehydrogenase recombination method, and the latter by high performance liquid chromatography HPLC, the results are shown in Table 2.
  • the content of pQQ in the culture supernatant was increased by more than 20% compared with the conventional medium, and the activity of sorbitol dehydrogenase per unit volume was increased by more than 10% compared with the conventional medium, and the specific gravity of the yeast extract in the fermentation medium was effectively reduced.
  • the cost of fermentation was increased by more than 20% compared with the conventional medium, and the activity of sorbitol dehydrogenase per unit volume was increased by more than 10% compared with the conventional medium, and the specific gravity of the yeast extract in the fermentation medium was effectively reduced.
  • Example 4 Effect of sodium glutamate addition on the synthesis of coenzyme pQQ and SLDH by fermentation of Gluconobacter oxydans in different time periods
  • the fermentation medium of different components was prepared to investigate the effects of sodium glutamate addition on the synthesis of coenzyme pQQ and SLDH by fermentation of Gluconobacter oxydans in different time periods.
  • the fermentation medium is formulated as: D-sorbitol 50g/L, yeast extract 5g/L, KH 2 PO 4 5g/L, K 2 HPO 4 5g/L, sodium glutamate 1g/L, solvent deionized Water, pH is natural; under the same conditions, no 1 g / L sodium glutamate was added as a control.
  • the seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 was inoculated to the fermentation medium at a volume concentration of 2%, and cultured at 28 ° C, 150 rpm for 24 h, wherein every 6 h was sampled and centrifuged to detect the relative pQQ in the supernatant.
  • the content and the relative volume of the enzyme activity of SLDH are shown in Table 3.
  • Example 5 Comparison of the activity of 6NSL catalyzed by resting cells of Gluconobacter oxydans obtained by adding sodium glutamate to the medium
  • the fermentation medium of different components was prepared, and the effect of partial replacement of yeast extract by sodium glutamate on the synthesis of sorbitol dehydrogenase coenzyme pQQ by fermentation of Gluconobacter oxydans was investigated.
  • the fermentation medium formula of different nitrogen sources is: D-sorbitol 50g/L, yeast extract (30g/L, 20g/L, 15g/L, 10g/L, 5g/L), KH 2 PO 4 5g/ L, K 2 HPO 4 5g / L, sodium glutamate 1g / L, the solvent is deionized water, the pH value is natural; under the same conditions, without adding 1g / L sodium glutamate as a control.
  • the seed solution of Gluconobacter oxydans CCTCC No. M 208069 of Example 1 was inoculated to the fermentation medium at a volume concentration of 2%, fermented at 28 ° C, 150 rpm for 24 h, the fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded to collect the oxidized Portuguese.
  • the cells were allowed to rest in the cellulase, and the cells were washed with an equal volume of water, centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the cells were collected to catalyze the synthesis of 6NSL.
  • the resting cell viability obtained by the high-concentration yeast extract (5-30 g/L) fermentation medium of sodium increased by more than 10%, and at the same time, the concentration of 5 g/L yeast extract was increased by adding 1 g/L of sodium glutamate.
  • the cumulative concentration of the substrate 6NSL reached 53.6g/L, which was higher than the accumulation at the concentration of 30g/L yeast extract (45.8g/L).

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Abstract

一种促进氧化葡糖杆菌发酵产D-山梨醇脱氢酶及吡咯喹啉醌的方法，所述方法为将氧化葡糖杆菌接种至发酵培养基，在28～32℃、150～180rpm条件下发酵培养6～24h，将发酵液离心，收集湿菌体，获得含D-山梨醇脱氢酶和吡咯喹啉醌的菌体细胞。所述方法促进了辅酶pQQ的合成及单位体积D-山梨醇脱氢酶的酶活，用该方法培养获得的氧化葡糖杆菌生物转化合成米格列醇前体6-脱氧-6-氨基(N-羟乙基)-α-L-呋喃山梨糖(6NSL)，产物6NSL的转化进程增加了21～35％，生物转化步骤周期由48h缩短至36h。此外，在相同底物浓度下(60g/L)，产物6NSL的累积浓度提高了10g/L以上。

Description

一种促进氧化葡糖杆菌合成山梨醇脱氢酶及辅酶吡咯喹啉醌的方法 技术领域
本发明涉及发酵工程和生物转化领域，具体涉及到氧化葡糖杆菌发酵培养及细胞膜D-山梨醇脱氢酶(SLDH)及其辅酶pQQ的制备，以及该菌种生物转化合成米格列醇中间体6-脱氧-6-氨基(N-羟乙基)-α-L-呋喃山梨糖(6NSL)中的应用。
背景技术
米格列醇[(2R,3R,4R,5S)-2-羟甲基-1-(2-羟乙基)-3,4,5-哌啶三醇，miglitol]是Bayer公司开发的一种降糖药物，通过抑制小肠黏膜表皮细胞的α-糖苷酶活力来减少糖的吸收，具有安全有效、耐受性良好和毒副作用小等优点。
目前合成米格列醇常用的方法是化学生物组合法，该工艺的关键催化步骤需要利用位于氧化葡糖杆菌(Gluconobacter oxydans)细胞膜上D-山梨醇脱氢酶(D-sorbitol dehydrogenase，EC 1.1.99.22)在其辅酶吡咯并喹啉醌(Pyrroloquinoline quinine，pQQ)协同下，对底物N-羟乙基葡萄糖胺进行4位羟基的选择性不对称氧化，获得的中间产物直接耦合化学加氢环化合成米格列醇，最后化学加氢生产米格列醇(图1)。该方法简化了生产工艺，提高了产物得率，降低了合成成本。其中生物法催化合成中间体6NSL是生产米格列醇的关键步骤，主要分为两个步骤：第一步，氧化葡糖杆菌的发酵生产高活力的山梨醇脱氢酶；第二步，利用静息细胞催化N-羟乙基葡萄糖胺合成米格列醇关键合成前体6NSL。此外，基于膜山梨醇脱氢酶(SLDH)特定的不完全 氧化高效催化特性，氧化葡糖杆菌已被广泛应用于催化合成二羟基丙酮，维生素C等其他多元醇的选择性氧化催化合成工艺中。
专利CN 101302549A公开了由山梨醇、酵母抽提物等组成的培养基培养氧化葡糖杆菌制备相应的静息细胞生产米格列醇的工艺，但酵母抽提物的质量百分比高达2.4％，发酵成本所占比重大。此外，US4806650，US5401645等专利公开了以N-羟乙基葡萄糖胺为底物用化学生物组合法合成米格列醇的生产工艺，但缺乏对发酵过程中直接影响米格列醇生物转化能力的关键参数，如单位体积膜山梨醇脱氢酶活力及其辅酶pQQ含量的测定及监控。本发明根据氧化葡糖杆菌发酵合成膜山梨醇脱氢酶的产酶特性，首次提出谷氨酸钠的添加及部分替代酵母抽提物能够有效促进辅酶pQQ的合成，并进一步提高山梨醇脱氢酶的发酵体积酶活，最终有效提高单位发酵体积获得菌体转化合成6NSL的时空产率，降低了米格列醇合成的生产成本。此外，应用此发酵培养基配方获得的高活力氧化葡糖杆菌山梨醇脱氢酶不完全氧化作用不仅仅限于米格列醇的合成，也包括其他多元醇，如二羟基丙酮，维生素C等关键手性化合物的催化合成。
发明内容
为了解决发酵生产氧化葡糖杆菌静息细胞工艺中发酵培养基酵母抽提物所占比重大、成本高的限制因素，本发明根据氧化葡糖杆菌的发酵代谢特征及膜山梨醇脱氢酶及其辅酶pQQ的合成代谢通路，提出了一种廉价高效的发酵培养基，添加少量的谷氨酸钠部分替代酵母抽提物，在降低发酵成本的同时能够进一步提高山梨醇脱氢酶的发酵体积酶活，为后续静息细胞催化合成米格列醇中间体6NSL提供了良好的基础。
为实现上述目的，本发明采用的技术方案：
本发明提出了一种促进氧化葡糖杆菌发酵产D-山梨醇脱氢酶 (SLDH)及吡咯喹啉醌(pQQ)的方法，将氧化葡糖杆菌(Gluconobacter oxydans)接种至发酵培养基，在28～32℃、150～180rpm条件下发酵培养6～24h，将发酵液离心，收集湿菌体，获得含D-山梨醇脱氢酶和吡咯喹啉醌的菌体细胞；所述发酵培养基组成为：D-山梨醇50～80g/L、酵母抽提物0.5～30g/L、KH 2PO 45g/L、K 2HPO 4 5g/L、氨基酸0.2～2g/L，溶剂为去离子水，pH6.5。
进一步，优选所述发酵培养基中氨基酸为谷氨酸钠、亮氨酸、酪氨酸或脯氨酸。优选所述发酵培养基中氨基酸浓度为0.5～1g/L，更优选1g/L。
进一步，优选所述氨基酸为谷氨酸钠，在发酵过程中向发酵培养基中添加谷氨酸钠并部分替代酵母抽提物。
进一步，优选所述酵母抽提物浓度为1～7g/L，更优选5g/L。
进一步，所述发酵培养基组成为：D-山梨醇50g/L、酵母抽提物5g/L、KH 2PO 45g/L、K 2HPO 45g/L、谷氨酸钠1g/L，溶剂为去离子水，pH 6.5。
进一步，所述氧化葡糖杆菌在发酵培养前，先进行斜面活化及种子培养，然后再将种子液以体积浓度5-15％的接种量接入发酵培养基进行发酵。
进一步，所述斜面活化培养是将氧化葡糖杆菌接种至斜面培养基，28℃恒温培养箱内培养3～5天，待斜面培养基中CaCO 3变透明光亮，肉眼目测无染菌即可接种，获得斜面菌体；斜面培养基组成为：酵母抽提物2～10g/L、碳酸钙5～20g/L、葡萄糖10～60g/L、琼脂24g/L，溶剂为去离子水，pH值自然，优选斜面培养基组成为：酵母抽提物5g/L、碳酸钙5g/L、葡萄糖50g/L、琼脂24g/L，溶剂为去离子水，pH值自然。
进一步，所述种子培养是将斜面菌体接种至种子培养基，在28℃、225rpm摇床培养24～48h，OD 600≥8达到转接标准，镜检菌形呈短 杆状，着色深，无杂菌，获得种子液；所述种子培养基组成为：D-山梨醇30～60g/L、酵母抽提物10～30g/L、KH 2PO 4 1～5g/L、K 2HPO 4 0.1～1g/L，溶剂为去离子水，pH值pH 4.0～7.0，优选种子培养基组成为：D-山梨醇50g/L、酵母抽提物20g/L、KH 2PO 4 5g/L、K 2HPO 4 5g/L，溶剂为去离子水，pH值6.5。
进一步，所述氧化葡糖杆菌优选为氧化葡糖杆菌(Gluconobacter oxydans)由中国典型培养物保藏中心(CCTCC)保藏，保藏编号为No.M 208069，专利公开号为CN101591681A。
本发明中，在搅拌式反应器发酵培养过程中，相对饱和溶氧需要控制在5％～30％，待搅拌转速下降，培养基中山梨醇基本耗尽时，终止发酵，发酵周期控制为14～16h；发酵末期细胞镜检：菌形呈短杆状，着色深，无杂菌。
本发明在制备氧化葡糖杆菌静息细胞时，还涉及菌体的收集及清洗：将发酵所得的氧化葡糖杆菌进行陶瓷微滤膜过滤收集，在过滤过程中加入超纯水洗涤菌体至滤出液无色透明。
本发明将发酵制备的氧化葡糖杆菌静息细胞用于催化生产米格列醇前体6NSL，优选的操作步骤为：以N-羟乙基葡萄糖胺为底物，加入MgSO 4·7H 2O，随后添加静息细胞(以湿菌体重量计)，以去离子水为反应介质，调pH值至4.5～5.5，构成反应体系，在5～30℃、400～600rpm条件下转化反应36h，取转化反应液，获得6NSL；所述反应体系中，底物终浓度40～100g/L，MgSO 4·7H 2O终浓度1～5g/L，静息细胞终浓度20～100g/L。
与现有技术相比，本发明有益效果主要体现在：
本发明针对氧化葡糖杆菌的发酵特性及其膜定位的D-山梨醇脱酶的催化特征出发，通过发酵培养基中谷氨酸钠的少量添加并部分替换酵母抽提物的组分，显著降低了氧化葡糖杆菌发酵产酶的生产成本。 在此基础上，还进一步促进了辅酶pQQ的合成及单位体积D-山梨醇脱氢酶的酶活，用该方法培养获得的氧化葡糖杆菌生物转化合成米格列醇前体6NSL，产物6NSL的转化进程增加了21-35％，缩短了生物转化步骤周期，静息细胞转化过程由48h缩短至36h，此外，在相同底物浓度下(60g/L)，产物6NSL的累积浓度提高了10g/L以上。本发明的提出有效降低了化学生物组合法生产米格列醇工艺中关键工段生物催化合成6NSL的生产成本，并有效提升了米格列醇前体6NSL的时空产率，为工业化大规模生产米格列醇奠定了基础。
附图说明
图1化学生物组合法合成米格列醇工艺路线。
具体实施方式
以下是本发明的实施例，本发明的实施例目的在于更加具体的说明本项发明，但本发明提供的发酵培养基培养获得的氧化葡糖杆菌静息细胞高活力D-山梨醇脱氢酶及其辅酶pQQ不仅仅应用于米格列醇前体6NSL的合成，也可以应用于其他多元醇的选择性氧化催化工艺中。
实施例1
将氧化葡糖杆菌(Gluconobacter oxydans)CCTCC No.M 208069，专利公开号CN101591681A)接种至斜面培养基，28℃恒温培养箱内培养3～5天，获得斜面菌体；斜面培养基组成为：酵母抽提物5g/L、碳酸钙5g/L、葡萄糖50g/L、琼脂24g/L，溶剂为去离子水，pH值自然。
将斜面菌体接种至种子培养基，在28℃、225rpm摇床培养48h，获得氧化葡糖杆菌CCTCC No.M 208069种子液；所述种子培养基组成为：D-山梨醇50g/L、酵母抽提物20g/L、KH 2PO 4 5g/L、K 2HPO 4 5 g/L，溶剂为去离子水，pH值6.5。
实施例2：发酵培养基中氨基酸的种类对氧化葡糖杆菌SLDH酶活力的影响
配制不同组分的发酵培养基，考察不同氨基酸对发酵培养氧化葡糖杆菌合成山梨醇脱氢酶的影响。不同氨基酸的发酵培养基配方为：D-山梨醇50g/L、酵母抽提物20g/L、KH 2PO 4 5g/L、K 2HPO 4 5g/L，1g/L(谷氨酸钠、亮氨酸、酪氨酸和脯氨酸)，溶剂为去离子水，pH值6.5；同样条件下，以不添加任何氨基酸作为对照。
将实施例1氧化葡糖杆菌CCTCC No.M 208069种子液以体积浓度2％接种至发酵培养基，在28℃、150rpm下发酵培养24h，发酵液离心，所得菌体用于检测山梨醇脱氢酶SLDH相对活力。
酶活力单位(U)定义为在20g/L山梨醇浓度下，30℃，pH 6.0的磷酸缓冲液体系中，每分钟催化D-山梨醇生成1.0μmoL L-山梨糖的所需要的酶量。D-山梨醇及L-山梨糖浓度通过高效液相色谱(HPLC)测定。
SLDH酶活检测操作流程：取发酵液20mL离心10000rpm，10min，弃上清，洗涤一遍，离心弃上清，配制缓冲液100mM PBS，再将20g/L山梨醇和5g/L七水硫酸镁溶于缓冲液，重新调pH至6.0，建立10mL的反应体系，28℃反应30min，取样1mL离心进行液相检测分析山梨醇的转化率及山梨糖产物得率；液相检测条件为：流动相为5mM H 2SO 4溶液；流速0.6mL/min；进样量20μL；柱温60℃，时间12min。
结果如表1所示。
表1 不同氨基酸的添加对氧化葡糖杆菌产SLDH的影响
氨基酸种类 SLDH相对酶活力(％)
空白 100
谷氨酸钠 121
亮氨酸 112
酪氨酸 101
脯氨酸 102
结果分析：如表1所示，向培养基中添加1g/L不同类型的氨基酸，摇瓶发酵培养24h后，相较于不添加，其单位体积山梨醇脱氢酶相对酶活力有着不同程度地变化。其中当添加1g/L的酪氨酸和脯氨酸时，SLDH的活力几乎没有变化；而当添加谷氨酸钠和亮氨酸时，相较于空白对照组，其SLDH的酶活力分别提高21％和12％，且谷氨酸钠的提高幅度较大。这表明通过1g/L谷氨酸钠的添加能够使得单位体积山梨醇脱氢酶相对活力较对照培养基提高20％以上。
实施例3：谷氨酸钠添加对发酵培养氧化葡糖杆菌合成辅酶pQQ及SLDH的影响
配制不同组分的发酵培养基，考察谷氨酸钠部分替代酵母抽提物对发酵培养氧化葡糖杆菌合成山梨醇脱氢酶以及辅酶pQQ的影响。不同氮源的发酵培养基配方为：D-山梨醇50g/L、酵母抽提物(30g/L，20g/L，15g/L，10g/L，5g/L)、KH 2PO 4 5g/L、K 2HPO 4 5g/L，1g/L谷氨酸钠，溶剂为去离子水，pH值6.5；同样条件下，以不添加1g/L谷氨酸钠作为对照。
将实施例1氧化葡糖杆菌CCTCC No.M 208069种子液以体积浓度2％接种至发酵培养基，在28℃、150rpm下发酵培养24h，发酵液离心，上清液用于检测pQQ的相对累积浓度，菌体用于检测SLDH相对活力，其中前者通过葡萄糖脱氢酶重组酶法测定，而后者通过高效液相色谱HPLC检测，结果如表2所示。
表2 谷氨酸钠的添加对不同酵母抽提物浓度下pQQ合成及SLDH活力影响
Figure PCTCN2019073044-appb-000001
结果分析：如表2所示，摇瓶发酵培养24h后，比较在不同酵母抽提物的发酵培养基中添加1g/L的谷氨酸钠，24h发酵培养氧化葡糖杆菌后，上清中pQQ的浓度、单位体积山梨醇脱氢酶酶活力较空白对照组均有不同程度的显著提高，当酵母抽提物浓度降低至5g/L时，1g/L谷氨酸钠的添加能够保证发酵培养上清辅酶pQQ的含量较常规培养基提高20％以上，单位体积山梨醇脱氢酶活力较常规培养基提高10％以上，同时有效地降低了发酵培养基中酵母抽提物的比重，降低了发酵成本。
实施例4：谷氨酸钠添加在不同时间段对发酵培养氧化葡糖杆菌合成辅酶pQQ及SLDH的影响
配制不同组分的发酵培养基，考察谷氨酸钠的添加在不同时间段对发酵培养氧化葡糖杆菌合成辅酶pQQ及SLDH的影响。发酵培养基配方为：D-山梨醇50g/L、酵母抽提物5g/L、KH 2PO 4 5g/L、K 2HPO 45g/L、谷氨酸钠1g/L，溶剂为去离子水，pH值自然；同样条件下，以不添加1g/L谷氨酸钠作为对照。
将实施例1氧化葡糖杆菌CCTCC No.M 208069种子液以体积浓度2％接种至发酵培养基，在28℃、150rpm下发酵培养24h，其中每隔6h取样，离心，检测上清中pQQ相对含量及SLDH的相对体积酶活，结果如表3所示。
表3 发酵培养基中辅酶pQQ、SLDH随培养时间的累积情况
Figure PCTCN2019073044-appb-000002
结果分析：如表3所示，摇瓶发酵培养24h后，在不同时间段，相较于发酵培养基中添加1g/L的谷氨酸钠，其发酵培养上清辅酶pQQ的含量、单位体积山梨醇脱氢酶酶活力较空白对照组均有不同程度的显著提高，当酵母抽提物浓度为5g/L时，1g/L谷氨酸钠的添加，能使不同时间段辅酶PQQ的合成量较对照培养基提高30％以上，保证不同时间段的单位体积山梨醇脱氢酶活力较对照培养基提高10％以上。
实施例5：培养基中添加谷氨酸钠发酵所得氧化葡糖杆菌静息细胞催化合成6NSL活力比较
配制不同组分的发酵培养基，考察谷氨酸钠部分替代酵母抽提物对发酵培养氧化葡糖杆菌合成山梨醇脱氢酶辅酶pQQ的影响。不同氮源的发酵培养基配方为：D-山梨醇50g/L、酵母抽提物(30g/L，20g/L，15g/L，10g/L，5g/L)、KH 2PO 4 5g/L、K 2HPO 4 5g/L、谷氨酸钠1g/L，溶剂为去离子水，pH值自然；同样条件下，以不添加1g/L谷氨酸钠作为对照。
将实施例1氧化葡糖杆菌CCTCC No.M 208069种子液以体积浓度2％接种至发酵培养基，在28℃、150rpm下发酵培养24h，将发酵液10000rpm离心10min，弃上清，收集氧化葡糖杆菌静息细胞，加入等体积清水洗涤菌体，10000rpm离心10min弃上清，收集细胞，催化合成6NSL。
转化体系(总体积为50mL)：有挡板摇瓶50mL/500mL，添加终浓度80g/L N-羟乙基葡萄糖胺、终浓度5g/L MgSO 4·7H 2O，75g/L湿 菌体，以去离子水为反应介质，建立催化反应体系。具体反应条件如下：温度15℃；转速220rpm；用2M NaOH调碱，使转化液的pH值维持在4.5～5.0。转化24h后停止转化，取转化液10000rpm离心10min，取上清并通过HPLC的检测方法，6NSL的累积浓度如表4所示：
表4 谷氨酸钠的添加对发酵培养获得氧化葡糖杆菌静息细胞催化合成6NSL累积浓度比较
Figure PCTCN2019073044-appb-000003
结果分析：如表4所示，比较在不同酵母抽提物的发酵培养基中添加1g/L的谷氨酸钠，24h发酵培养氧化葡糖杆菌后，获得的静息细胞催化底物N-羟乙基葡萄糖胺合成米格列醇前体6NSL的累积浓度较空白对照组有不同程度的提高。单位体积下，当酵母抽提物浓度降低至5g/L时，添加1g/L谷氨酸钠的培养基发酵获得的氧化葡糖杆菌静息细胞催化合成6NSL的累积浓度较不添加谷氨酸钠的高浓度酵母抽提物(5～30g/L)发酵培养基获得的静息细胞活力提高10％以上，同时在5g/L酵母抽提物浓度下，通过添加1g/L谷氨酸钠，底物6NSL的累积浓度达到53.6g/L，高于在30g/L酵母抽提物浓度下的累积情况(45.8g/L)，结果提示通过添加1g/L廉价的谷氨酸钠，能够有效替代发酵培养基中的酵母抽提物，在保证菌体高效转化能力的前提下，有效降低了发酵成本。

Claims (10)

  1. 一种促进氧化葡糖杆菌发酵产山梨醇脱氢酶及吡咯喹啉醌的方法，其特征在于，所述方法为：将氧化葡糖杆菌接种至发酵培养基，在28～32℃、150～180rpm条件下发酵培养6～24h，将发酵液离心，收集湿菌体，获得含D-山梨醇脱氢酶和吡咯喹啉醌的菌体细胞；所述发酵培养基组成为：D-山梨醇50～80g/L、酵母抽提物0.5～30g/L、KH 2PO 4 5g/L、K 2HPO 4 5g/L、氨基酸0.2～2g/L，溶剂为去离子水，pH6.5。
  2. 如权利要求1所述的方法，其特征在于，所述发酵培养基中氨基酸为谷氨酸钠、亮氨酸、酪氨酸或脯氨酸。
  3. 如权利要求2所述的方法，其特征在于，所述发酵培养基中氨基酸浓度为0.5～1g/L。
  4. 如权利要求2所述的方法，其特征在于，所述发酵培养基中氨基酸为谷氨酸钠。
  5. 如权利要求1所述的方法，其特征在于，所述酵母抽提物浓度为1～7g/L。
  6. 如权利要求1所述的方法，其特征在于，所述发酵培养基组成为：D-山梨醇50g/L、酵母抽提物5g/L、KH 2PO 4 5g/L、K 2HPO 4 5g/L、谷氨酸钠1g/L，溶剂为去离子水，pH6.5。
  7. 如权利要求1所述的方法，其特征在于，所述氧化葡糖杆菌在发酵培养前，先进行斜面活化及种子培养，然后再将种子液以体积浓度5-15％的接种量接入发酵培养基进行发酵。
  8. 如权利要求7所述的方法，其特征在于，所述斜面活化培养是将氧化葡糖杆菌接种至斜面培养基，28℃恒温培养箱内培养3～5天，获得斜面菌体；斜面培养基组成为：酵母抽提物2～10g/L、碳酸钙5～ 20g/L、葡萄糖10～60g/L、琼脂24g/L，溶剂为去离子水，pH值自然。
  9. 如权利要求8所述的方法，其特征在于，所述种子培养是将斜面菌体接种至种子培养基，在28℃、225～235rpm摇床培养24～48h，获得种子液；所述种子培养基组成为：D-山梨醇30～60g/L、酵母抽提物10～30g/L、KH 2PO 4 1～5g/L、K 2HPO 4 0.1～1g/L，溶剂为去离子水，pH值pH 4.0～7.0。
  10. 如权利要求1所述的方法，其特征在于，所述氧化葡糖杆菌为氧化葡糖杆菌(Gluconobacter oxydans)CCTCC No.M 208069。
PCT/CN2019/073044 2018-01-26 2019-01-24 一种促进氧化葡糖杆菌合成山梨醇脱氢酶及辅酶吡咯喹啉醌的方法 WO2019144920A1 (zh)

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