WO2021056683A1 - 一株产脂肪酶菌株及其应用 - Google Patents

一株产脂肪酶菌株及其应用 Download PDF

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WO2021056683A1
WO2021056683A1 PCT/CN2019/114830 CN2019114830W WO2021056683A1 WO 2021056683 A1 WO2021056683 A1 WO 2021056683A1 CN 2019114830 W CN2019114830 W CN 2019114830W WO 2021056683 A1 WO2021056683 A1 WO 2021056683A1
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vitamin
strain
palmitate
application according
bacillus subtilis
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English (en)
French (fr)
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王立梅
齐曼婷
徐田甜
季亚美
李政洪
齐斌
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常熟理工学院
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Priority to JP2022518955A priority Critical patent/JP7376186B2/ja
Priority to US17/754,062 priority patent/US20220333149A1/en
Publication of WO2021056683A1 publication Critical patent/WO2021056683A1/zh

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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    • C12N1/20—Bacteria; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • the invention relates to a lipase-producing strain and its application in the enzymatic synthesis of vitamin A palmitate, and belongs to the technology in the field of industrial microorganisms.
  • Vitamin A palmitate is currently one of the most commonly used and most widely used vitamin A series products. Vitamin A palmitate can not only help maintain normal visual function, but also participate in various metabolic activities to maintain the health of the organism. Generally used as an additive in industries such as food, cosmetics, and medicine. At present, there are mainly chemical methods and enzymatic methods for the synthesis of vitamin A palmitate. The chemical method to synthesize vitamin A palmitate has problems such as environmental pollution and equipment corrosion, while the enzymatic method has less pollution, high space-time yield and low cost. Therefore, the research of vitamin A palmitate enzymatic synthesis technology is becoming more and more active.
  • Bacillus subtilis lipases mainly include LipA and LipB.
  • LipA can hydrolyze long-chain fatty acids.
  • Lip A has no lid structure and has a small molecular weight, so it is considered to be one of the smallest ⁇ / ⁇ sheet hydrolases.
  • the structure of Lip A is very similar to lipase B derived from Candida antarctica, while the current enzymatic synthesis of vitamin A palmitate mostly uses Novozymes 435 (Candida antarctica lipase B).
  • Novozymes 435 Candida antarctica lipase B.
  • Mimp paste is mainly made from twine shrimp produced from the sea and fermented naturally with salt for about one month.
  • sesame paste It is rich in protein, chitin and fat. It is a common seasoning in my country and Southeast Asia. The microbial diversity and composition of sesame paste are very complicated, and it is suitable for the screening of lipase-producing strains. At present, there is no report of screening lipase-producing strains from sesame shrimp paste and using this strain to develop organic phase whole-cell transformation method to produce vitamin A palmitate.
  • the first objective of the present invention is to provide a lipase-producing strain.
  • a lipase-producing strain classified as Bacillus subtilis (Bacillus subtilis) CS1802, deposited in the China Type Culture Collection, address: Wuhan, China, Wuhan University, deposit number CCTCC M2018262, preservation date May 2018 10th.
  • strains were separated and obtained from naturally fermented sesame paste by the Fermentation Engineering Center of Changshu Institute of Technology.
  • Bacillus subtilis CS1802 The physical and chemical properties of Bacillus subtilis CS1802 of the present invention are as follows:
  • Morphology It grows on the screening medium for 1d and can form obvious colonies.
  • the colony shape is irregular or wrinkled, Gram-positive bacteria, rod-shaped.
  • the optimum growth temperature is around 30°C, aerobic; the optimum growth pH is around 7.
  • Another object of the present invention is to provide the application of the above-mentioned strains in the enzymatic synthesis of vitamin A palmitate.
  • the present invention provides an application method for producing vitamin A palmitate using a whole-cell transformation method, which specifically includes the following steps:
  • step (1) The strains cultured in step (1) are connected to the fermentation medium for shaking fermentation; the fermentation broth is centrifuged, the precipitate is discarded, and the supernatant is taken, that is, the fermented bacterial broth.
  • the temperature of shaking culture and fermentation culture is 30°C.
  • fermentation medium components are:
  • the olive oil emulsion preparation method is as follows: the olive oil emulsifier PVA is mixed with olive oil in a volume ratio of 3:1, and ultrasonic emulsification is used.
  • vitamin A and palmitic acid are dissolved in an organic solvent according to a mass ratio of 1:1; the concentration of vitamin A and palmitic acid is 10-25 g/L; preferably 15 g/L.
  • the organic solvent is preferably n-hexane.
  • bacteria are inserted into the organic phase system and fermented for 0.5 to 2 hours; preferably 1 hour.
  • the present invention provides a Bacillus subtilis strain that can be used for enzymatic synthesis of vitamin A palmitate; the strain is derived from traditional natural fermented foods and has broad application prospects in the food industry; and the strain is used on solid medium for beef extract peptone It grows well, is easy to cultivate and store.
  • the whole cell transformation method under the condition of 15g/L vitamin A and palmitic acid substrate concentration, the output of vitamin A palmitate is 15.35mg/mL, and the conversion rate is 76.75%.
  • Figure 1 shows the microscopic morphology of Bacillus subtilis CS1802 after Gram staining.
  • Figure 2 is a phylogenetic tree of Bacillus subtilis (Bacillus subtilis) CS1802.
  • Figure 3 shows the addition amount of transformed cells of Bacillus subtilis (Bacillus subtilis) CS1802 whole-cell method to produce vitamin A palmitate.
  • Figure 4 shows the conversion time of Bacillus subtilis (Bacillus subtilis) CS1802 whole-cell method to produce vitamin A palmitate.
  • Figure 5 shows the substrate concentration of vitamin A palmitate produced by Bacillus subtilis CS1802 whole-cell method.
  • the biological material involved in the present invention is classified and named: Bacillus subtilis (Bacillus subtilis) CS1802, deposited in the China Type Culture Collection (CCTCC), address: Wuhan. Wuhan University, China, deposit number CCTCC NO: M2018262, The preservation date is May 10, 2018.
  • This example illustrates the screening, purification and identification method of Bacillus subtilis CS1802.
  • the screened sample is the sesame shrimp paste from Lianyungang Haiwa Food Co., Ltd. Weigh 25g shrimp paste and 225mL physiological saline to prepare a bacterial suspension, and dilute the concentration to 10 -1 , 10 -2 , 10 -3 and 10 -respectively. 4 times. Spread the bacterial suspension stock solution, 10 -1 fold dilution, 10 -2 fold dilution, 10 -3 fold dilution and 10 -4 fold dilution on the primary screening medium, grow at 30°C for 1 to 2 days, and pick it A single colony that grows well is streaked and separated on the primary screening medium. Pick a single colony produced on the primary screening medium with an obvious transparent circle around it and inoculate it into the secondary screening medium, and culture it on a shaker at 30°C and 200r/min for 1 to 2 days.
  • Primary screening medium peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, tributyrin 2mL/L, agar powder 20g/L, distilled water to dilute the volume to 1000mL.
  • Screening medium Tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L , MgSO 4 7H 2 O 1g / L, KH 2 PO 4 0.5g / L, K 2 HPO 4 0.5g / L, Olive oil emulsion is 12mL/L, distilled water is fixed to 1000mL.
  • the preparation method of olive oil emulsion is: mixing olive oil emulsifier PVA with olive oil in a volume ratio of 3:1, and emulsifying with ultrasonic waves.
  • Morphology It grows on the screening medium for 1d and can form obvious colonies.
  • the colony shape is irregular or wrinkled, Gram-positive bacteria, rod-shaped.
  • Physiological and biochemical characteristics 7% NaCl growth, citrate utilization, oxidase, contact enzyme, VP determination and starch hydrolysis are all positive; can use sucrose, maltose, rhamnose, raffinose, glucose, maltose, N-acetamido Carbon sources such as glucose and colloidal chitin.
  • the optimum growth temperature is around 30°C, aerobic; the optimum growth pH is around 7.
  • This example specifically illustrates the application of the strain CS1802 in the fermentation of olive oil to produce lipase.
  • step (2) Put the cultured strains in step (1) into the fermentation medium, the inoculum amount is 2%, 30°C, shake culture for 14-24 hours. The fermentation broth was centrifuged, the precipitate was discarded, the supernatant was taken, and the lipase activity was determined to be 214.3 U/L.
  • composition of the fermentation medium is the same as the aforementioned re-screening medium.
  • the method for determining lipase activity is as follows: centrifuge the fermentation broth at 3000r for 10 min, take the supernatant as the sample to be tested and transfer it to the coated microwells pre-coated with lipase antibody, and add the sample to be tested and horseradish peroxidase ( HRP) labeled detection antibody, incubated at 37°C for 1 h and washed thoroughly. The color is developed with the substrate 3,3',5,5'-tetramethylbenzidine (TMB), which is converted into blue under the catalysis of HRP, and into the final yellow under the action of acid. Measure the OD value with a microplate reader at a wavelength of 450nm, and calculate the sample activity from the standard curve.
  • HRP horseradish peroxidase
  • the standard product is 0, 1.5, 3, 6, 12, 24 U/L enzyme solution prepared with pure lipase.
  • Enzyme activity is defined as: at 37°C, the amount of enzyme that decomposes substrates and produces 1 ⁇ mol of fatty acids per milligram of protein per hour is an enzyme activity unit U.
  • the lipase ELISA detection kit was purchased from Wuhan Purity Biotechnology Co., Ltd.
  • This example specifically illustrates the application of the whole cell transformation of strain CS1802 to produce vitamin A palmitate.
  • step (2) Put the cultured strains in step (1) into the fermentation medium, the inoculum amount is 2%, 30°C, shake culture for 14-24 hours. The fermentation broth is centrifuged, the precipitate is discarded, and the supernatant is taken.
  • composition of the fermentation medium is the same as the aforementioned re-screening medium.
  • the determination method of vitamin A palmitate is: high performance liquid method with external standard method for quantification.
  • the chromatographic conditions were: chromatographic column: Alltech C 18 (250x4.6 mm, 4.5 ⁇ m); mobile phase: 100% methanol; detector: Shimadzu 10A UV detector; detection wavelength: 327 nm; flow rate: 1 mL/min.
  • the conversion rate calculation formula is:
  • This example specifically illustrates the application of the whole cell transformation of strain CS1802 to produce vitamin A palmitate.
  • step (2) Put the cultured strains in step (1) into the fermentation medium, the inoculum amount is 2%, 30°C, shake culture for 14-24 hours. The fermentation broth is centrifuged, the precipitate is discarded, and the supernatant is taken.
  • This example specifically illustrates the application of the whole cell transformation of strain CS1802 to produce vitamin A palmitate.
  • step (2) Put the cultured strains in step (1) into the fermentation medium, the inoculum amount is 2%, 30°C, shake culture for 14-24 hours. The fermentation broth is centrifuged, the precipitate is discarded, and the supernatant is taken.

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Abstract

一株产脂肪酶菌株及其应用，其分类命名为枯草芽孢杆菌(Bacillus subtilis)CS1802，保藏编号为CCTCC NO：M2018262，该菌株可用于全细胞转化维生素A和棕榈酸生产维生素A棕榈酸酯。枯草芽孢杆菌(Bacillus subtilis)CS1802，来源于传统自然发酵食品，属于公认安全微生物。该菌株易于培养和保藏，通过全细胞转化维生素A和棕榈酸所得维生素A棕榈酸酯含量最高为15.35mg/L，转化效率最高为76.75％，该菌株为酶法合成维生素A棕榈酸酯提供新途径，具有重要应用前景。

Description

一株产脂肪酶菌株及其应用 技术领域
本发明涉及一株产脂肪酶菌株及其在酶法合成维生素A棕榈酸酯中的应用，属于工业微生物领域技术。
背景技术
维生素A棕榈酸酯是目前最常用的用途最为广泛的维生素A系列产品之一，维生素A棕榈酸酯不但能帮助维持正常视觉功能，还能参与各种代谢活动维持生物机体健康。一般作为添加剂应用于食品、化妆品、医药等行业。目前合成维生素A棕榈酸酯主要有化学法和酶法。化学方法合成维生素A棕榈酸酯存在环境污染、设备腐蚀等问题，而酶法则污染少，时空产率高，成本低。因此维生素A棕榈酸酯酶法合成技术的研究日趋活跃。
枯草芽孢杆菌脂肪酶主要有LipA和LipB，大量研究表明，LipA可以水解长链脂肪酸。Lip A没有盖子结构，并且分子量较小，因此被认为是最小的α/β折叠水解酶之一。通过结构对比发现，Lip A的结构与来源于南极假丝酵母(Candida antarctica)的脂肪酶B很相似，而目前酶法合成维生素A棕榈酸酯的方法大多采用诺维信435(Candida antarctica lipase B)固定化酶，但该酶价格昂贵，工业化生产应用成本太高。麻虾酱主要是由产自海洋的麻线虾加盐自然发酵一月左右制得，富含蛋白质、甲壳素及脂肪，是我国及东南亚地区的常用调味料。麻虾酱微生物多样性和构成非常复杂，适合用作产脂肪酶菌株的筛选。目前未有从麻虾酱中筛选产脂肪酶菌株并利用该菌株开发有机相全细胞转化法生产维生素A棕榈酸酯的报道。
发明内容
本发明的第一目的在于提供一株产脂肪酶菌株。
为了实现本发明的上述技术目的，本发明采用的技术方案是：
一株产脂肪酶菌株，其分类命名为枯草芽孢杆菌(Bacillus subtilis)CS1802，保藏于中国典型培养物保藏中心，地址：中国.武汉.武汉大学，保藏编号CCTCC M2018262，保藏日期为2018年5月10日。
上述菌株由常熟理工学院发酵工程中心从自然发酵的麻虾酱中分离获得。
本发明的枯草芽孢杆菌CS1802的理化性质如下：
形态学：在筛选培养基上生长1d，能形成明显的菌落。菌落形状不规则或有皱褶，革兰氏阳性菌，杆状。
培养特征：最适生长温度30℃左右，好氧；最适生长pH 7左右。
本发明的另一目的在于提供上述菌株在酶法合成维生素A棕榈酸酯中的应用。
本发明提供了一种利用全细胞转化法产维生素A棕榈酸酯的应用方式，具体包括如下步骤：
(1)将菌株CS1802接种至牛肉膏蛋白胨液体培养基中振荡培养；
(2)将步骤(1)所培养菌株接入发酵培养基中振荡发酵；发酵液进行离心，弃除沉淀，取上清液，即发酵好的菌液。
(3)将发酵好的菌液离心，将菌体接入有机相体系中发酵生产维生素A棕榈酸酯。
进一步的，振荡培养和发酵培养的温度为30℃。
进一步的，发酵培养基成分为：
胰蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，MgSO 4·7H 2O 1g/L，KH 2PO 4 0.5g/L，K 2HPO 40.5g/L，橄榄油乳化液12mL/L。所述橄榄油乳化液配制方法为：橄榄油乳化剂PVA与橄榄油以体积比3：1混合，利用超声波乳化。
进一步的，所述有机相体系为将维生素A和棕榈酸按照1：1的质量比溶于有机溶剂；所述维生素A和棕榈酸浓度为10～25g/L；优选15g/L。所述有机溶剂优选采用正己烷。
进一步的，将菌体接入有机相体系中发酵0.5～2h；优选1h。
本发明提供了一株可用于酶法合成维生素A棕榈酸酯的枯草芽孢杆菌；该菌株来源于传统的自然发酵食品，在食品行业具有广阔的应用前景；该菌株在牛肉膏蛋白胨固体培养基上生长良好，容易培养和保存，通过全细胞转化法，在15g/L维生素A和棕榈酸底物浓度的条件下，维生素A棕榈酸酯产量为15.35mg/mL，转化率为76.75％。
附图说明
图1为枯草芽孢杆菌(Bacillus subtilis)CS1802经革兰氏染色后的显微形态。
图2为枯草芽孢杆菌(Bacillus subtilis)CS1802的系统进化树。
图3为枯草芽孢杆菌(Bacillus subtilis)CS1802全细胞法产维生素A棕榈酸酯的转化菌体添加量。
图4为枯草芽孢杆菌(Bacillus subtilis)CS1802全细胞法产维生素A棕榈酸酯的转化时间。
图5为枯草芽孢杆菌(Bacillus subtilis)CS1802全细胞法产维生素A棕榈酸酯的底物浓度。
本发明涉及的生物材料，其分类命名为：枯草芽孢杆菌(Bacillus subtilis)CS1802，保藏于中国典型培养物保藏中心(简称CCTCC)，地址：中国.武汉.武汉大学，保藏编号CCTCC NO：M2018262，保藏日期为2018年5月10日。
具体实施方式
实施例1
本实施例说明枯草芽孢杆菌(Bacillus subtilis)CS1802的筛选纯化及鉴定方法。
筛选样品为连云港市海娃食品有限公司的麻虾酱，称取25g虾酱与225mL生理盐水制备菌悬液，同时将其浓度分别稀释至10 -1、10 -2、10 -3和10 -4倍。将菌悬液原液、10 -1倍稀释、10 -2倍稀释、10 -3倍稀释和10 -4倍稀释的菌液涂布于初筛培养基上，30℃生长1～2d后挑取生长良好的单一菌落在初筛培养基上划线分离。挑取初筛培养基上生产的周围有明显透明圈的单一菌落接种到复筛培养基中，30℃，200r/min摇床培养1～2d。
初筛培养基：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，三丁酸甘油酯2mL/L，琼脂粉20g/L，蒸馏水定容至1000mL。
复筛培养基：胰蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，MgSO 4 .7H 2O 1g/L，KH 2PO 4 0.5g/L，K 2HPO 4 0.5g/L，橄榄油乳化液12mL/L，蒸馏水定容至1000mL。橄榄油乳化液配制方法为：将橄榄油乳化剂PVA与橄榄油以体积比3：1混合，利用超声波乳化。
从初筛培养基平板上挑取一环菌与载玻片上水珠混合并过火。通过结晶紫初染，碘液酶染，乙醇脱色，番红复染后在显微镜下镜检，该菌为革兰氏阳性菌(图1)。
该菌株的理化性质如下：
形态学：在筛选培养基上生长1d，能形成明显的菌落。菌落形状不规则或有皱褶，革兰氏阳性菌，杆状。
生理生化特征：7％NaCl生长，柠檬酸盐利用，氧化酶，接触酶，V-P测定及淀粉水解均为阳性；能利用蔗糖、麦芽糖、鼠李糖、棉籽糖、葡萄糖、麦芽糖、N-乙酰氨基葡萄糖和胶体甲壳素等碳源。
培养特征：最适生长温度30℃左右，好氧；最适生长pH 7左右。
对上述菌株的16SrDNA部分的序列的测定及BLAST比对，后用MEGA 5.1构建N-J系统进化树分析，其16SrDNA序列如SEQ ID NO：1所示，系统进化树如图2所示，由此鉴定为枯草芽孢杆菌，经微生物保藏程序的鉴定和保藏，将其分类命名定为枯草芽孢杆菌(Bacillus subtilis)CS1802，其保藏编号为CCTCC NO：M2018262。
实施例2
本实施例具体说明菌株CS1802在利用橄榄油发酵产脂肪酶中的应用。
(1)将菌株CS1802接种至牛肉膏蛋白胨液体培养基中，30℃，振荡培养18～24h；
(2)将步骤(1)所培养菌株接入发酵培养基中，接种量为2％，30℃，振荡培养14～24h。发酵液进行离心，弃除沉淀，取上清液，测定其脂肪酶酶活为214.3U/L。
发酵培养基成分和前述复筛培养基相同。
脂肪酶酶活测定方法为：将发酵液3000r离心10min，取上清液为待测样品往预先包被脂肪酶抗体的包被微孔中，依次加入待测样品、辣根过氧化物酶(HRP)标记的检测抗体，经过37℃温育1h并彻底洗涤。用底物3,3',5,5'-四甲基联苯胺(TMB)显色，在HRP催化下转化成蓝色，并在酸的作用下转化为最终的黄色。用酶标仪在450nm波长下测定OD值，通过标准曲线计算样品活性。标准品为纯脂肪酶配制的0、1.5、3、6、12、24U/L酶溶液。酶活定义为：37℃条件下，每毫克蛋白每小时分解底物产生1μmol脂肪酸的酶量为一个酶活性单位U。脂肪酶ELISA检测试剂盒购自武汉纯度生物科技有限公司。
实施例3
本实施例具体说明菌株CS1802全细胞转化产维生素A棕榈酸酯的应用。
(1)将菌株CS1802接种至牛肉膏蛋白胨液体培养基中，30℃，振荡培养18～24h；
(2)将步骤(1)所培养菌株接入发酵培养基中，接种量为2％，30℃，振荡培养14～24h。发酵液进行离心，弃除沉淀，取上清液。
(3)将发酵好的菌液3000r/min离心5min，将菌体按5g/L，10g/L，15g/L，20g/L，25g/L，30g/L接入有机相体系(维生素A：棕榈酸＝10g：10g溶于1L正己烷)中发酵2h后测定维生素A棕榈酸酯的含量，并计算转化率。由图3可知，当菌体添加量为25g/L时转化效率最高为55.7％，维生素A棕榈酸酯含量为11.14mg/L。
发酵培养基成分和前述复筛培养基相同。
维生素A棕榈酸酯测定方法为：高效液相法，采用外标法定量。色谱条件为：色谱柱：Alltech C 18(250x4.6mm，4.5μm)；流动相：100％甲醇；检测器：岛津10A紫外检测器；检测波长：327nm；流速：1mL/min。
转化率计算公式为：
Figure PCTCN2019114830-appb-000001
实施例4
本实施例具体说明菌株CS1802全细胞转化产维生素A棕榈酸酯的应用。
(1)将菌株CS1802接种至牛肉膏蛋白胨液体培养基中，30℃，振荡培养18～24h；
(2)将步骤(1)所培养菌株接入发酵培养基中，接种量为2％，30℃，振荡培养14～24h。发酵液进行离心，弃除沉淀，取上清液。
(3)将发酵好的菌液3000r/min离心5min，将菌体按25g/L接入有机相体系(维生素A：棕榈酸＝10g：10g溶于1L正己烷)中发酵0.5h、1h、1.5h、2h、2.5h、3h后测定维生素A棕榈酸酯的含量，并计算转化率。由图4可知，当菌体添加量为25g/L，转化1h后转化效率最高为73.6％，维生素A棕榈酸酯含量为14.72mg/L。
实施例5
本实施例具体说明菌株CS1802全细胞转化产维生素A棕榈酸酯的应用。
(1)将菌株CS1802接种至牛肉膏蛋白胨液体培养基中，30℃，振荡培养18～24h；
(2)将步骤(1)所培养菌株接入发酵培养基中，接种量为2％，30℃，振荡培养14～24h。发酵液进行离心，弃除沉淀，取上清液。
(3)将发酵好的菌液3000r/min离心5min，将菌体按25g/L接入不同底物浓度有机相体系，即维生素A和棕榈酸浓度为5g:5g、10g:10g、15g:15g、20g:20g和25g:25g分别溶于1L正己烷中发酵1h后测定维生素A棕榈酸酯的含量，并计算转化率。由图5可知，当菌体添加量为25g/L，有机相体系为维生素A:棕榈酸＝15g:15g溶于1L培养基中转化1h后转化效率最高为76.75％，维生素A棕榈酸酯含量为15.35mg/L。

Claims (10)

  1. 一株产脂肪酶菌株，其特征在于，其分类命名为枯草芽孢杆菌(Bacillus subtilis)CS1802，保藏编号CCTCC NO：M2018262。
  2. 权利要求1所述菌株在酶法合成维生素A棕榈酸酯中的应用。
  3. 根据权利要求2所述的应用，其特征在于，采用全细胞转化法合成维生素A棕榈酸酯。
  4. 根据权利要求2或3所述的应用，其特征在于，包括如下步骤：
    (1)将菌株CS1802接种至牛肉膏蛋白胨液体培养基中振荡培养；
    (2)将步骤(1)获取的培养菌株接入发酵培养基中振荡发酵，取上清液，即菌液；
    (3)将发酵好的菌液离心，将菌体接入有机相体系中发酵生产维生素A棕榈酸酯。
  5. 根据权利要求4所述的应用，其特征在于，所述步骤(2)中，发酵培养基成分为：
    胰蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，MgSO 4·7H 2O 1g/L，KH 2PO 40.5g/L，K 2HPO 40.5g/L，橄榄油乳化液12mL/L。
  6. 根据权利要求5所述的应用，其特征在于，所述橄榄油乳化液配制方法为：将橄榄油乳化剂PVA与橄榄油以体积比3：1混合，利用超声波乳化。
  7. 根据权利要求4所述的应用，其特征在于，所述步骤(3)中，有机相体系为将维生素A和棕榈酸按照1：1的质量比溶于有机溶剂。
  8. 根据权利要求7所述的应用，其特征在于，所述维生素A和棕榈酸浓度为10～25g/L；优选15g/L。
  9. 根据权利要求7所述的应用，其特征在于，所述有机溶剂为正己烷。
  10. 根据权利要求4所述的应用，其特征在于，所述步骤(3)中，将菌体接入有机相体系中发酵0.5～2h；优选1h。
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