WO2017197993A1 - 一种液化沙雷氏菌及其转化合成洋茉莉醛的方法 - Google Patents

一种液化沙雷氏菌及其转化合成洋茉莉醛的方法 Download PDF

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WO2017197993A1
WO2017197993A1 PCT/CN2017/079320 CN2017079320W WO2017197993A1 WO 2017197993 A1 WO2017197993 A1 WO 2017197993A1 CN 2017079320 W CN2017079320 W CN 2017079320W WO 2017197993 A1 WO2017197993 A1 WO 2017197993A1
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zmt
serratia liquefaciens
producing
resin
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郑璞
邓志敏
陈鹏程
赵希景
赵明涛
邢晨光
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厦门欧米克生物科技有限公司
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Priority to EP17798557.9A priority Critical patent/EP3460047A4/en
Priority to US16/090,748 priority patent/US11332766B2/en
Publication of WO2017197993A1 publication Critical patent/WO2017197993A1/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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    • 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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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/425—Serratia

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  • the invention relates to a method for transforming and synthesizing jasmonic acid, and belongs to the field of biotechnology.
  • Heliotropin also known as piperonal, has a chemical name of 3,4-dioxymethylenebenzaldehyde, a molecular formula of C 8 H 6 O 3 , and a molecular weight of 150.14.
  • Jasmine aldehyde is widely found in essential oils, violets, camphor woods and hedgehogs. It has aromas similar to sunflower and cherry. Its flavor is powerful sweet floral, spicy and slightly bitter. It is the most important and most attractive in the world.
  • One of the spices One of the spices.
  • jasmine aldehyde In the perfume industry, you can use jasmine aldehyde as a starting material to prepare new jasmonaldehyde, piperonyl acetone, jasmononitrile, etc. These substances are widely used in cosmetics such as perfumes; in medicine, you can use jasmine aldehyde Raw materials for the preparation of antihypertensive, cerebrovascular diseases and anticancer drugs intermediates; in agriculture, can use jasmine aldehyde as a starting material to prepare synergistic powder and synergistic aldehydes; industrially used as a polishing agent. In the 1990s, the annual production of jasmine aldehyde in the world was around 550 tons, mainly produced by China, Japan, and Spain. By 2012, the annual production of jasmine aldehyde in the world reached 1,100 tons, and the world demand for jasmine aldehyde. The amount is increasing year by year.
  • the industrial production of jasmine aldehyde is mainly through the semi-synthesis method using scutellaria oil as raw material and the total synthesis method using catechol as raw material.
  • the process is mature, the environmental pollution is serious and the energy consumption is high.
  • the bio-fermentation and enzymatically produced perfume substances are considered to be natural fragrance substances, and are increasingly being used by people.
  • the first technical problem to be solved by the present invention is to provide a microorganism strain, Serratia liquefaciens ZMT-1, which can convert xanthine to produce jasmonaldehyde.
  • the Serratia liquefaciens ZMT-1 was deposited with the China Center for Type Culture Collection on April 5, 2016.
  • the deposit number is CCTCC NO: M 2016170, and the deposit address is Wuhan University, Wuhan, China.
  • the Serratia liquefaciens ZMT-1 is on the base inorganic salt medium plate, and the colony is light white, translucent, viscous, with convex in the middle and neat edges with a diameter of about 1 mm. Electron micrographs showed that the cells were straight-roded, rounded, no spores, no capsules, and flagella; the size was (0.5-0.8) ⁇ m ⁇ (0.5-2.0) ⁇ m.
  • the basic inorganic salt medium comprises: ammonium nitrate 1g/L, magnesium sulfate heptahydrate 0.5g/L, ammonium sulfate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 1.5g/L, chlorine Sodium 0.5g/L, pH 7.0.
  • the method for cultivating the Serratia liquefaciens ZMT-1 may be: inoculating the original strain in a slant medium, culturing at 25 to 37 ° C for 1 to 2 days for activation; and inactivating the strain and inoculating the seed medium at 25 Incubate for 24 to 36 hours under conditions of ⁇ 37 ° C and 150 to 220 rpm.
  • the seed culture medium peptone 1%, yeast powder 0.5%, sodium chloride 1%, glucose 1%, pH natural.
  • a second technical problem to be solved by the present invention is to provide a method for applying the Serratia liquefaciens ZMT-1 to bio-transformation of jasmonaldehyde by using xanthine as a raw material.
  • the seed liquid is inoculated to a fermentation medium having a liquid volume of 10 to 40% at a volume ratio of 2% to 10%, and the temperature is 22 to 37 ° C, and the rotation speed is 150 ⁇ . After incubation at 220 rpm for 24 to 36 hours, the substrate was added and the mixture was transformed under the same temperature and rotation conditions for 24-48 hours.
  • the fermentation medium comprises: ammonium nitrate 0.5-1 g/L, magnesium sulfate heptahydrate 0.1-0.5 g/L, ammonium sulfate 0.1-0.5 g/L, potassium dihydrogen phosphate 0.1. ⁇ 0.5g/L, dipotassium hydrogen phosphate 0.5 ⁇ 1.5g/L, sodium chloride 0.1 ⁇ 0.5g/L, yeast powder 0.1 ⁇ 1g/L, glucose 3 ⁇ 20g/L, pH 6.8 ⁇ 7.5.
  • the glucose may be replaced with fructose, or mannitol, or sucrose, or glycerol or maltose.
  • the amount of the substrate is 0.5 to 3 g/L.
  • the Serratia liquefaciens ZMT-1 seed solution is inoculated into a fermentation medium having a liquid volume of 20% by volume 5% at a temperature of 30 ° C and a rotation speed of 180 rpm. Under the conditions, after culturing for 24 to 36 hours, the substrate was added and transformed under the same temperature and rotation conditions for 48 hours.
  • a wet resin is added to each 100 mL of the conversion system, and the resin is a macroporous resin XAD-2 or HZ-802, and the adsorption is oscillated for 30 to 60 minutes.
  • the mixture was filtered, washed with ethyl acetate (1 to 2 times the volume of the resin), and the eluate was dried over anhydrous sodium sulfate.
  • the filtrate was concentrated in vacuo at 30 to 50 ° C and allowed to stand at 4 ° C in a refrigerator.
  • the invention screens a strain of serratia liquefaciens ZMT-1 which produces jasmonaldehyde from soil, has a wide conversion temperature of 22-37 ° C, and has mild reaction conditions, and can be obtained by using a complicated nitrogen source such as yeast powder in a short time.
  • the higher yield of jasmine aldehyde 160 ⁇ 500mg/L has a good application prospect.
  • Serratia liquefaciens ZMT-1 was deposited with the China Center for Type Culture Collection on April 5, 2016. The deposit number is CCTCC NO: M 2016170 and the deposit address is Wuhan University, China.
  • Figure 1 shows the gas phase mass spectrometry (GC-MS) identification of the conversion products.
  • Figure 2 shows the infrared spectral scanning identification of the product.
  • GC-MS gas chromatography-mass spectrometry
  • GC-MS conditions TSQ8000 mass spectrometer, carrier gas is helium, flow rate is 1mL/min, column: Agilent HP-5 (30m*0.25mm, 0.25 ⁇ m); detection procedure: initial temperature initial 110 °C was maintained for 1 min, raised to 120 ° C at 10 ° C / min for 15 min, then increased to 20 ° C / min to 250 ° C (10 min); injection volume: 0.1 ⁇ l.
  • Infrared spectrum scanning identification The infrared spectrum of the sample was measured by KBr tableting method, Nexus type Fourier transform infrared spectrometer (test resolution ⁇ 0.5cm -1 , scanning times 64 times, test range 378 ⁇ 4000cm -1 ).
  • the sieving still adopts the method of tube culture, and the strain that needs to be rescreened is transferred to the liquid fermentation medium for 24 to 36 hours, and then the substrate is added with xanthine oil 0.5-1.5 g/L, and converted at 30 ° C and 150-220 rpm.
  • 24 ⁇ 48h after the end of the transformation, take 2mL of the conversion solution and centrifuge to take the supernatant, carry out color reaction with 2,4-dinitrophenylhydrazine or analyze the conversion products by high performance liquid chromatography (HPLC) and gas phase detection (GC).
  • HPLC high performance liquid chromatography
  • GC gas phase detection
  • Gas chromatography-mass spectrometry was used to identify the product, and ZMT-1 was the most prominent for accumulating jasmonaldehyde.
  • Figure 1 shows the results of identification of the conversion solution by gas chromatography-mass spectrometry (GC-MS).
  • strain ZMT-1 was identified for morphological and physiological and biochemical characteristics according to the "Industrial Microbial Experimental Technology Manual” and “Bergey's Manual of Determinative Bacteriology” (1994).
  • Morphological description of the strain ZMT-1 on the basic inorganic salt plate, the colony is light white, translucent, viscous, with convex in the middle and neat edges with a diameter of about 1 mm. Electron micrographs showed that the cells were straight-roded, rounded, no spores, no capsules, and flagella; the size was (0.5-0.8) ⁇ m ⁇ (0.5-2.0) ⁇ m.
  • Facultative aerobic, Gram-negative can grow in the temperature range of 10 ⁇ 37 ° C, pH 4 ⁇ 9, containing 0% ⁇ 4% NaCl (w / v); can make the milk sputum, make gelatin liquefaction, Catalase can be produced, the urease reaction is weakly positive, no amylase is produced; H 2 S is not produced, the sputum reaction is negative, and the nitrate reduction is positive. Its physiological and biochemical characteristics are strong tolerance to temperature, pH and NaCl.
  • the genomic DNA of the strain ZMT-1 was extracted and screened according to the method of bacterial genomic DNA extraction kit (Shanghai Jierui Bioengineering Co., Ltd.), and the universal primers for bacteria (upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer 1492R: 5 '-GGTTACCTTGTTACGACTT-3')
  • upstream primer 27F 5'-AGAGTTTGATCCTGGCTCAG-3'
  • downstream primer 1492R 5 '-GGTTACCTTGTTACGACTT-3'
  • the 16S rRNA gene sequence was amplified by PCR and sequenced by Shanghai Biotech Co., Ltd.
  • the 16S rRNA gene sequence fragment was submitted to GeneBank under the accession number KU999993.
  • the 16S rRNA gene sequence of ZMT-1 was compared with the 16S rRNA gene sequence of the relevant strains in GeneBank by the BLAST search program on the NCBI website (Table 1). The results showed that the 16S rDNA sequence of the strain ZMT-1 was in the GeneBank database.
  • the 16S rDNA sequence of the multiple strains of the genus Serratia is 98% to 99% homologous. 15 strains with higher homology were downloaded from http://rdp.cme.msu.ed for comparison, and the strain ZMT-1 was found to be closely related to the typical strain Serratia liquefaciens ATCC 27592.
  • strain ZMT-1 and Serratia liquefaciens ATCC 27592 differ in carbon source utilization (such as lactose, etc.), different in MR, and the same in VP reaction; in oxidase, contact enzyme, gelatin decomposition, nitrate reductase, hydrazine experiment The same is true; because strain ZMT-1 is a fermentation strain, arginine double hydrolase is negative, so it is not a format of Serratia; ZMT-1 metabolizes arabinose to produce acid, so it is not a sticky saray bacteria.
  • carbon source utilization such as lactose, etc.
  • strain ZMT-1 is considered to be a strain of Serratia liquefaciens and named as Serratia liquefaciens ZMT-1, which was deposited in China on April 5, 2016. Typical Culture Collection, under the accession number CCTCC NO: M 2016170.
  • ATCC 27592 indicates Serratia liquefaciens ATCC 27592; +, indicating positive; w, indicating weak positive; -, indicating negative.
  • Fermentation medium composition ammonium nitrate 1g / L, magnesium sulfate heptahydrate 0.5g / L, ammonium sulfate 0.5g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1.5g / L, sodium chloride 0.5g /L, yeast powder 0.5g / L, glucose 5g / L, pH 6.8 ⁇ 7.5.
  • the liquid loading amounts were 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, and 350 mL, respectively. Incubate at 30 ° C, 180 rpm for 24 h. A 0.9 g/L substrate was added and transformed under the same culture conditions for 36 h, and the product concentration was determined according to liquid phase detection. The results are shown in Table 3.
  • Serratia liquefaciens, ZMT-1 seed solution was inoculated with 5% inoculum, respectively, and a fermentation medium containing glucose, fructose, mannitol, sucrose, glycerol and maltose as carbon sources. Fermentation conversion was carried out in the same manner as in Example 2, and the results are shown in Table 4.
  • the strain ZMT-1 seed solution was inoculated into a 500 mL flask with a 5% inoculum, and the liquid volume was 200 mL. Incubate at 30 ° C, 180 rpm for 30 h. A 1.8 g/L substrate was added for conversion, and the final yield of jasmonaldehyde was 524 mg/L.

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Abstract

提供了一株液化沙雷氏菌（Serratia liquefaciens）ZMT-1，其保藏编号为CCTCC NO:M2016170，以及利用该液化沙雷氏菌菌株ZMT-1或其产的酶为催化剂，以黄樟油素为原料生产洋茉莉醛的方法。

Description

一种液化沙雷氏菌及其转化合成洋茉莉醛的方法 技术领域
本发明涉及一种液化沙雷氏菌及其转化合成洋茉莉醛的方法，属于生物技术领域。
背景技术
洋茉莉醛(Heliotropin)又称胡椒醛(Piperonal)，化学名称为3，4-二氧亚甲基苯甲醛，分子式C8H6O3，分子量150.14。在19世纪中叶，Rud.Fittig和W.H.Mielch在研究生物碱胡椒碱的水解时发现该物质。洋茉莉醛广泛存在于香精油、紫罗兰、樟脑木以及刺槐中，具有类似葵花和樱桃的香气，其口味是有力的甜花香，辛香和微苦，是世界上最主要的且最吸引人的香料之一。
在香料行业中，可以利用洋茉莉醛为起始原料制备新洋茉莉醛，胡椒基丙酮，洋茉莉腈等，这些物质广泛的应用于香水等化妆品中；在医药上，可以利用洋茉莉醛为原料制备抗高血压、脑血管疾病以及抗癌药物的中间体；在农业上，可以利用洋茉莉醛为起始原料制备增效散以及增效醛等；在工业上常用作抛光剂。上世纪90年代，世界洋茉莉醛年产量在550吨左右，主要是由中国、日本、西班牙三个国家生产，到2012年，世界洋茉莉醛年产量达到1100吨，世界对洋茉莉醛的需求量在逐年增加。
目前洋茉莉醛的工业生产主要是通过以黄樟油素为原料的半合成法和以邻苯二酚为原料的全合成法，虽然工艺成熟，但环境污染严重，并且耗能较多。随着人们对物质生产过程中的关注及欧洲国家和美国法律对食品添加物质的要求，生物发酵法及酶法生产的的香料物质，被认为是天然的的香料物质，越来越受到人们的青睐。现阶段工业上还没有大规模的生物法生产洋茉莉醛的报道。
发明内容
本发明要解决的第一个技术问题是提供一株可以转化黄樟油素生产洋茉莉醛的微生物菌株，液化沙雷氏菌ZMT-1。
所述液化沙雷氏菌(Serratia liquefaciens)ZMT-1已于2016年4月5日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2016170，保藏地址为中国武汉武汉大学。
所述液化沙雷氏菌ZMT-1在基础无机盐培养基平板上，菌落呈浅白色，半透明的，粘稠的，中间凸起，边缘整齐，直径1mm左右。电镜照片表明，菌体细胞为直杆状，端圆，不产生芽孢，无荚膜，有鞭毛；大小为(0.5～0.8)μm×(0.5-2.0)μm。兼性需氧，革兰氏阴性，能够在温度范围10～37℃、pH 4～9、含有0％～4％的NaCl的条件下生长；能够较好利用葡萄糖、蔗糖、甘露醇、果糖、麦芽糖、甘油；能够利用铵盐、硝酸盐以及酵母粉、蛋白胨等 复杂氮源。
所述基础无机盐培养基含：硝酸铵1g/L，七水硫酸镁0.5g/L，硫酸铵0.5g/L，磷酸二氢钾0.5g/L，磷酸氢二钾1.5g/L，氯化钠0.5g/L，pH 7.0。
所述液化沙雷氏菌ZMT-1的培养方法，可以是将原始菌株接种于斜面培养基，25～37℃培养1～2天，进行活化；将菌株活化后接种于种子培养基，在25～37℃、150～220rpm条件下震荡培养24～36h。所述种子培养基：蛋白胨1％，酵母粉0.5％，氯化钠1％，葡萄糖1％，pH自然。
本发明要解决的第二个技术问题是提供一种应用于所述液化沙雷氏菌Serratia liquefaciens ZMT-1，以黄樟油素为原料，生物转化生产洋茉莉醛的方法。
在本发明的一种实施方式中，是将种子液按体积比2％～10％接种到装液量为10～40％的发酵培养基中，在温度为22～37℃，转速为150～220rpm的条件下，培养24～36h后，加底物，相同温度、转速条件下转化24～48h。
在本发明的一种实施方式中，所述发酵培养基含有：硝酸铵0.5～1g/L，七水硫酸镁0.1～0.5g/L，硫酸铵0.1～0.5g/L，磷酸二氢钾0.1～0.5g/L，磷酸氢二钾0.5～1.5g/L，氯化钠0.1～0.5g/L，酵母粉0.1～1g/L，葡萄糖3～20g/L，pH 6.8～7.5。其中，所述葡萄糖可以用果糖、或甘露醇、或蔗糖、或甘油或麦芽糖代替。底物的用量为0.5～3g/L。
在本发明的一种实施方式中，将液化沙雷氏菌ZMT-1种子液按体积比5％接种到装液量为20％的发酵培养基中，在温度为30℃，转速为180rpm的条件下，培养24～36h后，加底物，相同温度、转速条件下转化48h。
在本发明的一种实施方式中，转化结束后，向每100mL转化体系中添加湿树脂5～50g，所述树脂为大孔树脂XAD-2或HZ-802，震荡吸附30～60min，将树脂过滤出，用1～2倍于树脂体积的乙酸乙酯洗脱，洗脱液加无水硫酸钠脱水后，滤液30～50℃真空浓缩，于4℃冰箱静置结晶。
本发明从土壤中筛选出一株产洋茉莉醛的菌株Serratia liquefaciens ZMT-1，具有广泛的转化温度22～37℃，并且反应条件温和，可以利用酵母粉等复杂氮源，短时间内可以获得较高的洋茉莉醛产量(160～500mg/L)，具有良好的应用前景。
生物材料保藏
液化沙雷氏菌(Serratia liquefaciens)ZMT-1，于2016年4月5日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2016170，保藏地址为中国武汉大学。
附图说明
图1为转化产物气相质谱联用(GC-MS)鉴定。
图2为产物的红外光谱扫描鉴定。
具体实施方式
(GC-MS)分析产物：取转化液2mL进行离心，取上清，过0.45um微孔滤膜，进液相或者取转化液2mL，用等体积的乙酸乙酯进行萃取，静置，取上层有机相，加无水硫酸钠除去多余水分，用0.45um有机滤膜进行过滤处理，然后进行气质联用(GC-MS)分析产物。气质联用(GC-MS)条件：TSQ8000质谱仪，载气为氦气，流速为1mL/min，色谱柱：安捷伦HP-5(30m*0.25mm,0.25μm)；检测程序：初始温度初始110℃维持1min，以10℃/min升至120℃后维持15min，再以20℃/min升至250℃(10min)；进样体积：0.1μl。扫描程序：电子冲击波70eV，频率1scan/0.2s，m/e：40～400单位。进样口温度270℃，传输线温度280℃。
采用HPLC分析转化液中产物的成分，如用waters 1525高效液相色谱仪流动相：甲醇：水：冰醋酸＝55:45:0.05；进样量：10uL；流速1mL/min；检测波长：245nm；柱温：30℃；检测器为DAD；色谱柱为Amethyst C18-H反向柱(5μm，4.6mm×250mm)。
红外光谱扫描鉴定：采用KBr压片法，Nexus型傅立叶变换红外光谱仪(测试分辨率≦0.5cm-1，扫描次数为64次，测试范围为378～4000cm-1)测样品红外谱图。
实施例1菌株Serratia liquefaciens ZMT-1的筛选与鉴定
(1)微生物菌株的筛选：
从厦门、无锡、抚远县西藏林芝、湖北以及河南等地采集25份土样，经过富集处理，取样涂平板，然后挑取单菌落296个于平板划线，菌株生长完全后，于试管中进行转化培养，转化结束后，取2mL转化液离心取上清，并与2,4-二硝基苯肼进行反应，出现橙红色的菌株进行复筛。复筛依旧采取试管培养的方法，将需要复筛的菌株转接于液体发酵培养基培养24～36h，然后加入底物黄樟油素0.5～1.5g/L，30℃、150～220rpm下转化24～48h，转化结束后，取2mL转化液离心取上清，与2,4-二硝基苯肼进行显色反应或者利用高效液相色谱(HPLC)、气相检测(GC)分析转化产物以及气质联用(GC-MS)鉴定产物，其中ZMT-1为积累洋茉莉醛最显著。图1为转化液用气相质谱联用(GC-MS)鉴定的结果。
(2)微生物菌株的鉴定
对菌株ZMT-1按《工业微生物实验技术手册》以及《Bergey’s Manual of Determinative Bacteriology》(1994)进行形态学以及生理生化特征的鉴定。
菌株形态学描述：ZMT-1在基础无机盐平板上，菌落呈浅白色，半透明的，粘稠的，中间凸起，边缘整齐，直径1mm左右。电镜照片表明，菌体细胞为直杆状，端圆，不产生芽孢，无荚膜，有鞭毛；大小为(0.5～0.8)μm×(0.5-2.0)μm。兼性需氧，革兰氏阴性，能够在温度范围10～37℃、pH 4～9、含有0％～4％的NaCl(w/v)时生长；可使牛奶胨化，使明胶液 化，可产生过氧化氢酶，脲酶反应弱阳性，不产生淀粉酶；不产生H2S，吲哚反应阴性，硝酸盐还原阳性。其生理生化的显著特性是对温度、pH、NaCl的耐受性较强。
按照细菌基因组DNA抽提试剂盒(上海捷瑞生物工程有限公司)方法提取筛选菌株ZMT-1的基因组DNA，以细菌通用引物(上游引物27F：5'-AGAGTTTGATCCTGGCTCAG-3'；下游引物1492R：5'-GGTTACCTTGTTACGACTT-3')通过PCR扩增其16S rRNA基因序列，委托上海生工有限公司进行测序，测得的16S rRNA基因序列片段提交至GeneBank,登记号为KU999993。ZMT-1的16S rRNA基因序列与NCBI网站中用BLAST检索程序检索GeneBank中相关菌株的16S rRNA基因序列进行同源性对比(表1)，结果表明菌株ZMT-1的16S rDNA序列与GeneBank数据库中沙雷氏菌属的多株菌的16S rDNA序列的同源性在98％～99％。在从http://rdp.cme.msu.ed网站下载15个同源性较高的菌株进行比对，得出菌株ZMT-1与典型菌株Serratia liquefaciens ATCC 27592亲缘关系最近。
菌株ZMT-1与Serratia liquefaciens ATCC 27592在碳源利用(如乳糖等)方面有所不同、M-R方面不同，V-P反应方面相同；在氧化酶、接触酶、明胶分解、硝酸盐还原酶、吲哚实验等方面完全相同；又因菌株ZMT-1为发酵型菌株，精氨酸双水解酶阴性，所以其不是格式沙雷氏菌；ZMT-1代谢阿拉伯糖产酸，所以其不是粘质沙雷氏菌。
综上，认为菌株ZMT-1为一株液化沙雷氏菌(Serratia liquefaciens)，并命名为液化沙雷氏菌(Serratia liquefaciens)ZMT-1，此菌已于2016年4月5日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2016170。
表1同源性分析表
Figure PCTCN2017079320-appb-000001
Figure PCTCN2017079320-appb-000002
表2生理生化特性对照表
Figure PCTCN2017079320-appb-000003
Figure PCTCN2017079320-appb-000004
ATCC 27592表示Serratia liquefaciens ATCC 27592；+，表示阳性；w，表示弱阳性；—，表示阴性。
实施例2
以葡萄糖为单一碳源，菌株ZMT-1种子液以5％接种量分别接种于6个500mL三角瓶中。发酵培养基组成：硝酸铵1g/L，七水硫酸镁0.5g/L，硫酸铵0.5g/L，磷酸二氢钾0.5g/L，磷酸氢二钾1.5g/L，氯化钠0.5g/L，酵母粉0.5g/L，葡萄糖5g/L，pH 6.8～7.5。装液量分别为50mL、100mL、150mL、200mL、250mL、300mL、350mL。30℃、180rpm下震荡培养24h。添加0.9g/L底物，于相同培养条件下转化36h，产物浓度根据液相检测测定，结果如表3。
表3不同装液量对转化的影响
装液量(mL) 50 100 150 200 250 300 350
36h产量(mg/L) 150 183 198 201 192 200 205
48h产量(mg/L) 162 205 214 227 226 223 225
实施例3
液化沙雷氏菌，ZMT-1种子液以5％接种量，分别接入以葡萄糖、果糖、甘露醇、蔗糖、甘油、麦芽糖为碳源的发酵培养基。按实施例2的方法进行发酵转化，结果如表4。
表4不同碳源转化情况
碳源 产量(mg/L)
葡萄糖 204
果糖 191
甘露醇 167
蔗糖 196
甘油 171
麦芽糖 185
实施例4
以葡萄糖为单一碳源，菌株ZMT-1种子液以5％接种量接种于500mL三角瓶中，装液量为200mL。30℃、180rpm下震荡培养30h。添加1.8g/L底物进行转化，最终洋茉莉醛产量524mg/L。向转化体系中添加树脂湿重/体系体积为5％～50％的大孔树脂XAD-2或HZ-802，震荡吸附30～60min，将树脂过滤出，用两倍于树脂体积的乙酸乙酯在35～45℃条件下进行洗脱，加无水硫酸钠静置脱水8～10h，滤液真空浓缩至洋茉莉醛含量250～300g/L，用自来水冲洗蒸馏瓶使其冷却，然后用2～5倍浓缩液体积的冷水冲洗浓缩液，于4℃冰箱静置结晶，低温干燥后，得到约210mg晶体样品。图2为产物的红外光谱鉴定结果，经红外测定，为洋茉莉醛。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (9)

  1. 一株液化沙雷氏菌(Serratia liquefaciens)ZMT-1，保藏编号为CCTCC NO:M2016170。
  2. 一种生产洋茉莉醛的方法，其特征在于，以黄樟油素为原料，以液化沙雷氏菌ZMT-1，保藏编号为CCTCC NO:M2016170或其产的酶为催化剂。
  3. 根据权利要求2所述生产洋茉莉的方法，其特征在于，是将液化沙雷氏菌ZMT-1种子液按体积比2％～10％接种到装液量为10～40％的发酵培养基中，在温度为22～37℃，转速为150～220rpm的条件下，培养24～36h后，加底物，相同温度、转速条件下转化24～48h。
  4. 根据权利要求3所述生产洋茉莉的方法，其特征在于，所述发酵培养基含有：硝酸铵0.5～1g/L，七水硫酸镁0.1～0.5g/L，硫酸铵0.1～0.5g/L，磷酸二氢钾0.1～0.5g/L，磷酸氢二钾0.5～1.5g/L，氯化钠0.1～0.5g/L，酵母粉0.1～1g/L，葡萄糖3～20g/L，pH6.8～7.5。
  5. 根据权利要求4所述生产洋茉莉的方法，其特征在于，所述葡萄糖用果糖、或甘露醇、或蔗糖、甘油或麦芽糖代替。
  6. 根据权利要求3所述生产洋茉莉的方法，其特征在于，底物的用量为0.5～3g/L。
  7. 根据权利要求3～6任一所述生产洋茉莉的方法，其特征在于，将液化沙雷氏菌ZMT-1种子液按体积比5％接种到装液量为20％的发酵培养基中，在温度为30℃，转速为180rpm的条件下，培养24～36h后，加底物，相同温度、转速条件下转化48h。
  8. 根据权利要求3所述生产洋茉莉的方法，其特征在于，转化结束后，向每100mL转化体系中添加湿树脂5～50g，所述树脂为大孔树脂XAD-2或HZ-802，震荡吸附30～60min，将树脂过滤出，用1～2倍于树脂体积的乙酸乙酯洗脱，洗脱液加无水硫酸钠脱水后，滤液30～50℃真空浓缩，于4℃冰箱静置结晶。
  9. 如权利要求1所述液化沙雷氏菌ZMT-1的应用，其特征在于，所述应用的领域包括食品、饲料、化妆品、香精香料、化学和医药领域。
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