WO2023004983A1 - 一株柠檬明串珠菌菌株及其应用 - Google Patents

一株柠檬明串珠菌菌株及其应用 Download PDF

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WO2023004983A1
WO2023004983A1 PCT/CN2021/120964 CN2021120964W WO2023004983A1 WO 2023004983 A1 WO2023004983 A1 WO 2023004983A1 CN 2021120964 W CN2021120964 W CN 2021120964W WO 2023004983 A1 WO2023004983 A1 WO 2023004983A1
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strain
leuconostoc
sucrase
fructan sucrase
fructan
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PCT/CN2021/120964
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吴正钧
刘振民
韩瑨
王晓花
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光明乳业股份有限公司
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Priority to AU2021441277A priority Critical patent/AU2021441277B2/en
Publication of WO2023004983A1 publication Critical patent/WO2023004983A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1055—Levansucrase (2.4.1.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/0101—Levansucrase (2.4.1.10)

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  • the invention relates to the technical field of microorganisms, in particular to a leuconostoc strain and application thereof.
  • Levan is a kind of fructose macromolecular polymer derived from plants or microorganisms. Its molecules usually contain a large number of ⁇ -(2,6) fructosidic bonds in the main chain and a small amount of ⁇ -(2,1) fructosidic bonds. composed of chains. Studies have shown that Levan derived from microorganisms has anti-tumor, anti-diabetic, immune-enhancing, and blood-lipid-lowering functions. In addition, Levan can also be used in the preparation of nanomaterials and drug carriers. In order to improve the purity of the product Levan, levansucrase is usually extracted from microorganisms, and then reacted with a substrate to obtain higher quality Levan.
  • fructan sucrase a bacterial strain with safe source and diversified products (fructan sucrase) to satisfy the market.
  • the present invention provides a strain of Leuconostoc citreum and application thereof.
  • the present invention provides the following technical solutions:
  • the invention provides a strain of Leuconostoc citreum, which is classified as Leuconostoc citreum and whose preservation number is CGMCC NO.6431.
  • fructan sucrase coding genes there are two consecutively arranged and normally expressed fructan sucrase coding genes on the genome of the strain.
  • fructan sucrase coding gene codes and expresses fructan sucrase with three different molecular weights.
  • the molecular weights of the fructan sucrase are 130kDa, 90kDa and 80kDa respectively.
  • the invention also provides an application of Leuconostoc citricum in the preparation of fructan sucrase.
  • the strain produced three kinds of natural fructan sucrase with different molecular weights by single fermentation.
  • the present invention discloses a strain of Leuconostoc citrea for the first time.
  • Two fructan sucrase encoding genes that can be normally expressed and have low homology with known similar genes are continuously arranged on the genome of the strain.
  • the fruit polysaccharide of the present application The homology of the sugar sucrase coding gene with known similar genes is less than 30%. More importantly, these two fructan sucrase encoding genes can encode and express natural fructan sucrase enzymes with three different molecular weights, so that Leuconostoc citrate can obtain three natural fruit sucrase enzymes with different molecular weights in a single fermentation.
  • the polysaccharide sucrase greatly improves the preparation efficiency of the levan sucrase, and the above-mentioned characteristics make the technical scheme have significant technical advantages, and thus has a good application prospect in the field of levan sucrase preparation.
  • Figure 1 is the distribution map of genes 1291 and 1292 on the genome of CGMCC NO.6431;
  • Fig. 2 is the SDS-PAGE gel picture of protein 1291 and 1292;
  • Fig. 3 is a homology comparison diagram of genes 1291 and 1292;
  • Fig. 4 is the in situ activity detection result of fructan sucrase in fermented liquid
  • Figure 5 is the nuclear magnetic resonance spectrum of the polysaccharide sample.
  • the application provides a strain of Leuconostoc citreum and its application.
  • Leuconostoc citreum is CGMCC NO.6431.
  • the classification of the strain is named Leuconostoc citreum, which was preserved in the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on August 13, 2012.
  • CGMCC General Microbiology Center
  • Leuconostoc citrea has two fructan sucrase coding genes that are arranged continuously and normally expressed.
  • the homology between the fructan sucrase coding gene and known similar genes is less than 30%.
  • levan sucrase can encode and express three levan sucrase enzymes with different molecular weights.
  • the molecular weights of fructan sucrase are 130kDa, 90kDa and 80kDa respectively.
  • the above-mentioned technical scheme discloses a strain of Leuconostoc citrate for the first time, and two fructan sucrase enzymes that can be normally expressed and have low homology to known similar genes are continuously arranged on the genome of the strain Encoding gene, more importantly, these two fructan sucrase encoding genes can encode and express three kinds of natural fructan sucrase with different molecular weights, so that Leuconostoc citrate can obtain three kinds of different molecular weights in a single fermentation
  • the natural fructan sucrase greatly improves the production efficiency of fructan sucrase, and the above-mentioned characteristics make the technical solution have significant technical advantages, so it has a good application prospect in the field of fructan sucrase preparation.
  • a method for determining the gene encoding fructan sucrase specifically comprising the following:
  • the bacterial solution was taken out, cooled in ice, centrifuged at 5000 rpm at 4°C for 15 minutes, the bacterial cells were collected, washed once with the bacteriostasis buffer, poured out the bacteriostasis buffer, and frozen at -80°C.
  • the maximum flow rate is 0.5mL/min per 1mL column volume
  • wash the column with washing buffer until no protein is eluted then wash the elution buffer to elute the target protein.
  • the elution buffer containing the target protein is desalted with a desalting column, and the eluate is collected to obtain the purified target protein.
  • the sample to be tested was mixed with Native laoding, loaded on a 4-20% SDS-PAGE gradient gel (Nanjing GenScript Biotechnology Co., Ltd., China), and electrophoresed at 110V for 1.5h.
  • Half of the obtained protein gel was used for Coomassie brilliant blue staining, and the other half was soaked twice with NaAc solution (20mM, pH5.5), and then placed in NaAc solution (20mM, pH5.5) containing 5% sucrose and 0.3 ⁇ NaN3 After reacting at 30°C for 48 hours, the number and position of the bands of the in situ reaction product were observed in a dark room to confirm the number and molecular weight of the fructan sucrase synthesized by Leuconostoc citrea.
  • Leuconostoc citreum (Leuconostoc citreum) CGMCC NO.6431 has two consecutive and normally expressed fructan sucrase coding genes (1291 and 1292).
  • Embodiment 2 Homology comparison of fructan sucrase coding gene
  • Gene 1291 and 1292 were compared with other known similar genes (CAD48195.1, AAO14618.1, WP010237336, AAB97111.1, AC15886.1, BAA04475, AAC36458.1, CBJ48143.1, CCM43846.1, CPR14579. 1. EHD23269.1, AAT81165.1, AAL09386.1, the sequences are all from the NCBI website) for homology comparison, the results are shown in Figure 3, the homology of genes 1291 and 1292 with known similar genes is ⁇ 30%, It shows that the two fructan sucrase coding genes have significant uniqueness.
  • sucrose 28 °C of aerobic cultures were taken out for 24 h, and a single colony was picked with an inoculation loop and put into 20 mL of M17 liquid medium containing 2% (w/v) sucrose ( Merck Co., Germany), 28°C, 180rpm shaker culture for 24h, take out the culture, centrifuge at 9,000rpm for 10 minutes, discard the supernatant, wash the bacteria twice with sterile distilled water, suspend with the original culture volume of sterile distilled water, The seeds used for fermentation were obtained, and the bacterial concentration of the seed solution was detected to be 1 ⁇ 10 9 CFU/mL.
  • Leuconostoc citricum seeds were aseptically inoculated at an inoculum size of 3% (v/v) in a tomato juice medium containing 15% (v/v) sucrose, pH 6.5, and cultured at 30° C. and 200 rpm for 96 hours to obtain a fermentation broth.
  • the in situ activity of fructan sucrase in the fermentation broth was detected, and the results are shown in Figure 4.
  • Three obvious polysaccharide bands were found in the in situ reaction of SDS PAGE, indicating that the fermentation broth contained three kinds of fructans with different molecular weights. Sucrase, its molecular weight is 130kDa, 90kDa and 80kDa respectively.
  • the fructan sucrase gene of Leuconostoc citrus CGMCC NO.6431 can encode and express three different molecular weight fructan sucrase enzymes, the molecular weights of which are 130kDa, 90kDa and 80kDa, respectively.
  • thermostable levansucrase from Bacillus sp.TH4-2 capable of producing high molecular weight levan at high temperature[J].Journal of Biotechnology,2002,99( 2): 111-119.
  • the fructan sucrase coding gene of the present application has a homology of less than 30% with known similar genes. Moreover, the fructan sucrase produced by the fermentation of Leuconostoc citrate not only has many kinds, but the fructan sucrase with a molecular weight of 130kDa is currently the largest known levan sucrase with natural activity.
  • fructan sucrase the crude enzyme extract was dissolved in distilled water and loaded on the AKTA protein purification system (GE Company, USA), packing: Sephacryl S-300 High Resolution, flow rate: 1mL/min, column Volume: 120mL, detector: UV detector, collect the eluate with higher 280nm signal according to the UV signal, and freeze-dry. Take 5 mg of each sample and add it to NaAc solution (20mM, pH 5.5) containing 5% sucrose and 0.3 ⁇ NaN3, react at 30°C for 48h, and then measure Whether there is free glucose production, if so, the sample is fructan sucrase.
  • the fermented liquid prepared in Example 3 was centrifuged at 8000 g for 10 min to obtain the supernatant, and freeze-dried to obtain a crude enzyme extract, which was separated to obtain three fructan sucrase samples.

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Abstract

提供了一株柠檬明串珠菌菌株及其应用，该菌株的分类命名为柠檬明串珠菌Leuconostoc citreum，保藏编号为CGMCC NO.6431。该菌株的基因组上有两个连续排列且正常表达的果聚糖蔗糖酶编码基因。该果聚糖蔗糖酶编码基因编码表达三个分子量分别为130kDa、90kDa和80kDa的果聚糖蔗糖酶。该柠檬明串珠菌单次发酵即可获得三种不同分子量的天然的果聚糖蔗糖酶，提高了果聚糖蔗糖酶的制备效率。

Description

一株柠檬明串珠菌菌株及其应用 技术领域
本发明涉及微生物技术领域，具体是一株柠檬明串珠菌菌株及其应用。
背景技术
Levan是一类来源于植物或微生物的果糖大分子聚合物，其分子中通常含有大量β-(2，6)果糖苷键组成的主链及少量含有大量β-(2，1)果糖苷键组成的支链。研究表明，微生物来源的Levan具有抗肿瘤、抗糖尿病、增强免疫、降血脂等功能，此外，Levan还可用于纳米材料及药物载体的制备。为了提高产物Levan的纯度，通常会将果聚糖蔗糖酶(levansucrase)从微生物中提取出来，再与底物反应来获得更高品质的Levan。然而，目前已知的产果聚糖蔗糖酶的微生物较少，并且少数微生物还有致病性，此外，几乎所有的果聚糖蔗糖酶产生菌只能合成一种分子量大小的果聚糖蔗糖酶，因此，寻找一株来源安全、产物(果聚糖蔗糖酶)多样化的菌株来满足市场成为本领域急需解决的问题。
发明内容
基于上述技术问题，本发明提供了一株柠檬明串珠菌(Leuconostoc citreum)菌株及其应用。
为实现上述目的，本发明提供如下技术方案：
本发明提供了一株柠檬明串珠菌菌株，该菌株的分类命名为柠檬明串珠菌Leuconostoc citreum，保藏编号为CGMCC NO.6431。
进一步地，所述菌株的基因组上有两个连续排列且正常表达的果聚糖蔗糖酶编码基因。
进一步地，所述果聚糖蔗糖酶编码基因编码表达三个不同分子量的果聚糖蔗糖酶。
进一步地，所述果聚糖蔗糖酶的分子量分别为130kDa、90kDa和80kDa。
本发明还提供了一种柠檬明串珠菌在果聚糖蔗糖酶制备方面的应用。
进一步地，该菌株单次发酵制备三种不同分子量的天然果聚糖蔗糖酶。
与现有技术相比，本发明的有益效果是：
本发明首次公开了一株柠檬明串珠菌，该菌株的基因组上连续排列了两个可正常表达、与已知同类基因的低同源性的果聚糖蔗糖酶编码基因，本申请的果聚糖蔗糖酶编码基因与已知同类基因的同源性＜30％。更重要的是，这两个果聚糖蔗糖酶编码基因可编码表达三种不同分子量的天然的果聚糖蔗糖酶，使柠檬明串珠菌单次发酵即可获得三种不同分子量的天然的果聚糖蔗糖酶，大大提高了果聚糖蔗糖酶的制备效率，上述特点使本技术方案具有显著的技术优势，因而在果聚糖蔗糖酶制备领域具有良好的应用前景。
附图说明
图1为基因1291和1292在CGMCC NO.6431基因组上的分布图；
图2为蛋白1291和1292的SDS-PAGE胶图；
图3为基因1291和1292同源性比较图；
图4为发酵液中果聚糖蔗糖酶的原位活性检测结果；
图5为多糖样品的核磁共振图谱。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本申请提供了一株柠檬明串珠菌(Leuconostoc citreum)菌株及其应用。
具体的，柠檬明串珠菌(Leuconostoc citreum)的保藏号为CGMCC NO.6431。该菌株的分类命名为柠檬明串珠菌Leuconostoc citreum，该菌种于2012年8月13日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101。
进一步地，柠檬明串珠菌的基因组有两个连续排列且正常表达的果聚糖蔗糖酶编码基因。
进一步地，果聚糖蔗糖酶编码基因与已知同类基因的同源性＜30％。
进一步地，果聚糖蔗糖酶编码基因可编码表达三个不同分子量的果聚糖蔗糖酶。
进一步地，果聚糖蔗糖酶分子量分别为130kDa、90kDa和80kDa。
在另一个具体的实施方式中，柠檬明串珠菌在果聚糖蔗糖酶制备方面的应用。
进一步地，柠檬明串珠菌单次发酵可制备三种不同分子量的天然果聚糖蔗糖酶。
与现有技术相比，上述技术方案首次披露了一株柠檬明串珠菌，该菌株的基因组上连续排列了两个可正常表达、与已知同类基因的低同源性的果聚糖蔗糖酶编码基因，更重要的是，这两个果聚糖蔗糖酶编码基因可编码表达三种不同分子量的天然的果聚糖蔗糖酶，使柠檬明串珠菌单次发酵即可获得三种不同分子量的天然的果聚糖蔗糖酶，大大提高了果聚糖蔗糖酶的制备效率，上述特点使本技术方案具有显著的技术优势，因而在果聚糖蔗糖酶制备领域具有良好的应用前景。
下面通过实施例进一步说明上述具体实施方式，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
下述实施例中，所有原料均为市售，并均符合相关的国家标准。
实施例1
一种确定果聚糖蔗糖酶编码基因的方法，具体包括如下：
对柠檬明串珠菌(Leuconostoc citreum)CGMCC NO.6431进行了全基因组测序，从全基因组序列中发现2个连续排列的果聚糖蔗糖酶编码基因，分别命名为1291和1292(见图1)，其具体序列如下。
1291基因序列：
Figure PCTCN2021120964-appb-000001
Figure PCTCN2021120964-appb-000002
1292基因序列：
Figure PCTCN2021120964-appb-000003
Figure PCTCN2021120964-appb-000004
为进一步证明上述1291和1292的果聚糖蔗糖酶编码基因是否可正常表达，设计了异源表达实验：
1)大肠杆菌的蛋白表达
鉴定正确的表达质粒转化感受态E.coli BL21(DE3)或Transrosetta，挑取3个单菌落分别接种于5mL LB培养基(含相应抗生素)，37℃培养过夜，取出。将过夜培养物分别接种于800mL LB培养基(含相应抗生素)，37℃培养5h(OD600≈0.5)，转移至25℃降温0.5h，加入IPTG至终浓度0.2mM，25℃表达16h。表达完成后取出菌液于冰中冷却，4℃，5000rpm离心15min，收集菌体，用破菌缓冲液洗涤1次，倒出破菌缓冲液将菌体冻存于-80℃。
2)蛋白的分离与纯化
取出-80℃冻存的菌体，按菌体：溶液＝1：5的比例将菌体均匀重悬于破菌缓冲液中，使用超高压破菌仪(破菌压力1000bar)破碎细菌三次至半透明状，再将菌液18000rpm离心0.5h，取上清转移至烧杯中，使用蠕动泵上样至事先平衡完毕的HisTrap FF 5mL或1mL柱上，使用破菌缓冲液冲洗柱子至无蛋白洗出(最大流速为每1mL柱体积0.5mL/min)，再换用冲淋缓冲液冲洗柱子至无蛋白洗出，最后使用洗脱缓冲液洗脱出目的蛋白。将含有目的蛋白的洗脱缓冲液用脱盐柱进行脱盐，收集淋洗液，得到纯化完成的目的蛋白。
对上述目的蛋白进行SDS PAGE电泳和原位活性检测，方法如下：
将待测样品与Native laoding混合，上样于4-20％SDS-PAGE梯度胶(南京金斯瑞生物科技有限公司，中国)，并在110V电压下电泳1.5h。所得的蛋白胶一半用于考马斯亮蓝染色，另外一半用NaAc溶液(20mM、pH5.5)浸洗2次后，置于含5％蔗糖、0.3‰NaN3的NaAc溶液(20mM、pH5.5)中，30℃反应48h后，于暗室观察原位反应产物的条带数量和位置，以此确认柠檬明串珠菌合成的果聚糖蔗糖酶的数量和分子量。
结果如图2所示，在SDS PAGE胶上发现有基因1291和1292表达的蛋白条带，说明这两个果聚糖蔗糖酶编码基因均可正常表达。
结论：柠檬明串珠菌(Leuconostoc citreum)CGMCC NO.6431的基因组上有两个连续排列且正常表达的果聚糖蔗糖酶编码基因(1291和1292)。
实施例2果聚糖蔗糖酶编码基因同源性比较
采用mega7软件将基因1291和1292与其他已知的同类基因(CAD48195.1、AAO14618.1、WP010237336、AAB97111.1、AC15886.1、BAA04475、AAC36458.1、CBJ48143.1、CCM43846.1、CPR14579.1、EHD23269.1、AAT81165.1、AAL09386.1，序列均来自NCBI网站)进行同源性比较，结果如图3所示，基因1291和1292与已知同类基因的同源性＜30％，表明这两个果聚糖蔗糖酶编码基因具有显著的独特性。
实施例3发酵液中果聚糖蔗糖酶的检测
1、材料与方法
(a)种子(发酵菌种)的制备：将柠檬明串珠菌(Leuconostoc citreum)CGMCC NO.6431的冻干粉用少量无菌蒸馏水溶解，用接种环取一环划线于含2％(w/v)蔗糖的M17琼脂培养基(Merck Co.德国)上，28℃好氧培养24h取出，用接种环挑取单菌落放入20mL含2％(w/v)蔗糖的M17液体培养基(Merck Co.德国)，28℃180rpm摇床培养24h取出，培养物9，000rpm离 心10分钟，弃去上清，菌体用无菌蒸馏水洗涤2次后，用原培养体积的无菌蒸馏水悬浮，得到发酵用的种子，经检测，种子液的菌浓度为1×10 9CFU/mL。
(b)番茄汁蔗糖培养基的制备：成熟番茄清洗，去皮，榨汁机压榨，100目纱布过滤取汁，煮沸5min，8000g离心10min取上清，加入15％(w/v)蔗糖加热溶解后冷却至室温，以食用级碱调节pH至6.5，121℃灭菌20min，冷却至室温，即得无菌的番茄汁蔗糖培养基。
(c)发酵液中果聚糖蔗糖酶的原位活性检测：取发酵液50mL 15000g离心30min，取上清，缓慢加入硫酸铵直至饱和度达到60％，冷藏过夜，20000g、4℃离心30min取沉淀，溶解于少量蒸馏水中，装入截留分子量为1000Da的透析袋内，置于1‰CaCl 2溶液中低温透析24h，期间换水5次，取出冻干得冻干粉。称量上述冻干样品5mg溶于0.5mL PBS中，再Native laoding混合，上样于4-20％SDS-PAGE梯度胶(南京金斯瑞生物科技有限公司，中国)，并在110V电压下电泳1.5h。所得的蛋白胶一半用于考马斯亮蓝染色，另外一半用NaAc溶液(20mM、pH5.5)浸洗2次后，置于含5％蔗糖、0.3‰NaN 3的NaAc溶液(20mM、pH5.5)中，30℃反应48h后，于暗室观察原位反应产物的条带数量和位置，以此确认柠檬明串珠菌合成的果聚糖蔗糖酶的数量和分子量。
2、发酵液中果聚糖蔗糖酶的检测
将柠檬明串珠菌种子以接种量3％(v/v)无菌接种于含蔗糖15％(v/v)，pH为6.5的番茄汁培养基，30℃、200rpm培养96h得发酵液。对发酵液中果聚糖蔗糖酶的原位活性进行检测，结果如图4所示，SDS PAGE原位反应发现有三个明显的多糖条带，表明发酵液内含有三种分子量不同的果聚糖蔗糖酶，其分子量分别为130kDa、90kDa和80kDa。
结论：柠檬明串珠菌CGMCC NO.6431的果聚糖蔗糖酶编码基因可编码表达三个不同分子量的果聚糖蔗糖酶，其分子量分别为130kDa、90kDa和 80kDa。
效果实施例1果聚糖蔗糖酶的分子量比较
通过文献检索，对本发明公开的三种果聚糖蔗糖酶的分子量与已知报道进行比较，结果如表1所示。
表1不同来源的果聚糖蔗糖酶的分子量比较
Figure PCTCN2021120964-appb-000005
数据来源：
[1]Ben Ammar Y,Matsubara T,Ito K,et al.Characterization of a thermostable levansucrase from Bacillus sp.TH4-2 capable of producing high molecular weight levan at high temperature[J].Journal of Biotechnology,2002,99(2):111-119.
[2]van,Hijum,Aft S,et al.Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri.[J].Microbiology,2004,150:621-630.
[3]Tieking M,Ehrmann M A,Vogel R F,et al.Molecular and functional characterization of a levansucrase from the sourdough isolate Lactobacillus sanfranciscensis TMW 1.392.[J].Applied Microbiology and Biotechnology,2005,66(6):655-663.
[4]Sangmanee S,Nakapong S,Kuttiyawong K,et al.Production and Immobilization of Levansucrase[J].Chiang Mai Journal of Science,2015,42(1):44-51.
通过比较后发现，本申请的果聚糖蔗糖酶编码基因与已知同类基因的同 源性＜30％。而且，由柠檬明串珠菌发酵产生的果聚糖蔗糖酶不但种类多，其中130kDa分子量的果聚糖蔗糖酶是目前已知最大的有天然活性的果聚糖蔗糖酶。
效果实施例3果聚糖蔗糖酶的制备
1、材料与方法
(a)果聚糖蔗糖酶的分离：将粗酶提取物溶解于蒸馏水中上样于AKTA蛋白纯化系统(GE公司，美国)，填料：Sephacryl S-300 High Resolution，流速：1mL/min，柱体积：120mL，检测器：UV检测器，根据UV信号收集280nm信号较高的洗脱液，冻干。各样品取5mg加入含5％蔗糖、0.3‰NaN3的NaAc溶液(20mM、pH5.5)中，30℃反应48h后，用葡萄糖检测试剂盒(中生北控生物科技股份有限公司，中国)测定是否有游离葡萄糖生成，若有则该样品为果聚糖蔗糖酶。
2、果聚糖蔗糖酶的制备
将实施例3制备获得的发酵液8000g离心10min取上清，冷冻干燥得粗酶提取物，对该提取物进行分离后即得三个果聚糖蔗糖酶样品。
效果实施例3果聚糖蔗糖酶样品的活性验证
将效果实施例2制备获得的三个果聚糖蔗糖酶样品20mg分别溶解于含5％蔗糖、0.3‰NaN 3的NaAc溶液(20mM、pH5.5)，30℃反应48h后，15，000g离心10min，取上清液，加入3倍体积于上清液体积的无水乙醇，静置过夜，15，000g离心10min，收集沉淀物并溶于水后，真空冷冻干燥即得三个多糖样品。将三个多糖样品按10mg/mL的浓度分别完全溶解于重水中，并应用核磁共振波谱仪(Avance III 400MHz，Bruker公司，德国)进行核磁共振(NMR)测定，结果发现，三个多糖样品的NMR图谱一致(如图5所示)，所有多糖样品的NMR-1H谱和NMR-13C谱的化学位移与果聚糖levan标准品吻合。
结论：由柠檬明串珠菌(Leuconostoc citreum)CGMCC NO.6431发酵提取获得的三种不同分子量的果聚糖蔗糖酶均有活性，可用于合成果聚糖levan。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。

Claims (6)

  1. 一株柠檬明串珠菌菌株，其特征在于，该菌株的分类命名为柠檬明串珠菌Leuconostoc citreum，保藏编号为CGMCC NO.6431。
  2. 根据权利要求1所述柠檬明串珠菌菌株，其特征在于，所述菌株的基因组上有两个连续排列且正常表达的果聚糖蔗糖酶编码基因。
  3. 根据权利要求2所述柠檬明串珠菌菌株，其特征在于，所述果聚糖蔗糖酶编码基因编码表达三个不同分子量的果聚糖蔗糖酶。
  4. 根据权利要求3所述柠檬明串珠菌菌株，其特征在于，所述果聚糖蔗糖酶的分子量分别为130kDa、90kDa和80kDa。
  5. 一种根据权利要求1-4任一项所述柠檬明串珠菌在果聚糖蔗糖酶制备方面的应用。
  6. 根据权利要求5所述柠檬明串珠菌在果聚糖蔗糖酶制备方面的应用，其特征在于，该菌株单次发酵制备三种不同分子量的天然果聚糖蔗糖酶。
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