CN111849809B - 一种可降解菊粉果聚糖的菌株及其应用 - Google Patents
一种可降解菊粉果聚糖的菌株及其应用 Download PDFInfo
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Abstract
本发明涉及一种可降解菊粉果聚糖的菌株及其应用,该菌株系是从腐烂菊芋块茎中对菌株进行分离和纯化。本发明所述的株系通过酶活测定证明其具有较高外切菊粉酶酶活,具有很好地降解菊芋中菊糖的性能。
Description
技术领域
本发明涉及一种可降解菊粉果聚糖的Glutamicibacter mishrai NJAU-1及其应用。
背景技术
菊芋(Helianthus tuberosus.L)最早起源于北美,于17世纪被引入欧亚大陆,而在20 世纪早期已经成为了一种较为常见的农作物(Seiler et al,2006)。菊芋是一种适应性特别广的植物,生存能力特别强,能够在盐碱地和海岸滩涂这些盐浓度较高,绝大部分植物所不能生长的地方进行大范围种植(Long et al,2010;隆小华等,2005)。菊芋发达的根系使得它伸展的区域范围更广,并且即使是在风沙危害较大的沙地不采取任何的措施下种植菊芋,在45天干旱无雨的恶劣条件下它依然能够正常的生长(孔涛等,2009)。除了能够抵抗盐胁迫、抗风沙外,菊芋对高温环境也具有较好的适应性,是一种繁殖能力特别强的植物。这些特征使得它被引入我国之后,被广泛地种植于一些较为贫瘠的地区。其较好的耐盐性,也让它成为了可供选择的具有较好开发前景的盐土植物之一,在江苏沿海滩涂盐碱地可以进行大面积种植。
菊粉(Inulin,又称菊糖),主要存在于菊芋、菊苣、蒲公英、牛蒡和朝鲜蓟等植物体的根部或茎部,是一种来源丰富的可再生资源。通过菊粉酶的催化作用,以菊粉为原料,可以生产高果糖浆、低聚果糖、乳酸、柠檬酸、乙醇、单细胞蛋白、单细胞油脂和丁醇等精细化学品。利用酶法生产果糖和低聚果糖,条件温和,工艺简单,成本低廉,安全性高,无污染。因此,利用酶法生产果糖和低聚果糖更适用于工业化生产。
菊粉酶是一种天然的果聚糖水解酶,外切型菊粉酶是唯一的一步法生产高果糖浆的重要酶种,内切型菊粉酶是生产低聚果糖的重要酶种。利用菊粉酶水解菊芋生产果糖和低聚果糖,具有工艺简单,转化率高,成本低,无污染等优点。菊粉酶还可用于发酵生产生物乙醇。因此,菊粉酶的开发利用对开发新的食品工业原料,解决粮食危机,能源危机等具有重要的意义。
由于微生物繁殖速度快,代谢旺盛,来源丰富,且成本低廉,工业上多用微生物来进行菊粉酶的生产。现阶段国内外对菊粉酶的研究主要集中在产菊粉酶微生物资源的开发与利用上。已发现的能合成菊粉酶的微生物有很多种,包括霉菌、细菌、酵母等均能生产菊粉酶。但是目前已有的能够分泌菊粉酶菌株的活性均较低,无法实现进一步产业化应用的需要,因而,高产菊粉酶微生物的筛选具有重要的工业生产意义。
发明内容
本发明的目的在于针对工业化大规模果糖生产问题,提供一种可降解菊粉果聚糖菌株系 Glutamicibacter mishrai NJAU-1,该菌株是一种新株系,具有产酶活较高的外切菊粉酶的特点。
本发明的目的可通过如下技术方案实现:
一株可降解菊粉果聚糖的菌株菌株脂肪性谷氨酸杆菌(Glutamicibactermishrai)NJAU-1,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2020 年4月28号,保藏编号为CGMCC No.19750。
本发明所述的菌株NJAU-1在制备外切菊粉酶中的应用。
本发明所述的菌株NJAU-1在生产果糖中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明的可降解菊粉果聚糖菌株菌株脂肪性谷氨酸杆菌(Glutamicibactermishrai)NJAU-1具有较高的菊粉酶活性的特点,该菌能够提高菊粉转换效率,提高果糖产量,可用于实际生产。
附图说明
图1、降解菊粉果聚糖菌株系Glutamicibacter mishrai NJAU-1在ZS培养基上的菌落形态;
图2、降解菊粉果聚糖菌株系Glutamicibacter mishrai NJAU-1的16S rDNA部分序列分析***发育树状图;括号中的数字表示GenBank序列号
图3、静息细胞NJAU-1与菊粉培养1h后的(A),和菊粉空白对照(B)的糖HPLC图; F:果糖;FOSs:低聚果糖
生物材料保藏信息
NJAU-1,分类命名为脂肪性谷氨酸杆菌(Glutamicibacter mishrai),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏日期为2020年4月28号,保藏编号为CGMCC No.19750。
具体实施方式
实施例1
1、降解菊粉果聚糖菌株系Glutamicibacter mishrai NJAU-1的分离和纯化
取腐烂菊芋块茎组织用研钵研磨成浆,静置一段时间后,吸取上清液100μl于100mlZS 液体培养基中,封口,37℃,200r/min摇床培养,培养3d。吸取菌液,梯度稀释10-3-10-5后,分别取100μL涂布于ZS培养基上,37℃培养1-2d。挑取单菌落在ZS培养基上划线,ZS培养基的配方如(表1)所示,倒置后在37℃下黑暗培养。筛选出长势较好的菌种,根据菌种形态和培养基的颜色变化判断菌种。
表1 ZS培养基配方(/L)
利用该分离纯化的方法,经过若干代反复纯化,在固体培养基ZS(表1)中划线培养,得到多株纯菌,本实施可分离纯化到NJAU-1。
NJAU-1形态特征:本菌株在菌落形态上,菌落是光滑的,圆形较小、发亮半透明、白色和淡黄色。在ZS平板上生长3~5天后的直径为0.1~0.5mm;培养特性:该菌的最适生长条件为:pH=7,温度37℃,转速200r/min;生理特性:细胞呈革兰氏染色阳性,严格有氧。
2、降解菊粉果聚糖菌株系Glutamicibacter mishrai NJAU-1的16S rDNA序列测序
筛出NJAU-1的单菌落,然后进行PCR特异性扩增,PCR扩增产物在1.0%的琼脂糖凝胶电泳上检验后,用于序列测定,该菌株16SrDNA序列如SEQ ID NO.1所示。
从GenBank数据库中获取参比菌株,应用软件MegAlign对分离菌株和参比菌株的16S rDNA部分序列进行了分析,构建了分离菌株与标准菌株的***发育关系图,得到反映待测菌株与参比菌株之间进化与发育关系的***发育树,如图2所示,从而确定分离出来的降解菊粉果聚糖菌株系Glutamicibacter mishrai NJAU-1为谷氨酸杆菌属(Glutamicibacter)的新菌株。
实施例2测定Glutamicibacter mishrai NJAU-1的产菊粉酶酶活
将分离纯化获得的Glutamicibacter mishrai NJAU-1菌种由平板接种到装有30mLZS液体培养基的100mL三角瓶中,在37℃,200r/min摇床振荡培养24h,使OD﹥1。取3ml悬浊液(3个重复)进行4℃,12000r/min离心10min,弃去上清液,收集菌体沉淀,用pH=7.0的磷酸盐缓冲液洗涤,洗去培养液,之后离心收集菌体沉淀,重复洗涤三次至上清液透明,弃去上清液。
内切菊粉酶活力的测定:将离心后的菌体沉淀用pH为7.0的磷酸盐缓冲液(加入2%菊粉底物)定量至3mL吹悬混匀。在37℃,200r/min培养1d。将悬浊液进行4℃,12000r/min 离心10min,收集上清液,取上清液200μL,加入1.5mL DNS试剂+1.8mL水,混匀后,沸水浴反应5min(精确计时),冷水冷却后,用蒸馏水在试管中定容至25mL,测定520nm处的吸光度。通过果糖标准曲线可以计算得出样品溶液中的糖含量。对照组除不接种菌株外,处理相同。
外切菊粉酶活力的测定:将离心后的菌体沉淀用pH为7.0的磷酸盐缓冲液(加入2%蔗糖底物)定量至3mL吹悬混匀。在37℃,200r/min培养1d。将悬浊液进行4℃,12000r/min 离心10min,收集上清液,取上清液200μL,加入1.5mL DNS试剂+1.8mL水,混匀后,沸水浴反应5min(精确计时),冷水冷却后,用蒸馏水在试管中定容至25mL,测定520nm处的吸光度。通过果糖标准曲线可以计算得出样品溶液中的糖含量。对照组除不接种菌株外,处理相同。
一个单位的酶活性被定义为每小时产生1μmol果糖所需的酶量。计算I/S<1,判断菊粉酶为外切菊粉酶。如表4所示,该菌株具有较高的外切菊粉酶酶活。
表2磷酸盐缓冲液(/L)
表3 DNS试剂(/L)
表4 DNS法菊粉酶酶活
实施例3
将离心后的菌体沉淀用pH为7.0的磷酸盐缓冲液(加入2%菊粉底物)定量至3mL吹悬混匀,在37℃,200r/min培养1h。将悬浊液进行4℃,12000r/min离心10min,收集上清液,通过高效液相色谱进行糖含量测定,结果如图3。
一个单位的酶活性被定义为每小时产生1μmol果糖所需的酶量。如表5所示,表明该菌株有较强的外切菊粉酶酶活。
表5 HPLC法菊粉酶酶活
序列表
<110> 南京农业大学
<120> 一种可降解菊粉果聚糖的菌株及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1448
<212> DNA
<213> 脂肪性谷氨酸杆菌(Glutamicibacter mishrai )
<400> 1
gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gatgaagccc tgcttgcagg 60
gtggattagt ggcgaacggg tgagtaacac gtgagtaacc tgcccccgac tctgggataa 120
gcccgggaaa ctgggtctaa taccggatat gacttcctac tgcatggtgg gttgttgaaa 180
gatttatcgg tgggggatgg actcgcggcc tatcagcttg ttggtgaggt aatggctcac 240
caaggcgacg acgggtagcc ggcctgagag ggtgaccggc cacactggga ctgagacacg 300
gcccagactc ctacgggagg cagcagtggg gaatattgca caatgggcgg aagcctgatg 360
cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa acctctttca gtagggaaga 420
agcgaaagtg acggtacctg cagaagaagc gccggctaac tacgtgccag cagccgcggt 480
aatacgtagg gcgcaagcgt tatccggatt tattgggcgt aaagagctcg taggcggttt 540
gtcgcgtctg ccgtgaaagt ccgaggctca acctcggatc tgcggtgggt acgggcagac 600
tagagtgatg taggggagac tggaattcct ggtgtagcgg tgaaatgcgc agatatcagg 660
aggaacaccg atggcgaagg caggtctctg ggcatttact gacgctgagg agcgaaagca 720
tggggagcga acaggattag ataccctggt agtccatgcc gtaaacgttg ggcactaggt 780
gtgggggaca ttccacgttt tccgcgccgt agctaacgca ttaagtgccc cgcctgggga 840
gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag cggcggagca 900
tgcggattaa ttcgatgcaa cgcgaagaac cttaccaagg cttgacatgt gccagaccgc 960
tccagagatg gggtttccct tcggggctgg ttcacaggtg gtgcatggtt gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca accctcgttc catgttgcca 1080
gcacgtagtg gtggggactc atgggagact gccggggtca actcggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgtctt gggcttcacg catgctacaa tggccggtac 1200
aatgggttgc gatactgtga ggtggagcta atccctaaaa gccggtctca gttcggattg 1260
gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta atcgcagatc agcaacgctg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca agtcacgaaa gttggtaaca 1380
cccgaagccg atggcctaac caccttgtgt ggggggagtc gtcgaaggtg ggactggcga 1440
ttgggact 1448
Claims (3)
1.一株可降解菊粉果聚糖的菌株脂肪性谷氨酸杆菌(Glutamicibacter mishrai)NJAU-1,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2020年4月28号,保藏编号为CGMCC No.19750。
2.权利要求1所述的NJAU-1在制备外切菊粉酶中的应用。
3.权利要求1所述的NJAU-1在生产果糖中的应用。
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