CN111826308B - 一株海洋沉积物来源的几丁质高效降解菌及其应用 - Google Patents
一株海洋沉积物来源的几丁质高效降解菌及其应用 Download PDFInfo
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Abstract
本发明涉及一株海洋沉积物来源的几丁质高效降解菌及其应用，其特征在于：该几丁质高效降解菌为莱茵海默氏菌SX46，该莱茵海默氏菌SX46保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为：CGMCC No.19597，保藏日期为2020年04月22日；与现有技术相比，本发明具有如下优点：本发明提供的菌株SX46为莱茵海默氏菌属的新种，本发明提供的菌株SX46能够在成本低的培养基中良好生长，且产生的几丁质酶具有很高活性，可高效的将胶体几丁质转化为N‑乙酰‑D‑氨基葡萄糖(GlcNac)，转化率高达90％。
Description
技术领域
本发明属于应用微生物技术领域，尤其是涉及一种适合海洋沉积物来源的几丁质高效降解菌及其应用。
背景技术
几丁质(chitin)又称甲壳素，是N-乙酰-β-D-氨基葡萄糖(GlcNac)通过β-1,4-糖苷键连接起来的直链多聚物，是海洋环境含量最丰富的可再生资源，每年有超过1011吨几丁质形成，其降解主要通过海洋微生物分泌的几丁质酶(chitinase)系降解体系完成。几丁质酶降解几丁质产生高附加值几丁质寡糖、几丁质单糖及其衍生物等，具有良好的组织兼容性、生物降解性以及调节免疫力、抗菌、诱导植物抗病、促进植物生长和抗癌等生物活性，在医药行业、食品工业、环境、农业和养殖业等众多领域有着广泛的应用前景。
虽然海洋环境是巨大的微生物资源库，然而海洋环境来源的几丁质酶产生菌的数量要远低于陆地环境。缺乏高效的分离方法是造成海洋环境来源的几丁质酶产生菌获得率低的主要原因。目前，我国不但缺乏适应于海洋沉积物环境的几丁质酶产生菌的高效分离，而且缺少高产高活性几丁质酶的菌株，这也从而导致利用微生物来源几丁质酶制备高附加值产物的相关专利更加稀少。
发明内容
本发明所要解决的第一个技术问题是针对现有技术的现状提供一种海洋沉积物来源的几丁质高效降解菌。
本发明所要解决的第二个技术问题是针对现有技术的现状提供一种上述几丁质高效降解菌的应用。
本发明解决上述第一个技术问题所采用的技术方案为：该一株海洋沉积物来源的几丁质高效降解菌，其特征在于：该几丁质高效降解菌为莱茵海默氏菌Rheinheimera spSX46，该莱茵海默氏菌Rheinheimera sp SX46保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为：CGMCC No.19597，保藏日期为2020年04月22日莱茵海默氏菌。
本发明还提供一种富集几丁质高效降解菌的富集方法，其特征在于：通过采用富集培养基进行几丁质高效降解菌的富集，所述富集培养基中的氮源为(NH4)2SO4、KNO3和酵母提取物，碳源为几丁质粉末，所述富集培养基配方为：Na2HPO4，6.0g/L；KH2PO4，3.0g/L；NH4Cl，1.0g/L；NaCl，0.5g/L；酵母提取物，0.05g/L；几丁质粉末，10.0g/L，调节培养基的pH为7.0。
本发明还提供一种筛选几丁质高效降解菌的筛选方法，其特征在于：通过采用筛选培养基进行几丁质高效降解菌的筛选，所述筛选培养基中的氮源为(NH4)2SO4、KNO3和酵母提取物，碳源为几丁质粉末，并添加有胶体几丁质。
为解决第二个技术问题，本发明提供一种几丁质高效降解菌在生产几丁质酶中的应用，其特征在于：将所述莱茵海默氏菌SX46接种至以胶体几丁质为唯一碳源的发酵培养基中，其中胶体几丁质的含量为10～15g/L。
与现有技术相比，本发明具有如下优点：本发明提供的菌株SX46为莱茵海默氏菌属的新种，本发明提供的菌株SX46能够在成本低的培养基中良好生长，且产生的几丁质酶具有很高活性，可高效地将胶体几丁质转化为N-乙酰-D-氨基葡萄糖(GlcNac)，转化率高达90％，本发明符合绿色安全的环保理念，实现了海洋微生物资源最大利用化，通过对常规方法进行创新性设计，获得海洋环境未曾报道的高产高活性几丁质酶的菌株；利用该菌株产生的几丁质酶可高效的将几丁质转化为有应用价值的N-乙酰-D-氨基葡萄糖(GlcNac)。
保藏说明
1、莱茵海默氏菌SX46，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址是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04月22日，保藏号为CGMCC No.19597。
附图说明
图1是实施例1中第3代几丁质降解菌的富集培养体系的结果图；
图2是实施例1中菌株SX46在筛选平板上产生的透明圈的结果图；
图3是实施例4中GlcNac的产量的结果图。
具体实施方式
以下通过结合附图、序列表及实施例对本发明作进一步说明。
实施例1
(1)几丁质降解菌的富集培养
将5g沉积物转移至含100mL富集培养基的锥形瓶中，8℃、150转/min培养30天，即为第1代富集培养物；取1mL第1代富集无接种至含100mL富集培养基的锥形瓶中，继续震荡培养14天，即获得第2代富集培养物；取1mL第2代富集培养物接种至含100mL富集培养基的锥形瓶中，继续震荡培养14天，即获得第3代富集培养物，所述沉积物来源自大洋50航次采集的海洋沉积物。
所述的富集培养基配方为(g/L)：Na2HPO4，6.0；KH2PO4，3.0；NH4Cl，1.0；NaCl，0.5；酵母提取物，0.05g；几丁质粉末，10.0g，调节培养基的pH为7.0。
如图1所示，经3代富集培养后，富集液变浑浊，粉末几丁质被部分降解，说明富集物中几丁质降解菌已达到一定数量。
(2)几丁质降解菌的筛选与纯化
用富集培养基将上述第3代富集培养物进行系列梯度稀释后，涂布于固体筛选培养基，至于20℃培养箱中培养。挑取菌落周围出现透明圈的菌落进行3次划线纯化，得到能够降解几丁质降解的纯菌株SX46。
所述的固体鉴别培养基配方为(g/L)：Na2HPO4，6.0g；KH2PO4，3.0g；NH4Cl，1.0g；NaCl，0.5g；酵母提取物，0.05g；几丁质粉末，5g；胶体几丁质，10g。琼脂粉，15g。调节培养基的pH为7.0，鉴别培养基中的几丁质被菌株SX46降解后形成透明圈(图2)。
实施例2：几丁质降解菌SX46的分子鉴定及命名保藏
(1)菌株分子鉴定
采用常规的分子克隆法进行菌株SX46 16S rRNA基因扩增与测序，测序结果提交EzTaxon网站，确定菌株的分类地位。
(2)菌株16S rDNA基因序列及菌株保藏信息
所述菌株SX46的16S rRNA基因序列如序列表所示，基因全长为1453bp。EzTaxon网站同源性分析表明，该菌与Rheinheimera aquimaris(莱茵海默氏菌)的同源性最高(98.55％)，为潜在新种。
实施例3:菌株SX46几丁质酶粗酶的制备及活力测定
(1)菌株SX46发酵培养
挑取SX46单菌落接种于10mL发酵培养基，20℃、150r/min震荡培养至菌液OD600＝0.8～1.0作为种子液；取种子液按1:100比例接种于发酵培养基，20℃、150转/min震荡培养5d后，3000转/min离心5min，收集清液。
上述发酵培养基的培养为(g/L)：Na2HPO4，6.0g；KH2PO4，3.0g；NH4Cl，1.0g；NaCl，0.5g；酵母提取物，0.1g；胶体几丁质，10g，调节培养基的pH为7.0。
(2)几丁质酶粗酶的制备
在上述步骤制备的上清液中加入80％终浓度硫酸铵，4℃震荡30min后，4℃、10000g离心20min，收集沉淀；沉淀用2ml 50mM KH2PO4缓冲液(pH5.0)充分溶解后转移至透析袋(MWVO:6～8kDa)；将透析袋置于装有50mM KH2PO4缓冲液的容器中，4℃透析12-24h，透析过程中每隔2h更换一次透析液。透析完毕后，收集透析袋中的溶液即为几丁质酶粗酶溶液。
(3)几丁质酶粗酶活力测定
采用DNS法测定几丁质酶粗酶液的活力。DNS法测定几丁质酶活力具体操作步骤为：取1.5mL几丁质粗酶液与1.5mL含1.0％胶体几丁质的磷酸缓冲液(0.1M，pH 5.5)混合，40℃孵育30min，煮沸5min终止酶促反应，冰浴冷却，10000转/min离心5min，取上清液1mL与1mL DNS试剂，混匀，沸水浴10min后，冰浴冷却，离心后取上清液在波长540nm下测吸光度。以N-乙酰氨基葡萄糖做标准对照，步骤同上，所不同的是反应前进行煮沸灭活。酶活力单位(U)定义为：在40℃下每分钟产生1μmol还原糖所需的酶量。酶活计算公式为：酶活力(U/mL)＝m×N×1000/(M×T×V)。公式中：m为N-乙酰-D-氨基葡萄糖量，N为稀释倍数，M为N-乙酰-D-氨基葡萄糖分子量，T为反应时间，V为酶液体积。
结果表明，菌株培养5d后，几丁质酶粗酶活力高达150U/mL，具有很强的酶活性。
实施例4:利用菌株SX46产生的几丁质酶生产GlcNac
(1)GlcNAc转化反应体系
该转化体系含2％(w/v)终浓度胶体几丁质，0.5U/mL几丁质酶粗酶液，50mMKH2PO4缓冲液(pH5.0)。将上述反应体系至于37℃恒温环境中震荡孵育96h，每隔2小时取样1次，测定反应体系中GlcNAc的浓度。
(2)GlcNAc产量测定
使用高效液相测定上述反应体系中GkcNac的浓度。使用30cm×7.8mm规格的层析柱(Aminex HPX-87H)。上样后，以5mM H2SO4为流动相洗脱，流速为0.8m/min。通过比较样品中的GlcNAc峰面积与标准品GlcNAc(Sigma产品)峰面积的比值得出所述GlcNAc转化体系中的GlcNAc含量。GlcNAc产量(％)计算公式为：溶液中释放的GlcNAc量(mg)×100/胶体几丁质起始浓度(mg)×100。
结果表明(图3)，利用菌株SX46制备的几丁质粗酶液可将胶体α-chitin转化为GlcNac，12h内75％的胶体几丁质被转化为GlcNa；GlcNac最高转化效率发生在反应后第24h，此时90％的胶体几丁质转化为GlcNac。
序列表
<110> 浙江万里学院
<120> 一株海洋沉积物来源的几丁质高效降解菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1452
<212> DNA
<213> SX46
<400> 1
gtggccgggc agggccttac acatgcaagt cgagcgaatg agggtagctt gctacctgat 60
ttagcggcgg acgggtgagt aatgtatagg gagctgcccg atagaggggg ataccagttg 120
gaaacgactg ttaataccgc ataatgtcta cggaccaaag tgtgggacct tcgggccaca 180
tgctatcgga tgcacctata tgggattagc tagttggtgg ggtaacggct caccaaggcg 240
acgatcccta gctggtttga gaggatgatc agccacactg gaactgagac acggtccaga 300
ctcctacggg aggcagcagt ggggaatatt ggacaatggg cgcaagcctg atccagccat 360
gccgcgtgtg tgaagaaggc cttcgggttg taaagcactt tcagcgagga ggaagggtgt 420
tgtgttaata gcacagcatt ttgacgttac tcgcagaaga agcaccggct aactccgtgc 480
cagcagccgc ggtaatacgg agggtgcaag cgttaatcgg aattactggg cgtaaagcgc 540
acgtaggcgg tgtgttaagt tggatgtgaa agccccgggc tcaacctggg aattgcattc 600
aaaactggca cgctagagta tgtgagaggg gggtagaatt ccaagtgtag cggtgaaatg 660
cgtagagatt tggaggaata ccagtggcga aggcggcccc ctggcacaat actgacgctc 720
aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg 780
atgtctacta gctgttcgtg gtcttgtact gtgagtagcg cagctaacgc actaagtaga 840
ccgcctgggg agtacggtcg caagattaaa actcaaatga attgacgggg gcccgcacaa 900
gcggtggagc atgtggttta attcgacgca acgcgaagaa ccttacctac tcttgacatc 960
tagcgaagat tgcagagatg cagttgtgcc ttcgggaacg ctaagacagg tgctgcatgg 1020
ctgtcgtcag ctcgtgttgt gaaatgttgg gttaagtccc gcaacgagcg caacccttat 1080
ccttagttgc cagcacgtaa tggtgggaac tctagggaga ctgccggtga taaaccggag 1140
gaaggtgggg acgacgtcaa gtcatcatgg cccttacgag tagggctaca cacgtgctac 1200
aatggtacgt acagagggag gcaagctggc gacagtgagc ggatctctta aagcgtatcg 1260
tagtccggat tggagtctgc aactcgactc catgaagtcg gaatcgctag taatcgcaaa 1320
tcagaatgtt gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg 1380
agtgggttgc aaaagaagta ggtagcttaa ccttcgggag ggcgccttac ccacctttgg 1440
gatttccagg tg 1452
Claims (4)
1.一株海洋沉积物来源的几丁质高效降解菌，其特征在于：该几丁质高效降解菌为莱茵海默氏菌Rheinheimera sp SX46，该莱茵海默氏菌Rheinheimera sp SX46保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为：CGMCC No.19597，保藏日期为2020年04月22日莱茵海默氏菌。
2.一种富集如权利要求1所述的几丁质高效降解菌的富集方法，其特征在于：通过采用富集培养基进行几丁质高效降解菌的富集，所述富集培养基中的氮源为(NH4)2SO4、KNO3和酵母提取物碳源为几丁质粉末，所述富集培养基配方为：Na2HPO4，6.0g/L；KH2PO4，3.0g/L；NH4Cl，1.0g/L；NaCl，0.5g/L；酵母提取物，0.05g/L；几丁质粉末，10.0g/L，调节培养基的pH为7.0。
3.一种如权利要求1所述的几丁质高效降解菌在生产几丁质酶中的应用，其特征在于：将所述莱茵海默氏菌SX46接种至以胶体几丁质为唯一碳源的发酵培养基中，其中胶体几丁质的含量为10～15g/L。
4.根据权利要求3所述的几丁质高效降解菌在生产几丁质酶中的应用，其特征在于：所述发酵培养基配方为Na2HPO4，6.0g/L；KH2PO4，3.0g/L；NH4Cl，1.0g/L；NaCl，0.5g/L；酵母提取物，0.1g/L；胶体几丁质，10g/L，调节培养基的pH为7.0。
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