CN106244494A - 一株地芽孢杆菌菌株及其应用 - Google Patents
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Abstract
本发明公开了一株地芽孢杆菌菌株Geobacillus sp. YM6,其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.12089;本发明涉及的Geobacillus sp. YM6菌株能以羽毛粉作为唯一碳氮源,将菌株接入有完整羽毛的发酵培养基中,发酵培养48h后,羽毛、羽枝绝大部分被降解,降解羽毛角蛋白效果显著;该菌株所产角蛋白酶最适作用温度为65‑70℃,为高温中性角蛋白酶,最适pH为7.0,可应用于羽毛降解的相关生产领域。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株嗜热地芽孢杆菌菌株及其在降解羽毛中的应用。
背景技术
人们生活的环境中存在着大量的废弃羽毛,每年有数以百万计的羽毛得不到合理的利用。羽毛中含有丰富的角蛋白,若加以合理的利用,可使得角蛋白转化为能被利用的可溶性蛋白。但是由于其中的二硫键、氢键等高度交联,常见的水解酶像胃蛋白酶、胰蛋白酶等难以降解。
传统物理降解法原理是利用特殊温度、压力条件使羽毛水解为可溶性多肽或寡肽混合物,高压蒸汽可增加角蛋白的溶解性。该法工艺流程简单,将羽毛除杂、投入反应器后通入蒸汽,得到块状蛋白产物后烘干粉碎便可作为动物饲料。但是此方法多会破坏氨基酸分子键,产物稳定性差,若作为动物饲料,得到的蛋白粉口味较差,消化率低,限制了物理法的应用范围。
化学降解法利用酸、碱或氧化还原剂使羽毛中的角蛋白降解为可溶性蛋白、多肽或游离氨基酸。Kelly等利用Ca(OH)2处理鸡羽毛生产动物饲料,反应温度在150℃,羽毛角蛋白25min内溶解80%,反应后经碳酸化可回收54%的钙剂。利用NaOH的碱水解法可产生多种不同肽链,增加酰胺键和亚砜含量能改变角蛋白的化学性质,碱溶液在65℃反应,2~5h内可提出68%~82%的水溶性角蛋白,富含中间纤维和中间纤维丝成分,若将NaOH与Na2S联合溶解羽毛,40℃便能提取出角蛋白。氧化还原剂的使用也可降解羽毛,水、甘油和亚硫酸钠的组合可降解羽毛产生液体角蛋白,而巯基乙酸等物质的预处理可有效破坏二硫键。Schrooyen等选用2-巯基乙醇和尿素共同降解鸡羽毛,尿素起蛋白质溶胀作用,采用渗析除去巯基乙醇和尿素后发现,角蛋白多肽出现聚集现象,并且半胱氨酸残留物氧化形成凝胶,影响了角蛋白溶液的稳定性。改变实验条件发现在渗析前加入十二烷基硫酸钠(SDS)可阻止多肽链的聚集。但碱法效率不高,产物主要为多肽;酸法会腐蚀设备、污染环境,且复合氨基酸产率不高。
微生物途径来解决角蛋白难以降解这个问题一直以来都广受关注,早在十九世纪初,就发现一些生物能够分解角蛋白。迄今为止包裹细菌、真菌、放线菌等超过30几种微生物能降解角蛋白。随着分子生物学技术的发展,对角蛋白酶的研究重新成为热点。微生物降解法效果显著,即利用了蛋白资源,也有利于环境保护。因此微生物角蛋白酶降解法可使羽毛粉的营养价值显著提高,所获得经济效益显然要比普通的加工方法高得多。目前已经筛选和分离到很多能降解角蛋白的细菌,主要为芽孢杆菌。其他芽孢杆菌包括苏云金芽孢杆菌、地衣芽孢杆菌等。傅伟等人研究的苏云金芽孢杆菌和王政等人研究的地衣芽孢杆菌在分别发酵3d和5d后降解率分别是72.5%、84.06%。
高温环境有助于反应速度的加快,提高降解的效率。嗜热菌生长于高温环境,仅有极少数嗜热菌菌株具有降解羽毛的能力。此外,对具有角蛋白降解能力的嗜热菌的相关研究还非常有限,此类嗜热菌是开发应用角蛋白高温降解的重要微生物资源。
发明内容
本发明的目的是提供一株嗜热地芽孢杆菌菌株Geobacillus sp.YM6,已于2016年1月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNO.12089,保藏地址: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。
本发明中所述的地芽孢杆菌菌株Geobacillus sp.YM6具有以下微生物学特征:
1、形态学特征
菌落特征为菌落直径1.5-2.5毫米,边缘平整、菌体湿润、表面光滑、圆形半透明、形状较规则,颜色为乳白色。菌体形状为杆状,直径约1.5微米,长度4-8微米。
2、生理生化特征
菌株Geobacillus sp.YM6为革兰氏阳性菌,生长温度范围为42-72℃,最适生长温度为55℃;在7%的NaCl浓度中不生长,H2S反应显阴性,好氧生长,最适生长pH值为7.0。
3、本发明中地芽孢杆菌YM6碳源利用情况,使用杭州微生物试剂有限公司的肠杆菌科细菌生化鉴定管所得出;通过挑取单菌落穿刺至培养基中部,在55℃培养24h左右,通过培养基颜色的变化判断是阴性还是阳性,注:“+”表示阳性;“-”表示阴性。
本发明提供的Geobacillus sp.YM6与相似度最高菌株地芽孢杆菌YMTC 1049的表型特征比较结果见下表。
Geobacillus sp.YM6与地芽孢杆菌YMTC 1049( Genbank:AY191842.1)的特征比较
注:+:表示阳性,-:表示阴性,d:反应不同,11-89%菌株为阳性,v:一个菌株内特征不稳定。
本发明是通过以下技术方案实现的:
①样品采集于云南大理洱源65℃中性热泉的水样,取适量水样静置十分钟吸取上清液1mL进行梯度稀释至10-7,每个梯度取50μl涂布于初筛培养基平板上。在55℃条件下培养24h,挑取平板上长出的单菌落用于筛选羽毛降解菌株;
②将得到的单菌落分别接种到复筛培养基中,55℃震荡培养,48h后观察羽毛降解情况,并测定其中可溶性蛋白含量及羽毛的降解率,获得降解菌株;
③菌株的16SrDNA鉴定
提取菌株的总DNA,用细菌16SrDNA通用引物扩增目的片段,对片段进行回收、克隆、测序。对细菌16SrDNA基因片段序列使用NCBI的BLAST进行16SrDNA的相似性比对,结果显示与Geobacillus sp.YMTC 1049的同源性99%,初步认定它是地芽孢杆菌,命名为YM6。
所述初筛培养基的成分为:磷酸二氢钾1.5g,七水硫酸镁0.025g,氯化钙0.025g,七水硫酸亚铁0.015g,羽毛粉10g,琼脂20g,蒸馏水1000mL。
复筛培养基:不加琼脂的初筛培养基,以适量的羽毛代替羽毛粉。
本发明的优点和技术效果是:
1、本发明涉及的Geobacillus sp.YM6菌株能降解羽毛或羽毛粉;
2、本发明涉及的Geobacillus sp.YM6菌株不仅能在以羽毛为唯一的碳氮源的发酵培养基里生长,发酵24h后可见羽枝有明显脱落,48h羽枝完全脱落,且其中大部分被降解,脱毛及降解角蛋白效果显著;
3、本发明涉及的Geobacillus sp.YM6菌株为嗜热菌菌株,能在高温下降解羽毛,可应用于高温环境下的羽毛降解。
附图说明
图1为Geobacillus sp.YM6菌株对羽毛降解效果图,其中A图是羽毛降解前,B图是羽毛降解48h后。
具体实施方式
下面结合实施例对本发明进一步说明,下述实施例是说明性的,不是限定性的,不能以下述实施例来限定本发明的保护范围。
实施例1:地芽孢杆菌菌株Geobacillus sp.YM6的获得
①样品采集于云南大理洱源65℃中性热泉的水样,取适量水样静置十分钟吸取上清液1mL进行梯度稀释至10-7,每个梯度取100μl涂布于初筛培养基;在55℃条件下培养24h,选出长出菌落的平板;
初筛培养基的成分为:磷酸二氢钾1.5g,七水硫酸镁0.025g,氯化钙0.025g,七水硫酸亚铁0.015g,羽毛粉10g,琼脂15g,蒸馏水1000mL。
②将得到的单菌落分别接种到复筛培养基,37℃震荡培养, 48h后观察羽毛降解情况,并测定其中可溶性蛋白含量及羽毛的降解率,优选高效降解菌株;
复筛培养基:不加琼脂的初筛培养基,以1g的羽毛代替羽毛粉。
③菌株的16SrDNA鉴定(具体内容见实施例5)
提取菌株的总DNA,用细菌16SrDNA通用引物扩增目的片段,对片段进行回收、克隆、测序;对细菌16SrDNA基因片段序列使用NCBI的BLAST进行16S rDNA的相似性比对,结果显示与Geobacillus sp.YMTC 1049的同源性99%,初步认定它是地芽孢杆菌,命名为YM6。
实施例2:Geobacillus sp.YM6菌株对羽毛降解的失重率测定
(1)将鸡毛洗净、烘干、称重;
(2)将2g的鸡毛加入100mL的复筛培养基中,灭菌;
(3)将1mLGeobacillus sp.YM6菌液(OD600为0.4)接种入复筛培养基中,55℃震荡培养48h,观羽毛降解情况,降解效果如图1所示;
(4)将发酵液过滤,收集残渣并烘干至恒重,计算羽毛失重率;羽毛失重率计算公式为:;
(5)计算表明,羽毛失重率为65.7%。
实施例2:Geobacillus sp.YM6菌株产蛋白酶的最适温度测定
① 配制100mL筛选培养基,加入培养基体积1%的YM6菌液,55℃,培养24h,其中筛选培养基为:磷酸二氢钾1.5g、七水硫酸镁0.025g、氯化钙0.025g、七水硫酸亚铁0.015g、羽毛粉10g、蒸馏水1000mL。
②取8个1.5mLEP管,每管取发酵液1.5mL,小型离心机13000rpm,离心5min,吸取上清液1mL作为酶液;
③分别将酶液在温度梯度设为45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃的水浴锅中保温10分钟;然后按照国标SB/T 10317-1999蛋白酶活力测定方法,测定样品的酶活;
④结果表明该酶最适作用温度是65-70℃。
实施例3:Geobacillus sp.YM6菌株产蛋白酶的最适pH值测定
①依据常用缓冲液配制的配方,分别配制磷酸盐缓冲液、Tris-HCl缓冲液和碳酸盐缓冲液(pH梯度为5、6、7、8、9、10、11);
②将待测样品按体积比为1:10加入缓冲体系中,待测样品为实施例2中步骤的上清液1mL;
③将溶液在65℃的条件下,按照国标SB/T 10317-1999蛋白酶活力测定方法,测定样品的酶活;
④结果表明该酶的最适pH值是7。
实施例4 :Geobacillus sp YM6菌株降解羽毛过程中可溶性蛋白含量测定
①溶性蛋白含量的测定用考马斯亮蓝法;
②将100mL发酵培养基盛装于250mL的锥形瓶中,接种YM6,量为培养基体积的2%,55℃,150rpm震荡培养,分别于24h、32h、48h、72h取样,计算其中的可溶性蛋白的量;
③24、32、48和72小时,可溶性蛋白的含量分别为0.7 mg/mL、1.3 mg/mL、2.4 mg/mL和1.9 mg/mL;
④培养48小时的发酵液中蛋白质的含量最高为2.4 mg/mL。
实施例5:Geobacillus sp YM6菌株16S rDNA序列测定及分析
①在1.5mL的EP管中加入1mL过夜培养的YM6菌株菌液,于98℃加热10min裂解菌体;
②将加热所得的裂解液,放入小型离心机13000rpm,离心3min,吸取上清液5ul作为PCR扩增模板;
③采用16SrRNA通用引物进行PCR反应,上游引物(1492r)5'-GGT TAC CTT GTT ACGACT T-3',下游引物(8f)5'-AGA GTT TGA TCC TGG CTC AG-3';其中PCR反应的体系是模板5μl、上游引物 5μl、下游引物 5μl、Taq PCR Starmix 25μl和ddH2O 10μl。
PCR的扩增程序:
;
④ 电泳检测扩增产物16SrDNA片段,然后使用BioTeke®多功能DNA纯化回收试剂盒,进行片段胶回收;
⑤连接:目的片段8μl、solutionⅠ5μl、18T载体1μl在16℃下,连接5h;
⑥转化:将感受态DH5α从-80℃取出,插入冰盆中,待其融化后将全部的连接体系加入到感受态细胞中,冰浴30min;42℃热击90s,然后冰浴90s,加入950μl预冰的LB液体。37℃,100rpm培养1h;然后5000rpm,离心10min,弃上清,保留约200ul液体;将管壁上的固体吹打混匀后涂布加油Amp的LB平板上,过夜培养;
⑦随机挑取平板上的单菌落,进行菌落PCR验证,电泳检测是否有目的条带;
⑧选出目的条带对应的单菌落,然后使用Solarbio®质粒小提试剂盒进行质粒的提取,送至测序公司测序,序列见SEQ ID NO:1所示;
⑨使用NCBI的BLAST进行16S rDNA的相似性比对,结果显示与Geobacillus sp.YMTC1049的同源性99%,所以初步认定它是地芽孢杆菌属,命名为YM6。
序列表
<110> 昆明理工大学
<120> 一株地芽孢杆菌菌株及其应用
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1456
<212> DNA
<213> Geobacillus sp.YM6
<400> 1
gcgatgcact ctataatgca gtcgagcgga ccggatcgga gcttgctctg atttggtcag 60
cggcggacgg gtgagtaaca cgtgggcaac ctgcccgcaa gaccgggaca actccgggaa 120
accggagcta ataccggata acaccgaaga ccgcatggtc tttggttgaa aggcggcctt 180
tgggctgtca cttgcggatg ggcccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatgcgtagc cggcctgaga gggtgaccgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatgggcg aaagcctgac 360
ggagcgacgc cgcgtgagcg aagaaggcct tcgggtcgta aagctctgtt gtgagggacg 420
aaggagcgcc gttcgaagag ggcggcgcgg tgacggtacc tcacgagaaa gccccggcta 480
actacgtgcc agcagccgcg gtaatacgta gggggcgagc gttgtccgga attattgggc 540
gtaaagcgcg cgcaggcggt tccttaagtc tgatgtgaaa gcccacggct caaccgtgga 600
gggtcattgg aaactggggg acttgagtgc aggagagggg agcggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcggctctc tggcctgcaa 720
ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagagg ggtcacaccc tttagtgctg cagctaacgc 840
gataagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960
tcttgacatc ccctgacaac ccaagagatt gggcgttccc ccttcggggg gacagggtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc tcgcctctag ttgccagcat tcggttgggc actctagagg gactgccggc 1140
gacaagtcgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200
cacacgtgct acaatgggcg gtacaaaggg ctgcgaaccc gcgaggggga gcgaatccca 1260
aaaagccgct ctcagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaatcgct 1320
agtaatcgtg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380
tcacaccacg agagcttgca acacccgaag tcggtgaggt aacccttacg ggagccagcc 1440
gccgtagtgt cagagc 1456
<210> 2
<211> 19
<212> DNA
<213> 人工序列
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
agagtttgat cctggctcag 20
Claims (2)
1.一种地芽孢杆菌菌株Geobacillus sp.YM6,其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.12089。
2.权利要求1所述的地芽孢杆菌菌株在降解角蛋白中的应用。
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CN114958690A (zh) * | 2022-06-28 | 2022-08-30 | 广东海洋大学 | 一种芽孢杆菌及其筛选方法和在高效降解羽毛上的应用 |
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