CN105969689B - 产纤维素酶细菌及应用 - Google Patents
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Abstract
本发明涉及高效纤维素酶产生菌及产酶条件,具体的说是一株产纤维素酶细菌及产酶条件。细菌(Novosphingobium sp.)YIC32,已于2016年4月25日保藏于广东省微生物菌种保藏中心(简称GDMCC,地址为:广东省广州市先烈中路100号大院59号楼5楼),其保藏编号为GDMCC No.60031。本发明菌株生长速度快,纤维素酶活高,产酶能力强,不会对自然界的碳循环产生负面影响,不会对环境造成危害,是应用于秸秆腐熟生产有机肥料、秸秆还田、促进生态恢复和保护生态环境、饲料加工等行业的好材料。在农林废弃物回收利用过程中可发挥重要作用。
Description
技术领域
本发明涉及产纤维素酶,具体的说是一种产纤维素酶细菌及应用。
背景技术
纤维素是自然界中分布最为广泛、产量最为丰富的可再生资源,广泛存在于植物的根茎叶中。我国是农业大国,每年秸秆产量约为7亿吨左右,秸秆中的主要成分为纤维素。纤维素降解后可转化为生物燃料、优质饲料等物质。然而在自然界中,纤维素降解困难,主要源于其特殊的结构。纤维素是由葡萄糖通过β-(1,4)糖苷键连接而成的线状大分子聚合物。纤维素酶是能够将纤维素降解为葡萄糖的一类酶的总称,根据其催化功能分为三大类:1)葡聚糖内切酶(1,4-β-D-glucan glucanohydrolase):该酶能够随机切割纤维素多糖链内的无定型区,产生不同长度的寡糖和新链末端;2)葡聚糖外切酶(1,4-β-D-glucancellobilhydrolase):作用于纤维素还原性和非还原性的纤维素多糖链的末端,产生葡萄糖或纤维二糖;3)β-葡聚糖苷酶(β-1,4-glucosidase):该酶功能为水解纤维二糖产生葡萄糖。由于纤维素酶的三个组分经过协同作用,将大分子的纤维素降解为低分子量的寡糖、双糖或多糖,而被广泛应用于食品加工、酿造、造纸、饲料添加剂、纺织、医药等工农业领域。使得纤维素酶成为酶制剂工业中的一个新的增长点。纤维素酶主要由微生物产生,受制于纤维素酶制剂工业化生产的因素主要有菌种和酶活及产量,因而高酶活微生物的筛选和产酶条件的优化尤为重要。
发明内容
本发明的目的是提供一种高效产纤维素酶细菌及其应用。
为实现上述目的,本发明采用的技术方案为:
一种高效产纤维素酶细菌,细菌(Novosphingobium sp.)YIC32,已于2016年4月21日保藏于广东省微生物菌种保藏中心(简称GDMCC,地址为:广东省广州市先烈中路100号大院59号楼5楼),其保藏编号为GDMCC No.60031。
所述产纤维素酶细菌分离自粉碎的苹果树枝条自然发酵腐熟的木屑中,通过纤维素酶活的测定及菌种鉴定表明,该菌是一株产酶能力强、纤维素酶活高的细菌菌(Novosphingobium sp.)。对该菌进行16S rRNA基因序列分析,并通过在GenBank中比对发现,本发明的细菌扩增的16S rRNA序列与Novosphingobium panipatense SM16T(登录号:NR044210)相似性最高,为99%。从而将分离出来的细菌命 名为Novosphingobiumsp.YIC32。
所述细菌(Novosphingobium sp.)YIC32在生产纤维素酶中的应用。
进一步的说,将所述细菌(Novosphingobium sp.)YIC32于羧甲基纤维素钠产酶培养基中培养,获得纤维素酶。
所述羧甲基纤维素钠产酶培养基成分为:羧甲基纤维素钠5~15g、KH2PO4 1g、NaNO3 3~10g、KCl 0.5g、MgSO4 0.5g、蒸馏水l000ml、FeSO4﹒7H2O 0.01g,调节pH在5.5~6.0,调pH在5.5~6.0,于121℃,0.11Mpa下灭菌30min备用。
更进一步的说,将所述细菌(Novosphingobium sp.)YIC32在羧甲基纤维素钠产酶培养基中培养获得纤维素酶;其中,羧甲基纤维素钠产酶培养基的pH=5、羧甲基纤维素钠浓度为12.5g/L、NaNO3浓度为5g/L时纤维素内切酶酶活最高,为2.45±0.05IU/mL。pH=5、羧甲基纤维素钠浓度为12.5g/L、NaNO3浓度为3g/L时滤纸酶活、外切酶酶活最高,产酶能力最强,滤纸酶活达20.75±1.61IU/mL,外切酶产酶能力最强,酶活达47.72±0.24IU/mL。
本发明所具有优点:
1.本发明的细菌YIC32具有产纤维素酶活高的特性,该菌在纤维素筛选平板上降解圈/菌落直径达到4.0,在发酵培养基中酶活较高,滤纸酶活为12.37±0.13IU/mL,内切酶酶活为2.45±0.05IU/mL,外切酶酶活为23.60±0.887IU/mL。
2.经本发明优化后,该菌产酶能力明显提高,滤纸酶活为20.75±1.61IU/mL,内切酶酶活为2.45±0.05IU/mL,外切酶酶活为47.72±0.24IU/mL。
3.本发明菌株分离自秸秆腐熟发酵的秸秆中,属于Novosphingobium菌株,因而无毒无害,未经遗传改造,可放心应用到秸秆堆肥腐熟、秸秆还田、饲料加工、纤维素酶工程领域中。
附图说明
图1为本发明实施例提供的菌株YIC32产纤维素酶的刚果红染色前照片。
图2为本发明实施例提供的菌株YIC32产纤维素酶的刚果红染色照片。
图3为本发明实施例提供的菌株YIC32培养条件优化前后纤维素酶酶活。
具体实施方式
下面结合实施例对本发明作进一步说明。实施例旨在对本发明进行举例描述,而非以任何形式对本发明进行限制。
实施例1:菌株YIC32的获得
从粉碎的苹果树枝条堆肥腐熟物中采集约1g腐熟物,进行梯度稀释,从10-1~10-8,分别吸取100μL悬浊液进行涂布至羧甲基纤维素钠固体培养基,28℃倒置培养3-5天,分别挑取单菌落至PDA培养基平板,反复划线直至纯化后转接至PDA培养基斜面(参见图1)。将纯化后的菌株分别转接至羧甲基纤维素钠固体培养基,培养3天,测定菌落直径后,用1mg/ml的刚果红溶液染色1h,弃去染料,加入1mol/L的NaCl溶液脱色1h,然后用去离子水冲洗3-5遍后测定透明圈直径。一般说来,比值的大小与菌株降解纤维素能力大小有关。水解圈和菌株直径的比值越大,菌株降解纤维素的能力越强,因此比值大小的定性试验,直接反映了菌降解纤维素的的能力。因此,本研究选取了水解圈和菌株直径的比值最大的菌株YIC32作为研究对象。
羧甲基纤维素钠固体培养基:羧甲基纤维素钠5g、KH2PO4 1g、NaNO3 3g、KCl 0.5g、MgSO4 0.5g、蒸馏水l000ml、FeSO4﹒7H2O0.01g,琼脂粉15g。(调pH在5.5—6.0,于121℃,0.11Mpa下灭菌30min备用)。
PDA培养基:去皮马铃薯200g、葡萄糖20g、蒸馏水1000ml、1.5%琼脂。(自然pH值,于121℃,0.11Mpa下灭菌30min备用)。
1)菌株鉴定
将上述纯化获得的产纤维素酶活高的菌株进行菌落观察,在PDA固体培养基上,菌落表面湿润,黄绿色,为典型细菌的表型特征。
分子鉴定:为了确定本实施细菌菌株YIC32的系统进化地位,对其16S rRNA基因序列测定与系统发育分析。
首先提取该菌株的基因组DNA,并扩增其16S rRNA的基因序列,PCR引物为27F:(AGA GTT TGA TCM TGG CTC AG),1492R:(AGA GTT TGA TCM TGG CTC AG),将PCR产物进行测序并进行序列分析,表明,与Novosphingobium panipatense SM16T(登录号:NR044210)相似性最高为99%。
Novosphingobium sp.YIC32的16S rRNA基因序列:
ATGCCTACACATGCAGTCGAACGAGATCTTCGGAATCTAGTGGCGCACGGGTGCGTAACGCGTGGGAATCTGCCCTTGGGTTCGGAATAACAGTGAGAAATTACTGCTAATACCGGATGATGTCTTCGGACCAAAGATTTATCGCCCAGGGATGAGCCCGCGTAGGATTAGCTAGTTGGTGGGGTAATGGCCTACCAAGGCGACGATCCTTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTTACCAGGGATGATAATGACAGTACCTGGAGAATAAGCTCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGAGCTAGCGTTGTTCGG AATTACTGGGCGTAAAGCGCGCGTAGGCGGTTACTCAAGTCAGAGGTGAAAGCCCGGGGCTCAACCCCGGAACTGCCTTTGAAACTAGGTGACTAGAATCTTGGAGAGGTCAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAAGGCGACTGACTGGACAAGTATTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATAACTAGCTGTCCGGGTACTTGGTACTTGGGTGGCGCAGCTAACGCATTAAGTTATCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCTGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCGTTTGACATCCTCATCGCGGATTTGAGAGATCATTTCCTTCAGTTCGGCTGGATGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTCCTTAGTTGCCATCATTTGGTTGGGCACTCTAAGGAAACTGCCGGTGATAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACACGCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGCAGCAAGTGCGCGAGCACAAGCTAATCTCCAAAAGCCGTCTCAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGGCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCAGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGATTCACTCGAAGGCGTTGAGCTAACCCGCAAGGGAGGCAGGCGACCAC
综合以上鉴定结果,将菌株YIC32定名为Novosphingobium sp.;已于2016年4月21日保藏于广东省微生物菌种保藏中心(简称GDMCC,地址为:广东省广州市先烈中路100号大院59号楼5楼),其保藏编号为GDMCC No.60031。Novosphingobium sp.YIC32,简称菌株YIC32。
上述细菌菌菌株YIC32降解纤维素能力强,酶活高,未进行遗传改造。当菌体被释放入自然环境后,对人、动植物无害,不污染环境,不能够促进秸秆还田,农林废弃物的降解,在农业上有潜在的应用价值。
所得Novosphingobium sp.YIC32细菌菌株的实验室保藏方式:
短期保藏方式为,接种至PDA固体斜面,待菌落长好后保藏于4℃冰箱内,此保藏方式用于不超过3个月短期保藏;
长期保藏方式为:将细菌菌体加入终浓度为20%甘油中混匀并保藏至-80℃冰箱中,此保藏方式大约可保藏10年。
实施例2产酶能力及其他特性分析
1)菌株YIC32的产纤维素能力初筛分析
将上述获得细菌菌株YIC32点接至羧甲基纤维素钠培养基固体平板上,28℃培养5天,测定菌落直径后,用1mg/mL的刚果红溶液染色1h后,弃去染料,加入1mol/L氯化钠溶液脱色1h,测定透明圈直径。透明圈直径/菌落直径为4.0(参见图1,2),因此,该菌株有着很强的降解纤维素能力。
其中,羧甲基纤维素钠固体培养基成分:羧甲基纤维素钠5g、K2HPO41g、NaNO3 3g、KCl 0.5g、MgSO4 0.5g、蒸馏水l000ml、FeSO4 0.01g,琼脂15g(pH 5.5—6.0)。
2)菌株YIC32的纤维素酶活测定
对产纤维素酶菌株进行酶活的测定,酶活力的测定参考Miller G.L的DNS法,这种方法可以对菌株产纤维素酶活强度进行有效定量。酶活力定义在50℃条件下1mL粗酶液在1min内水解生成1.0μg的葡萄糖,称为1个纤维素酶酶活力单位(μg/min,IU)。
将上述获得菌株接种到50ml(100ml三角瓶)羧甲基纤维钠液体培养基中,于28℃、180r/min摇床培养,3d后取菌液,6000r/min离心10min,上清即为粗酶液。
其中,羧甲基纤维钠液体培养基为:羧甲基纤维素钠5g、K2HPO41g、NaNO3 3g、KCl0.5g、MgSO4 0.5g、蒸馏水l000ml、FeSO4 0.01g,琼脂15g(pH 5.5—6.0)。
对粗酶液进行三种酶活的测定:
①滤纸酶活的测定。将新华1号滤纸(1cm×3cm)卷成小卷,放进15ml EP管内,加入1.75mL HAc-NaAc缓冲液(pH=4.8),再加入0.25mL粗酶液,轻轻摇匀,使滤纸完全浸泡在液体中,50℃保温1h后,加入3ml DNS溶液,放于沸水浴中煮沸10min,用冷水立即终止反应。在波长为540nm下测定光吸收值(OD值)。
②内切酶活测定。移取含有0.5%CMC-Na的醋酸缓冲液1.75mL于试管中,加入粗酶液0.25mL,50℃保温30min,加入3ml DNS溶液,放于沸水浴中煮沸10min,置冷水中终止反应。在波长为540nm下测定光吸收值(OD值)。
③外切酶活测定。移取含有0.5%微晶纤维素的醋酸缓冲液1.75mL于试管中,加入酶液0.25mL,于50℃保温2h后,加入3ml DNS溶液,置沸水浴中煮沸10min,置冷水中终止反应。在波长为540nm下测定光吸收值(OD值)。
将上述三种酶活的测定的对照组选用经沸水煮10min后冷水冷却的上述粗酶液。每个样品做三个重复。测定结果显示菌株YIC32有着较高的酶活,滤纸酶活为12.37±0.13IU/mL,内切酶酶活为2.45±0.05IU/mL,外切酶酶活为23.60±0.89IU/mL(参见图3)。
实施例3菌株YIC32产纤维素酶培养条件的优化
纤维素酶的产生及分泌能力与菌的生长状态和培养环境(如碳、氮源配比、pH等)密切相关,本发明对发酵产酶过程中碳、氮源配比、pH进行了优化,最终使产酶能力显著提高。
优化的基础培养基为羧甲基纤维素钠培养基,由于羧甲基纤维素钠培养基配制完成要求pH 5.5-6.0,这可能不是YIC32产纤维素酶的最优pH,且碳源、氮源的配比都影响着产酶菌株的产酶能力,因此,本发明分别设置了pH=3,4,5,6,7,8一系列的pH培养条件、羧甲基纤维素钠培养基中羧甲基纤维素钠质量浓度百分比为0.25%,0.5%,0.75%,1%,1.25%一系列碳源浓度和NaNO3浓度为0.25%,0.3%,0.5%,1%,1.5%,2%一系列的氮源浓度,并进行了正交试验。
结果表明,羧甲基纤维素钠培养基的pH=5、培养基中羧甲基纤维素钠浓度为12.5g/L、培养基中NaNO3浓度为5g/L时纤维素内切酶酶活最高,为2.45±0.05IU/mL。pH=5、羧甲基纤维素钠浓度为12.5g/L、NaNO3浓度为3g/L时滤纸酶活、外切酶酶活最高,产酶能力最强,滤纸酶活达20.75±1.61IU/mL,外切酶产酶能力最强,酶活达47.72±0.24IU/mL。培养条件优化后,滤纸酶活、外切酶酶活分别比原始酶活增加了67.74%和102.2%(参见图3)。
Claims (4)
1.一种高效产纤维素酶细菌,其特征在于:细菌(Novosphingobium sp.)YIC32,已于2016年4月21日保藏于广东省微生物菌种保藏中心(简称GDMCC,地址为:广东省广州市先烈中路100号大院59号楼5楼),其保藏编号为GDMCC No. 60031。
2.按权利要求1所述的细菌的应用,其特征在于:所述细菌(Novosphingobium sp.)YIC32 在生产纤维素酶中的应用。
3.按权利要求2所述的细菌的应用,其特征在于:将所述细菌(Novosphingobium sp.)YIC32于羧甲基纤维素钠产酶培养基中培养获得纤维素酶。
4.按权利要求3所述的细菌的应用,其特征在于:所述羧甲基纤维素钠产酶培养基成分为:羧甲基纤维素钠5~15 g、KH2PO4 1 g、NaNO3 3~10 g、KCl 0.5 g、MgSO4 0.5 g、蒸馏水l000 ml、FeSO4﹒7H2O 0.01 g,调节pH在5.5~6.0。
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