实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

竹鼠肠道纤维素降解菌的分离鉴定及其产酶特性研究

  • 曾文婷 ,
  • 覃绍敏 ,
  • 吴健敏 ,
  • 梁小莉 ,
  • 白安斌 ,
  • 刘金凤 ,
  • 陈凤莲 ,
  • 秦树英 ,
  • 马玲
展开
  • 1. 广西大学动物科学技术学院, 南宁 530004;
    2. 广西壮族自治区兽医研究所, 广西兽医生物技术重点实验室, 南宁 530001;
    3. 广西农业职业技术学院动物科学技术学院, 南宁 530007
曾文婷(1996-),女,四川江油人,硕士研究生,预防兽医学专业。E-mail:zwt072996@163.com

收稿日期: 2020-12-24

  网络出版日期: 2021-07-06

基金资助

广西科技重大专项(桂科AA18118051);广西兽医生物技术重点实验室开放课题(19-50-40-B-06)

Isolation, Identification and Enzyme Production Characteristics of Cellulolytic Bacterium from Intestine of Bamboo Rats

  • ZENG Wengting ,
  • QIN Shaomin ,
  • WU Jianmin ,
  • LIANG Xiaoli ,
  • BAI Anbin ,
  • LIU Jinfeng ,
  • CHEN Fenglian ,
  • QIN Shuying ,
  • MA Ling
Expand
  • 1. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
    2. Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China;
    3. College of Animal Science and Technology, Guangxi University Guangxi Agricultural Vocational and Technical College, Nanning 530007, China

Received date: 2020-12-24

  Online published: 2021-07-06

Supported by

 

摘要

本试验旨在从竹鼠肠道内容物中分离筛选易培养、具有高纤维素酶活性的菌株。称取竹鼠肠道内容物1.0 g,以刚果红平板进行纤维素降解菌的初筛,再分别以羧甲基纤维素钠(CMC-Na)、微晶纤维素、D-水杨苷和滤纸为碳源,采用3,5-二硝基水杨酸(DNS)法测定菌株内切葡聚糖酶(Cen)、外切葡聚糖酶(Cex)、β-葡萄糖苷酶(BG)和滤纸酶(FPA)活性。采用生理生化试验和16S rDNA PCR方法进行菌株种属鉴定。结果显示:从竹鼠肠道内容物中分离获得4株产纤维素酶的需氧菌(GL-4、GL-5、GL-6和GL-8),其中GL-4的Cen和FPA活性最高,分别为49.32和25.13 U/mL;GL-8的Cex活性最高,为30.59 U/mL;GL-5的BG活性最高,为35.08 U/mL。对纤维素酶总活性最高的GL-4进行了培养条件优化,其在37 ℃、pH 8.0条件下培养24 h时,FPA活性最高。对产纤维素酶活性较高的3株菌(GL-4、GL-5和GL-8)进行种属鉴定,显示3株菌均为枯草芽孢杆菌。综上可知,本试验从竹鼠肠道内容物中分离到3株需氧枯草芽孢杆菌,这3株菌均具有较高的产酶能力;对菌株GL-4进行培养条件优化,得出其产酶最适条件为发酵时间24 h、发酵培养基pH 8和发酵温度37 ℃,可作为高粗纤维含量非粮型饲料开发利用的候选菌株。

本文引用格式

曾文婷 , 覃绍敏 , 吴健敏 , 梁小莉 , 白安斌 , 刘金凤 , 陈凤莲 , 秦树英 , 马玲 . 竹鼠肠道纤维素降解菌的分离鉴定及其产酶特性研究[J]. 动物营养学报, 2021 , 33(7) : 4142 -4152 . DOI: 10.3969/j.issn.1006-267x.2021.07.055

Abstract

This experiment aimed to isolate and screen bacterial strains with high cellulase activity from the intestinal contents of bamboo rats. Cellulose degrading bacteria in the 1.0 g intestinal contents of bamboo rats were preliminary screened by Congo red plate, and then used sodium carboxymethyl cellulose (CMC-Na), microcrystalline cellulose, D-salicylic acid and filter paper as carbon sources, to determine the activities of endoglucanase (Cen), exoglucanase (Cex), β-glucosidase (BG) and filter paper enzyme (FPA) of bacterial strains with 3,5-dinitrosalicylic acid (DNS) method. The species identification of bacterial strains through physiological and biochemical tests and 16S rDNA PCR method. The results showed that 4 cellulase-producing aerobic bacteria (GL-4, GL-5, GL-6 and GL-8) were isolated from the intestinal contents of bamboo rats. Among them, GL-4 had the highest Cen and FPA activities which were 49.32 and 25.13 U/mL, respectively; GL-8 had the highest Cex activity which was 30.59 U/mL; GL-5 had the highest BG activity which was 35.08 U/mL. The culture conditions of GL-4 which had the highest total cellulase activity were optimized. When the temperature was 37℃, the culture time was 24 h and the pH of the medium was 8.0, the GL-4 had the highest FPA activity. The species identification of 3 bacterial strains (GL-4, GL-5 and GL-8) with high cellulase activity showed that all bacterial strains were Bacillus subtilis. In conclusion, three strains of aerobic Bacillus subtilis are isolated from the intestinal contents of bamboo rats, and all the strains have high cellulase-producing capacity and are easy to cultivate. The optimal conditions for cellulase-producing of GL-4 which had the highest total cellulase activity are 24 h of culture time, 37℃ of culture temperature and pH 8 of medium. The GL-4 can be used as a candidate strain for the development and utilization of non-grain feeds with high crude fiber content.

参考文献

[1] JØRGENSEN H,KRISTENSEN J B,FELBY C.Enzymatic conversion of lignocellulose into fermentable sugars:challenges and opportunities[J].Biofuels,Bioproducts and Biorefining,2007,1(2):119-134.  
[2] 孙喆,鲍男,王涛,等.纤维素酶来源的研究进展[J].吉林畜牧兽医,2014,35(9):22-25. SUN Z,BAO N,WANG T,et al.Research progress on the source of cellulase[J].Jilin Animal Husbandry and Veterinary Medicine,2014,35(9):22-25.(in Chinese)
[3] 常娟,卢敏,尹清强,等.秸秆资源预处理研究进展[J].中国农学通报,2012,28(11):1-8. CHANG J,LU M,YIN Q Q,et al.Research progress of straw resource pretreatment[J].Chinese Agricultural Science Bulletin,2012,28(11):1-8.(in Chinese)
[4] 李收,杨平平.微生物发酵生产纤维素酶的研究进展[J].山东轻工业学院学报(自然科学版),2011,25(2):12-14. LI S,YANG P P.Research progress on the production of cellulase by microbial fermentation[J].Journal of Shandong Institute of Light Industry (Natural Science Edition),2011,25(2):12-14.(in Chinese)
[5] 陈小连,刘建新,王佳堃,等.瘤胃纤维分解菌多纤维素酶体及其类似物的研究进展[J].中国畜牧杂志,2009,45(3):50-53. CHEN X L,LIU J X,WANG J K,et al.Research progress of cellulosomes and their analogues of rumen cellulolytic bacteria[J].Chinese Journal of Animal Husbandry,2009,45(3):50-53.(in Chinese)
[6] 何静,李国伟,海勒,等.双峰驼粪便中纤维素分解菌的筛选及酶学特性分析[J].西北农业学报,2019,28(11):1750-1759. HE J,LI G W,HAI L,ET al.Screening of cellulolytic bacteria in the feces of bactrian camels and analysis of their enzymatic characteristics[J].Northwest Agricultural Journal,2019,28(11):1750-1759.(in Chinese)
[7] 张智,尹文哲,雅男,等.大熊猫粪便中纤维素降解菌的筛选及其产酶条件的优化[J].动物营养学报,2017,29(8):2817-2825. ZHANG Z,YIN W Z,YA N,et al.Screening of cellulose-degrading bacteria in giant panda feces and optimization of enzyme production conditions[J].Chinese Journal of Animal Nutrition,2017,29(8):2817-2825.(in Chinese)
[8] 樊程,李双江,李成磊,等.大熊猫肠道纤维素分解菌的分离鉴定及产酶性质[J].微生物学报,2012,52(9):1113-1121. FAN C,LI S J,LI C L,et al.Isolation and identification of cellulolytic bacteria in the intestine of giant panda and their enzyme-producing properties[J].Acta Microbiol Sinica,2012,52(9):1113-1121.(in Chinese)
[9] 杨伟平,孟凡旭,马丽,等.藏香猪源纤维素分解菌的分离鉴定及酶学特性分析[J].动物营养学报,2014,26(3):620-629. YANG W P,MENG F X,MA L,et al.Solation,identification and enzymatic characteristics analysis of cellulolytic bacterium from Tibetan pigs[J].Chinese Journal of Animal Nutrition,2014,26(3):620-629.(in Chinese)
[10] 卓智龙.银星竹鼠的生物学特性研究[D].硕士学位论文.南宁:广西师范大学,2012. ZHUO Z L.Study on the biological characteristics of the silver star bamboo rat[D].Master's Thesis.Nanning:Guangxi Normal University,2012.(in Chinese)
[11] LI R Q,FAN W,TIAN G,et al.The sequence and de novo assembly of the giant panda genome[J].Nature,2010,463(7279):311-317.  
[12] 宁俊平.竹鼠肠道兼性厌氧纤维素降解菌的分离、鉴定及其产酶活性研究[D].硕士学位论文.南宁:广西大学,2014. NING J P.Isolation,identification and enzyme-producing activity of facultative anaerobic cellulose-degrading bacteria in bamboo rat intestine[D].Master's Thesis.Nanning:Guangxi University,2014.(in Chinese)
[13] 曹晓燕.竹鼠肠道中厌氧纤维素降解菌的分离与鉴定[D].硕士学位论文.南宁:广西大学,2012. CAO X Y.Isolation and identification of anaerobic cellulose-degrading bacteria in bamboo rat intestines[D].Master's Thesis.Nanning:Guangxi University,2012.(in Chinese)
[14] 杨伟平.藏猪肠道细菌群落组成与纤维素分解菌的研究[D].博士学位论文.杨凌:西北农林科技大学,2015. YANG W P.Study on the composition of intestinal bacterial community and cellulolytic bacteria in Tibetan pigs[D].Ph.D. Thesis.Yangling:Northwest A&F University,2015.(in Chinese)
[15] 李日强,辛小芸,刘继青,等.天然秸秆纤维素分解菌的分离选育[J].上海环境科学,2002,21(1):8-11. LI R Q,XIN X Y,LIU J Q,et al.Isolation and screening on straw cellulose-decomposing microorganisms[J].Shanghai Environmental Sciences,2002,21(1):8-11.(in Chinese)
[16] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001. CAI M Y,DONG X Z.Manual of identification of common bacteria system[M].Beijing:Science Press,2001.(in Chinese)
[17] KREIG N R,HOLT J G,MURRAY R G E,et al.Bergey's manual of systematic bacteriology[M].New York:Springer Science & Business Media,1989:89-100.
[18] GHEZELBASH G R,MAJIDI S,ROAYAEI A M.Carboxymethyl-cellulase and filter-paperase activity of new strains isolated from Persian gulf[J].Microbiology Journal,2011,1(1):8-16.
[19] 沈琦,孙宏,王新,等.猪粪中嗜热纤维素降解菌的筛选及其除臭特性研究[J].中国畜牧杂志,2020,56(10):162-166. SHEN Q,SUN H,WANG X,et al.Study on the screening of thermophilic cellulose degrading bacteria in pig manure and its deodorizing characteristics[J].Chinese Journal of Animal Science,2020,56(10):162-166.(in Chinese)
[20] 朱宁.木质纤维素降解酶系在草本类生物质上的协作机制[D].博士学位论文.北京:中国农业大学,2016. ZHU N.Cooperative mechanism of lignocellulose degrading enzymes on herbaceous biomass[D].Ph.D. Thesis.Beijing:China Agricultural University,2016.(in Chinese)
[21] 殷晓敏,郑服丛,贺春萍,等.枯草芽孢杆菌B215生物学特性及对香蕉枯萎病的生防效果评价[J].热带作物学报,2010,31(8):1416-1419. YIN X M,ZHENG F C,HE C P,et al.Biological characteristics of Bacillus subtilis B215 and evaluation of its biocontrol effect on banana wilt[J].Chinese Journal of Tropical Crops,2010,31(8):1416-1419.(in Chinese)
[22] 王红兵,王荣柱,陆涛,等.一种海洋黑曲霉耐盐内切纤维素酶的分离纯化和酶学性质研究[J].高校化学工程学报,2016,30(2):410-416. WANG H B,WANG R Z,LU T,et al.Purification and characterization of a halotolerant endoglucanase from marine Aspergilla niger[J].Journal of Chemical Engineering of Chinese Universities,2016,30(2):410-416.(in Chinese)
[23] 刘阳,邓静,吴华昌,等.盐胁迫对枯草芽孢杆菌发酵代谢产物的影响[J].食品与发酵工业,2015,41(7):29-33. LIU Y,DENG J,WU H C,et al.The effect of salt stress on the fermentation metabolites of Bacillus subtilis[J].Food and Fermentation Industry,2015,41(7):29-33.(in Chinese)
[24] IRFAN M,TAYYAB A,HASAN F,et al.Production and characterization of organic solvent-tolerant cellulase from Bacillus amyloliquefaciens AK9 isolated from hot spring[J].Applied Biochemistry and Biotechnology,2017,182(4):1390-1402.  
[25] 崔海洋,程仕伟,黄田红,等.产纤维素酶的解淀粉芽孢杆菌分离鉴定及酶学性质研究[J].食品科学技术学报,2014,32(3):43-47,53. CUI H Y,CHEN S W,HUANG T H,et al.Screening and identifying of Bacillus amyloliquefaciens for cellulase production and its enzymatic characters[J].Journal of Food Science and Technology,2014,32(3):43-47,53.(in Chinese)
[26] 柴秀娟,李曹龙,孔德真,等.产纤维素酶菌株的筛选、鉴定及产酶条件的优化[J].生物技术通报,2014(9):164-170. CHAI X J,LI C L,KONG D Z,et al.Screening,identification of a cellulase-producing bacterium strain and its enzyme-producing conditions[J].Biotechnology Bulletin,2014(9):164-170.(in Chinese)
[27] GOUVEIA E R,BAPTISTA-NETO A,BADINO A C,Jr,et al.Optimisation of medium composition for clavulanic acid production by Streptomyces clavuligerus[J].Biotechnology Letters,2001,23(2):157-161.  
文章导航

/