中国牦牛瘤胃未培养微生物来源的纤维素降解酶的筛选、克隆和鉴定
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摘要
纤维素是世界上储量最丰富的可再生资源,为发展可再生生物燃料的提供了无尽的原料,但纤维素的糖化却一直是其利用过程中的瓶颈。为了寻找新型、高效的纤维素酶,本研究对中国牦牛瘤胃微生物纤维素降解酶资源进行了探索。
瘤胃样本细菌菌群分析显示,66.8%的细菌分布于硬壁菌门(Firmicutes)和27.9%的细菌分布于拟杆菌门(Bacteroidetes)。该样本中约10%的细菌为已知菌,包括了典型的瘤胃纤维素降解菌Ruminococcus albus, Butyrivibrio fibrisolvens和Fibrobacter succinogenes。其余约90%的细菌是不可培养微生物,其中很大一部分落入Ruminococcaceae科,Lachnospiraceae科和Prevotellaceae科,且与已知的瘤胃纤维素降解菌聚类在一起,提示它们可能为新型的不可培养纤维素降解菌。对其中的真菌菌群分析显示真菌含量约只有细菌的1%,含有具有纤维素降解能力的真菌Orpinomyces sp.。所以该瘤胃样本不仅含有已知纤维素降解菌,还可能含有许多不可培养的纤维素降解菌,提示了从中克隆到纤维素酶的高可能性。
基于该瘤胃样本中大部分微生物是不可培养的,我们使用宏基因组技术构建了中国牦牛瘤胃微生物的宏基因组文库,并对其进行功能筛选,获得了内切葡聚糖酶阳性克隆5个、外切葡聚糖酶阳性克隆24个、葡萄糖苷酶阳性克隆50个木糖苷酶阳性克隆27个和木聚糖酶阳性克隆6个。随后克隆和鉴定了其中的三个酶RuBGX2、RuBGX3和Ruce15B。
RuBGX2和RuBGX3均既有p-葡萄糖苷酶活性,又有p-木糖苷酶活性,属于糖苷水解酶家族3,具有48.9%的氨基酸序列一致性。他们的最适反应pH约为5左右,最适反应温度约为40℃,且对于p构型的糖苷底物均具有一定的广谱性,同时它既能水解纤维寡糖类底物,又能水解多种人工芳香基衍生底物。RuBGX2可水解聚合度为2、3、4的纤维寡糖,RuBGX3仅能水解聚合度为2、3的纤维寡糖。RuBGX2对1%的纤维二糖的转化率高达98.3%,对1%纤维四糖的转化率高达80%。RuBGX2和RuBGX3的β-葡萄糖苷酶活性使得他们可以降低纤维素水解中间产物的抑制作用来促进纤维素的水解,而β-木糖苷酶活性使得他们可以通过与木聚糖酶的协同作用来来促进半纤维素的水解。同源建模和分子对接结果显示RuBGX2和RuBGX3的β-葡萄糖苷酶活性和p-本糖苷酶活性来自于同一个活性中心,说明这两种活性是由底物的广谱特异性引起的。
Rucel5B既有内切葡聚糖酶活性,又有外切葡聚糖酶活性,属于糖苷水解酶家族5。它的内切葡聚糖酶活性高达219.7U/mg,外切葡聚糖酶活性高达52.9U/mg。Ruce15B的最适温度约60℃,在55℃处理1h后不丧失酶活,说明它具有比较高的热稳定性。底物特异性分析结果显示Ruce15B能够水解β-1,4葡萄糖苷键,但是不能水解β-1,3葡萄糖苷键β-1,6葡萄糖苷键和β-1,4木糖苷键,同时Ruce15B既能水解无定型纤维素(CMC, barley glucan, lichenan, PASC),又能水解晶体纤维素(微晶纤维素和滤纸avicel, filter paper)。进一步对Ruce15B的水解产物进行了色谱分析,结果显示它能从MuC的非还原端切割下纤维二糖,能够水解纤维四糖,avicel, filter paper和PASC并释放纤维二糖,说明了他的外切作用模式。而对可溶性无定型纤维素(CMC, barley glucan, lichenan)时间梯度降解结果显示在反应的开始阶段,产物的聚合度明显降低,提示Ruce15B具有内切的作用模式。
综上,本研究分析了牦牛瘤胃样本中微生物的多样性,建立了宏基因组文库功能筛选纤维素酶的平台,克隆并鉴定了两个β-葡萄糖苷酶/木糖苷酶RuBGX2和RuBGX3,和内切/外切葡聚糖酶Ruce15B。RuBGX2和RuBGX3不仅显示了葡萄糖苷酶和木糖苷酶两种活性,还显示了对纤维寡糖的高转化率和与木聚糖酶的协同作用；Ruce15B则显示出纤维素内切和外切作用模式,以及高活性和热稳定性的特质。这些研究结果都说明了瘤胃是新型纤维素降解酶的很好来源,这些纤维素降解酶在纤维素类生物质糖化的过程中具有很广阔的应用潜力。
Cellulose is the most abundant renewable biomass on earth. Saccharification of cellulose to fermentable sugar is an important process in conversion biomass to biofuels and chemicals and is the bottleneck of the application. To hunt for novel, high activity cellulases, the cellulases resources in yak (Bos grunniens) rumen were explored in this study.
The community analysis for rumen bacteria showed that66.8%bacteria belonged to Firmicutes and27.9%bacteria belonged to Bacteroidetes.10%bactera there were known species, including typical rumen cellulose-degrading bacteria Ruminococcus albus, Butyrivibrio fibrisolvens and Fibrobacter succinogenes. The other90%were uncultured, a part of which fell into Ruminococcaceae, Lachnospiraceae and Prevotellaceae, and clustered with several known rumen cellulose-degrading bacteria, suggesting they may be novel uncultured cellulose-degrading bacteria. The community analysis for rumen fungi showed that the abundance of fungi is only10%of that of bacteria, and there exist cellulose-degrading fungus Orpinomyces sp. So this rumen sample not only contains known cellulose-degrading microorganism, but also has great possibility to contain many novel uncultured cellulose-degrading microorganism.
Considering the majority of the rumen microorganisms were unculturable, we make use of metagenomic technology to construct cosmid library, and functionally screened for cellulases. The screening result5endoglucanase positive clones,24exoglucanase positive clones,50glucosidase positive clones,27xylosidase positive clones and6xylanase positive clones. Three of them, RuBGX2、RuBGX3and Rucel5B were cloned and characterized.
Both Rubgx2and Rubgx3have not only β-glucosidase activity, but also β-xylosidase activity, and belong to glycoside hydrolase family (GH)3. They shared48.9%amino acid sequence identity and belonged to glycoside hydrolase family3. Their optimal pH were about5, optimal temperature were about40℃. They had broad-spectrum activity towards β-configurational glycoside substrates, and can hydrolyze both cellooligosaccharide substrates and artificial aryl substrates. Their β-glucosidase activities enable the promotion of cellulose degradation by reducing inhibition of intermediate product, while their β-xylosidase activities enable the promotion of hemicellulose degradation through synergy effect with xylanase. Homology modeling and substrate docking results showed their β-glucosidase activity and β-xylosidase activity have the same active site, indicating these two kinds of activities are due to broad-spectrum specificity of substrates.
Ruce15B has both endoglucanase activity and exoglucanase activity, and belongs to GH5. Ruce15B's endoglucanase activity was219.7U/mg, and its exoglucanase activity was52.9U/mg. It exhibited optimal temperatures at about60℃and was thermostable at55℃, suggesting it's a thermostable enzyme. Ruce15B can hydrolyze β-1,4-glucosidic linkage, not β-1,3, β-1,6glucosidic linkage, or β-1,4xylosidic linkage. It was further confirmed that Ruce15B can cleave cellobiose from4-methylumbelliferyl-β-D-cellobiopyranoside, cellotetraose and insoluble cellulose (avicel, filter paper and PASC) through exo-mode of action. Time course degradation of soluble amorphous cellulose (CMC, barley glucan, lichenan) by Ruce15B showed that degree of polymerization of products was decreased rapidly at the beginning of the reaction, suggesting Ruce15B's endo-mode of action.
So this research analyzed the diversity of yak rumen microorganism community, constructed the metagenomic library platform to functional screen cellulase, clones and characterized two β-glucosidase/xylosidase RuBGX2and RuBGX3and endo/exoglucanase Ruce15B. RuBGX2and RuBGX3not only exhibited both β-glucosidase and β-xylosidase activity, but also exhibited high conversion on cellooligosaccharides and the synergy effect with xylanase. Rucel5B exhibited endo-and exo-mode in cellulose degradation, high activity and thermostability. These results indicated rumen is a good source for novel cellulose-degrading enzyme and these cellulases had huge potential in saccharification of cellulosic biomass.
瘤胃样本细菌菌群分析显示,66.8%的细菌分布于硬壁菌门(Firmicutes)和27.9%的细菌分布于拟杆菌门(Bacteroidetes)。该样本中约10%的细菌为已知菌,包括了典型的瘤胃纤维素降解菌Ruminococcus albus, Butyrivibrio fibrisolvens和Fibrobacter succinogenes。其余约90%的细菌是不可培养微生物,其中很大一部分落入Ruminococcaceae科,Lachnospiraceae科和Prevotellaceae科,且与已知的瘤胃纤维素降解菌聚类在一起,提示它们可能为新型的不可培养纤维素降解菌。对其中的真菌菌群分析显示真菌含量约只有细菌的1%,含有具有纤维素降解能力的真菌Orpinomyces sp.。所以该瘤胃样本不仅含有已知纤维素降解菌,还可能含有许多不可培养的纤维素降解菌,提示了从中克隆到纤维素酶的高可能性。
基于该瘤胃样本中大部分微生物是不可培养的,我们使用宏基因组技术构建了中国牦牛瘤胃微生物的宏基因组文库,并对其进行功能筛选,获得了内切葡聚糖酶阳性克隆5个、外切葡聚糖酶阳性克隆24个、葡萄糖苷酶阳性克隆50个木糖苷酶阳性克隆27个和木聚糖酶阳性克隆6个。随后克隆和鉴定了其中的三个酶RuBGX2、RuBGX3和Ruce15B。
RuBGX2和RuBGX3均既有p-葡萄糖苷酶活性,又有p-木糖苷酶活性,属于糖苷水解酶家族3,具有48.9%的氨基酸序列一致性。他们的最适反应pH约为5左右,最适反应温度约为40℃,且对于p构型的糖苷底物均具有一定的广谱性,同时它既能水解纤维寡糖类底物,又能水解多种人工芳香基衍生底物。RuBGX2可水解聚合度为2、3、4的纤维寡糖,RuBGX3仅能水解聚合度为2、3的纤维寡糖。RuBGX2对1%的纤维二糖的转化率高达98.3%,对1%纤维四糖的转化率高达80%。RuBGX2和RuBGX3的β-葡萄糖苷酶活性使得他们可以降低纤维素水解中间产物的抑制作用来促进纤维素的水解,而β-木糖苷酶活性使得他们可以通过与木聚糖酶的协同作用来来促进半纤维素的水解。同源建模和分子对接结果显示RuBGX2和RuBGX3的β-葡萄糖苷酶活性和p-本糖苷酶活性来自于同一个活性中心,说明这两种活性是由底物的广谱特异性引起的。
Rucel5B既有内切葡聚糖酶活性,又有外切葡聚糖酶活性,属于糖苷水解酶家族5。它的内切葡聚糖酶活性高达219.7U/mg,外切葡聚糖酶活性高达52.9U/mg。Ruce15B的最适温度约60℃,在55℃处理1h后不丧失酶活,说明它具有比较高的热稳定性。底物特异性分析结果显示Ruce15B能够水解β-1,4葡萄糖苷键,但是不能水解β-1,3葡萄糖苷键β-1,6葡萄糖苷键和β-1,4木糖苷键,同时Ruce15B既能水解无定型纤维素(CMC, barley glucan, lichenan, PASC),又能水解晶体纤维素(微晶纤维素和滤纸avicel, filter paper)。进一步对Ruce15B的水解产物进行了色谱分析,结果显示它能从MuC的非还原端切割下纤维二糖,能够水解纤维四糖,avicel, filter paper和PASC并释放纤维二糖,说明了他的外切作用模式。而对可溶性无定型纤维素(CMC, barley glucan, lichenan)时间梯度降解结果显示在反应的开始阶段,产物的聚合度明显降低,提示Ruce15B具有内切的作用模式。
综上,本研究分析了牦牛瘤胃样本中微生物的多样性,建立了宏基因组文库功能筛选纤维素酶的平台,克隆并鉴定了两个β-葡萄糖苷酶/木糖苷酶RuBGX2和RuBGX3,和内切/外切葡聚糖酶Ruce15B。RuBGX2和RuBGX3不仅显示了葡萄糖苷酶和木糖苷酶两种活性,还显示了对纤维寡糖的高转化率和与木聚糖酶的协同作用；Ruce15B则显示出纤维素内切和外切作用模式,以及高活性和热稳定性的特质。这些研究结果都说明了瘤胃是新型纤维素降解酶的很好来源,这些纤维素降解酶在纤维素类生物质糖化的过程中具有很广阔的应用潜力。
Cellulose is the most abundant renewable biomass on earth. Saccharification of cellulose to fermentable sugar is an important process in conversion biomass to biofuels and chemicals and is the bottleneck of the application. To hunt for novel, high activity cellulases, the cellulases resources in yak (Bos grunniens) rumen were explored in this study.
The community analysis for rumen bacteria showed that66.8%bacteria belonged to Firmicutes and27.9%bacteria belonged to Bacteroidetes.10%bactera there were known species, including typical rumen cellulose-degrading bacteria Ruminococcus albus, Butyrivibrio fibrisolvens and Fibrobacter succinogenes. The other90%were uncultured, a part of which fell into Ruminococcaceae, Lachnospiraceae and Prevotellaceae, and clustered with several known rumen cellulose-degrading bacteria, suggesting they may be novel uncultured cellulose-degrading bacteria. The community analysis for rumen fungi showed that the abundance of fungi is only10%of that of bacteria, and there exist cellulose-degrading fungus Orpinomyces sp. So this rumen sample not only contains known cellulose-degrading microorganism, but also has great possibility to contain many novel uncultured cellulose-degrading microorganism.
Considering the majority of the rumen microorganisms were unculturable, we make use of metagenomic technology to construct cosmid library, and functionally screened for cellulases. The screening result5endoglucanase positive clones,24exoglucanase positive clones,50glucosidase positive clones,27xylosidase positive clones and6xylanase positive clones. Three of them, RuBGX2、RuBGX3and Rucel5B were cloned and characterized.
Both Rubgx2and Rubgx3have not only β-glucosidase activity, but also β-xylosidase activity, and belong to glycoside hydrolase family (GH)3. They shared48.9%amino acid sequence identity and belonged to glycoside hydrolase family3. Their optimal pH were about5, optimal temperature were about40℃. They had broad-spectrum activity towards β-configurational glycoside substrates, and can hydrolyze both cellooligosaccharide substrates and artificial aryl substrates. Their β-glucosidase activities enable the promotion of cellulose degradation by reducing inhibition of intermediate product, while their β-xylosidase activities enable the promotion of hemicellulose degradation through synergy effect with xylanase. Homology modeling and substrate docking results showed their β-glucosidase activity and β-xylosidase activity have the same active site, indicating these two kinds of activities are due to broad-spectrum specificity of substrates.
Ruce15B has both endoglucanase activity and exoglucanase activity, and belongs to GH5. Ruce15B's endoglucanase activity was219.7U/mg, and its exoglucanase activity was52.9U/mg. It exhibited optimal temperatures at about60℃and was thermostable at55℃, suggesting it's a thermostable enzyme. Ruce15B can hydrolyze β-1,4-glucosidic linkage, not β-1,3, β-1,6glucosidic linkage, or β-1,4xylosidic linkage. It was further confirmed that Ruce15B can cleave cellobiose from4-methylumbelliferyl-β-D-cellobiopyranoside, cellotetraose and insoluble cellulose (avicel, filter paper and PASC) through exo-mode of action. Time course degradation of soluble amorphous cellulose (CMC, barley glucan, lichenan) by Ruce15B showed that degree of polymerization of products was decreased rapidly at the beginning of the reaction, suggesting Ruce15B's endo-mode of action.
So this research analyzed the diversity of yak rumen microorganism community, constructed the metagenomic library platform to functional screen cellulase, clones and characterized two β-glucosidase/xylosidase RuBGX2and RuBGX3and endo/exoglucanase Ruce15B. RuBGX2and RuBGX3not only exhibited both β-glucosidase and β-xylosidase activity, but also exhibited high conversion on cellooligosaccharides and the synergy effect with xylanase. Rucel5B exhibited endo-and exo-mode in cellulose degradation, high activity and thermostability. These results indicated rumen is a good source for novel cellulose-degrading enzyme and these cellulases had huge potential in saccharification of cellulosic biomass.
引文
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33. Kambourova, M., R. Mandeva, I. Fiume, L. Maurelli, M. Rossi, and A. Morana, Hydrolysis of xylan at high temperature by co-action of the xylanase from Anoxybacillus flavithermus BC and the β-xylosidase/a-arabinosidase from Sulfolobus solfataricus Oa. Journal of Applied Microbiology,2007.102(6):p. 1586-1593.
34. Lama, L., V. Calandrelli, A. Gambacorta, and B. Nicolaus, Purification and characterization of thermostable xylanase and [beta]-xylosidase by the thermophilic bacterium Bacillus thermantarcticus. Research in microbiology,2004. 155(4):p.283-289.
35. Varghese, J., M. Hrmova, and G. Fincher, Three-dimensional structure of a barley [beta]-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure,1999.7(2): p.179-190.
36. Kumar, N. and D. Deobagkar, Multifactional glucanases. Biotechnology Advances, 1996.14(1):p.1-15.
37. Fan, Z., K. Wagschal, W. Chen, M. Montross, C. Lee, and L. Yuan, Multimeric hemicellulases facilitate biomass conversion. Applied and Environmental Microbiology,2009.75:p.1754-1757.
38. Hong, S., J. Lee, K. Cho, R. Math, Y. Kim, S. Hong, Y. Cho, H. Kim, and H. Yun, Assembling a novel bifunctional cellulase-xylanase from Thermotoga maritima by end-to-end fusion. Biotechnology Letters,2006.28(22):p.1857-1862.
39. Khandeparker, R. and M. Numan, Bifunctional xylanases and their potential use in biotechnology. Journal of Industrial Microbiology and Biotechnology,2008.35(7): p.635-644.
40. Watanabe, Y., R. Moriyama, T. Matsuda, S. Shimizu, and K. Ohmiya, Purification and properties of the endo-1,4-[beta]-glucanase Ⅲ from Ruminococcus albus. Journal of Fermentation and Bioengineering,1992.73(1):p.54-57.
41. Liu, L., Y. Feng, C. Duan, H. Pang, J. Tang, and J. Feng, Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen. World Journal of Microbiology and Biotechnology,2009.25(6):p. 1035-1042.
42. Haki, G. and S. Rakshit, Developments in industrially important thermostable enzymes:a review. Bioresource Technology,2003.89(1):p.17-34.
43. Voget, S., H.L. Steele, and W.R. Streit, Characterization of a metagenome-derived halotolerant cellulase. Journal of biotechnology,2006.126(1):p.26-36.
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32. Chang, A., M. Scheer, A. Grote, I. Schomburg, and D. Schomburg, BRENDA, AMENDA and FRENDA the enzyme information system:new content and tools in 2009. Nucleic Acids Research,2009.37(Database issue):p. D588.
33. Kambourova, M., R. Mandeva, I. Fiume, L. Maurelli, M. Rossi, and A. Morana, Hydrolysis of xylan at high temperature by co-action of the xylanase from Anoxybacillus flavithermus BC and the β-xylosidase/a-arabinosidase from Sulfolobus solfataricus Oa. Journal of Applied Microbiology,2007.102(6):p. 1586-1593.
34. Lama, L., V. Calandrelli, A. Gambacorta, and B. Nicolaus, Purification and characterization of thermostable xylanase and [beta]-xylosidase by the thermophilic bacterium Bacillus thermantarcticus. Research in microbiology,2004. 155(4):p.283-289.
35. Varghese, J., M. Hrmova, and G. Fincher, Three-dimensional structure of a barley [beta]-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure,1999.7(2): p.179-190.
36. Kumar, N. and D. Deobagkar, Multifactional glucanases. Biotechnology Advances, 1996.14(1):p.1-15.
37. Fan, Z., K. Wagschal, W. Chen, M. Montross, C. Lee, and L. Yuan, Multimeric hemicellulases facilitate biomass conversion. Applied and Environmental Microbiology,2009.75:p.1754-1757.
38. Hong, S., J. Lee, K. Cho, R. Math, Y. Kim, S. Hong, Y. Cho, H. Kim, and H. Yun, Assembling a novel bifunctional cellulase-xylanase from Thermotoga maritima by end-to-end fusion. Biotechnology Letters,2006.28(22):p.1857-1862.
39. Khandeparker, R. and M. Numan, Bifunctional xylanases and their potential use in biotechnology. Journal of Industrial Microbiology and Biotechnology,2008.35(7): p.635-644.
40. Watanabe, Y., R. Moriyama, T. Matsuda, S. Shimizu, and K. Ohmiya, Purification and properties of the endo-1,4-[beta]-glucanase Ⅲ from Ruminococcus albus. Journal of Fermentation and Bioengineering,1992.73(1):p.54-57.
41. Liu, L., Y. Feng, C. Duan, H. Pang, J. Tang, and J. Feng, Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen. World Journal of Microbiology and Biotechnology,2009.25(6):p. 1035-1042.
42. Haki, G. and S. Rakshit, Developments in industrially important thermostable enzymes:a review. Bioresource Technology,2003.89(1):p.17-34.
43. Voget, S., H.L. Steele, and W.R. Streit, Characterization of a metagenome-derived halotolerant cellulase. Journal of biotechnology,2006.126(1):p.26-36.
1. Percival Zhang, Y.H., M.E. Himmel, and J.R. Mielenz, Outlook for cellulase improvement:screening and selection strategies. Biotechnology Advances,2006. 24(5):p.452-481.
2. Lynd, L.R., P.J. Weimer, W.H. van Zyl, and I.S. Pretorius, Microbial cellulose utilization:fundamentals and biotechnology. Microbiology and molecular biology reviews,2002.66(3):p.506.
3. Marchessault, R. and P. Sundararajan, Cellulose. The polysaccharides,1993.2:p. 11-95.
4. Wilson, J. and D. Mertens, Cell wall accessibility and cell structure limitations to microbial digestion of forage. Crop Science,1995.35(1):p.251-259.
5. Krause, D., S. Denman, R. Mackie, M. Morrison, A. Rae, G. Attwood, and C. McSweeney, Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics. Ferns Microbiology Reviews,2003.27(5):p. 663-693.
6. Teeri, T., Crystalline cellulose degradation:new insight into the function of cellobiohydrolases. Trends in Biotechnology,1997.15(5):p.160-167.
7. Teeri, T., A. Koivula, M. Linder, G. Wohlfahrt, C. Divne, and T. Jones, Trichoderma reesei cellobiohydrolases:why so efficient on crystalline cellulose? Biochemical Society Transactions,1998.26(2):p.173-178.
8. Esterbauer, H., W. Steiner, I. Labudova, A. Hermann, and M. Hayn, Production of Trichoderma cellulase in laboratory and pilot scale* 1. Bioresource Technology, 1991.36(1):p.51-65.
9. Nogawa, M., M. Goto, H. Okada, and Y. Morikawa, L-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei. Current Genetics, 2001.38(6):p.329-334.
10. Schulein, M., Enzymatic properties of cellulases from Humicola insolens. Journal of biotechnology,1997.57(1-3):p.71-81.
11.Broda, P., P. Birch, P. Brooks, and P. Sims, Lignocellulose degradation by Phanerochaete chrysosporium:gene families and gene expression for a complex process. Molecular microbiology.1996.19(5):p.923-932.
12. Chaudhary, P., N. Kumar, and D. Deobagkar, The glucanases of Cellulomonas. Biotechnology Advances,1997.15(2):p.315-331.
13. Bayer, E., E. Morag, and R. Lamed, The cellulosome--a treasure-trove for biotechnology. Trends in Biotechnology,1994.12(9):p.379-386.
14. Schwarz, W., The cellulosome and cellulose degradation by anaerobic bacteria. Applied Microbiology and Biotechnology,2001.56(5):p.634-649.
15. An, D., X. Dong, and Z. Dong, Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses. Anaerobe,2005.11(4):p.207-215.
16. Kamra, D.N., Rumen microbial ecosystem. Current Science,2005.89(1):p. 124-135.
17. Krause, D.O. and J.B. Russell, How Many Ruminal Bacteria Are There? Journal of Dairy Science,1996.79(8):p.1467-1475.
18. Lorenz, P., K. Liebeton, F. Niehaus, and J. Eck, Screening for novel enzymes for biocatalytic processes:accessing the metagenome as a resource of novel functional sequence space. Current opinion in biotechnology,2002.13(6):p. 572-577.