丝状真菌纤维素酶性质的研究以及酶水解条件的优化
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摘要
随着传统化石资源日益的枯竭,世界正在面临着严重的能源危机。所以,寻找可再生新型能源成为了目前世界广泛关注的热点。木质纤维索来源广泛、资源丰富、价格低廉并且大都来自农作物的废弃物。以这些废弃物为原料生产生物乙醇,不仅可以缓解能源危机,而且很大程度上减少了对环境的污染,特别是温室气体的排放。这种能够变废为宝,绿色友好型的能源生产方法已成为一种必然趋势。但是,以木质纤维素等非粮生物质生产乙醇的工艺普遍存在两方面的关键技术问题：纤维素酶生产成本高和酶解效率低、纤维索酶用量大。本论文主要通过针对金属离子对纤维素酶水解的影响以及消除其作用、酶液之间的协同作用和纤维素乙醇生产工艺中酶水解条件优化三方面的研究,从而能够提高纤维素酶水解效率,增加乙醇产率并降低乙醇生产成本。本论文的主要研究内容及结果如下
1金属离子对纤维素酶水解的影响以及消除其作用的研究
金属离子很大程程度上可以影响酶的活性,甚至在一些酶的活性中心中起决定性作用。在纤维素酶使用的过程中,往往会接触到各种金属。因此,本实验重点研究了金属离子对纤维素酶水解的影响,特别是Fe~(3+)针对斜卧青霉(Penicillium decumbens) JUA10-1菌株生产的纤维素酶活性的研究。首先确定了各种金属离子对纤维素酶水解是有影响的,但是影响的程度不一样。其中Fe~(3+)对纤维素酶水解的抑制作用最大。其次,进一步深入研究了Fe~(3+)对纤维素酶水解木质纤维素类底物抑制作用的机理,很可能在水解底物和纤维素酶两个方面发生了变化。一、纤维素的葡萄糖分子被Fe~(3+)氧化,氧化后的纤维素影响了纤维素酶的酶解；二、金属离子导致纤维素酶活性的失活。最后,我们根据推测的金属离子抑制机理,通过添加化学试剂二硫苏糖醇(DTT)或螯合剂(EDTA)的方法消除了Fe~(3+)对纤维素酶水解的抑制作用。2里氏木霉酶液与棘孢曲霉酶液之间协同作用的研究
在本章的实验研究中,我们首先从棘孢曲霉(Aspergillus aculeatus) ZLF酶液中纯化出β-葡萄糖苷酶。然后分别研究了棘孢曲霉酶液、β-葡萄糖苷酶与里氏木霉(Trichoderma reesei)酶液的协同作用。发现,ZLF酶液与β-葡萄糖苷酶都可以提高纤维索酶的水解作用,并且提高的效果是一样的。另外,从对复配酶的酶活性测定数据可得,β-葡萄糖苷酶活性的提高可以很大程度上提高纤维素酶的水解作用。此外,滤纸酶活的测定方法是以化学成分较为单一的滤纸为水解底物,而预处理后的木质纤维索化学成分和结构较为复杂。所以我们的实验证明滤纸酶活单位和预处理后的木质纤维素的酶解效率没有直接的关联。对于斜卧青霉(P. decumbens)来说,虽然斜卧青霉菌株分泌的纤维索酶中含有较高的β-葡萄糖苷酶活性,但是添加棘孢曲霉酶液依然可以提高纤维素酶的水解能力。但是,这种水解能力的提高只在脱木素木糖渣时才能表现出来,而对于木素含量较高的木糖渣来说,没有太明显的效果。
3纤维素乙醇工艺中酶水解条件的优化
纤维素酶水解效率低,用酶量大已成为以木质纤维素等非粮生物质为原料生产乙醇工艺的最大限制因素。为了解决这一问题,本实验侧重对酶蛋白负载量、水解底物两方面进行纤维素酶水解条件优化的研究,并同时对纤维索乙醇生产工艺进行了优化。一方面,分别以汽爆芦竹和碱处理木糖渣(DCCR)为水解底物,按照每克绝干底物对应不同酶蛋白负载量添加酶液,45℃糖化。结果表时不管是预处理的芦竹还是脱木素木糖渣,对于每克干物质来说都存在着酶负载量饱和点,90mg酶蛋白。纤维素酶水解中酶负载量大于饱和点时,就会造成酶液的浪费。另一方面,在乙醇生产工艺条件优化中,补料和酶液复配都可以很大程度上提高纤维素乙醇的产率。
The human society has been facing a severe energy crisis accompanying the depletion of traditional fossil fuels, making the search for renewable energy sources a focus of investigations. The lignocellulosic biomass is an excellent candidate energy source because it is abundantly distributed, generally cheap, and mostly from agricultural residues. Producing renewable energy from lignocellulosic biomass not only relieves the world from current energy crisis, but also significantly decreases environmental pollution, in particular the production of greenhouse gases. This environment friendly energy source has received widespread attention. However, two key technical problems still exist:the high cost and low efficiency of lignocellulose degrading enzymes, as well as the large dosage of enzymes required for lignocellulose degradation. This thesis documents the studies on three aspects aiming at improving cellulase efficiency, improvement of ethanol production and lowering ethanol production cost:1) identification of and relief from metal ion induced cellulase-catalyzed cellulose hydrolysis;2) identification of cellulase synergism;3) optimization of the cellulose hydrolysis process during lignocellulosic ethanol production. The results obtained from these studies include:
1The impact of metal ions on enzyme-catalyzed cellulose hydrolysis and the removal of metal ion-induced inhibition
Metal ions can largely impact, or even determine the activities of enzymes. During cellulase-catalyzed cellulose degradation, the presence of metal ions is inevitable. Our research investigated this metal ion impact on cellulase-catalyzed cellulose hydrolysis, in particular the impact of Fe3+on reactions catalyzed by cellulases from P. decumbens JUA10-1. We first identified the impact of metal ions on the activities of the cellulases, showing a varied degree of impact for different metal ions, out of which Fe3+leads to the highest level of inhibition. We subsequently investigated the mechanism underlying the inhibition of Fe3+on lignocellulose degradation catalyzed by cellulases from P. decumbens. The inhibitory effects are shown to be present on two different levels:Fe3+inhibits the reaction by reacting on the reducing ends of the substrate and improves the resistance to degradation; Fe3+also inhibits the catalytic abilities of cellulases by competing for enzyme binding with the substrate. Finally, we identified two approaches to abolish the inhibitory effect of Fe3+:the addition of reducing reagent DTT or chelating reagent EDTA.
2Synergism between cellulases from Trichoderma reeseil Penicillium decumbens and Aspergillus aculeatus
The P-glucosidase from A. aculeatus ZLF was purified to apparent homogeneity in this study. We further identified the synergistic effects between T. reesei cellulases or A. aculeatus cellulases and β-glucosidases. We found that both A. aculeatus cellulases show identical synergistic effects with cellulases from T. reesei. Our further investigations on the enzymatic activities of cellulase cocktails showed that the improvement of β-glucosidase activities leads to the increase of cellulose hydrolysis, while the filter paperase activity (FPA) of the cellulase cocktails stayed unchanged. The FPA was assayed by using filter paper as the substrate, which has a much simpler structure in comparison with natural substrates and more susceptible to enzymatic degradation. The FPA can therefore not represent the'true'abilities to hydrolyze cellulose for cellulases. For P. decumbens JUA10-1cellulases, although a higher level of β-glucosidase activity is present in the enzyme, supplementation of A. aculeatus ZLF cellulases can still improve the ability of the enzyme cocktails to hydrolyze cellulose. This synergistic effect is only obvious when delignined corncob residues serve as the substrate, while it is not apparent for substrates containing a higher level of lignin such as corncob residues.
3Optimization of the cellulose degradation process during lignocellulosic ethanol production
The low efficiency and high dosage requirement of cellulases have become the biggest problems during lignocellulosic ethanol production. We tried to optimize the cellulose degradation process by changing enzyme loading and substrates, in order to find solutions to these problems and optimize the lignocellulosic ethanol production process. We first used steam exploded Arundo donax Linn or delignined corncob residues as the substrate, changed the enzyme loadings, and carried out saccharification experiments at45℃. We found that both substrates and saccharification performance ceases to improve at an enzyme loading of90mg/g substrate, and the enzymes would be wasted when more enzymes were used. We further characterized the fed batch process and using cellulase cocktails for lignocellulosic ethanol production, and found both improve the production of lignocellulosic ethanol.
1金属离子对纤维素酶水解的影响以及消除其作用的研究
金属离子很大程程度上可以影响酶的活性,甚至在一些酶的活性中心中起决定性作用。在纤维素酶使用的过程中,往往会接触到各种金属。因此,本实验重点研究了金属离子对纤维素酶水解的影响,特别是Fe~(3+)针对斜卧青霉(Penicillium decumbens) JUA10-1菌株生产的纤维素酶活性的研究。首先确定了各种金属离子对纤维素酶水解是有影响的,但是影响的程度不一样。其中Fe~(3+)对纤维素酶水解的抑制作用最大。其次,进一步深入研究了Fe~(3+)对纤维素酶水解木质纤维素类底物抑制作用的机理,很可能在水解底物和纤维素酶两个方面发生了变化。一、纤维素的葡萄糖分子被Fe~(3+)氧化,氧化后的纤维素影响了纤维素酶的酶解；二、金属离子导致纤维素酶活性的失活。最后,我们根据推测的金属离子抑制机理,通过添加化学试剂二硫苏糖醇(DTT)或螯合剂(EDTA)的方法消除了Fe~(3+)对纤维素酶水解的抑制作用。2里氏木霉酶液与棘孢曲霉酶液之间协同作用的研究
在本章的实验研究中,我们首先从棘孢曲霉(Aspergillus aculeatus) ZLF酶液中纯化出β-葡萄糖苷酶。然后分别研究了棘孢曲霉酶液、β-葡萄糖苷酶与里氏木霉(Trichoderma reesei)酶液的协同作用。发现,ZLF酶液与β-葡萄糖苷酶都可以提高纤维索酶的水解作用,并且提高的效果是一样的。另外,从对复配酶的酶活性测定数据可得,β-葡萄糖苷酶活性的提高可以很大程度上提高纤维素酶的水解作用。此外,滤纸酶活的测定方法是以化学成分较为单一的滤纸为水解底物,而预处理后的木质纤维索化学成分和结构较为复杂。所以我们的实验证明滤纸酶活单位和预处理后的木质纤维素的酶解效率没有直接的关联。对于斜卧青霉(P. decumbens)来说,虽然斜卧青霉菌株分泌的纤维索酶中含有较高的β-葡萄糖苷酶活性,但是添加棘孢曲霉酶液依然可以提高纤维素酶的水解能力。但是,这种水解能力的提高只在脱木素木糖渣时才能表现出来,而对于木素含量较高的木糖渣来说,没有太明显的效果。
3纤维素乙醇工艺中酶水解条件的优化
纤维素酶水解效率低,用酶量大已成为以木质纤维素等非粮生物质为原料生产乙醇工艺的最大限制因素。为了解决这一问题,本实验侧重对酶蛋白负载量、水解底物两方面进行纤维素酶水解条件优化的研究,并同时对纤维索乙醇生产工艺进行了优化。一方面,分别以汽爆芦竹和碱处理木糖渣(DCCR)为水解底物,按照每克绝干底物对应不同酶蛋白负载量添加酶液,45℃糖化。结果表时不管是预处理的芦竹还是脱木素木糖渣,对于每克干物质来说都存在着酶负载量饱和点,90mg酶蛋白。纤维素酶水解中酶负载量大于饱和点时,就会造成酶液的浪费。另一方面,在乙醇生产工艺条件优化中,补料和酶液复配都可以很大程度上提高纤维素乙醇的产率。
The human society has been facing a severe energy crisis accompanying the depletion of traditional fossil fuels, making the search for renewable energy sources a focus of investigations. The lignocellulosic biomass is an excellent candidate energy source because it is abundantly distributed, generally cheap, and mostly from agricultural residues. Producing renewable energy from lignocellulosic biomass not only relieves the world from current energy crisis, but also significantly decreases environmental pollution, in particular the production of greenhouse gases. This environment friendly energy source has received widespread attention. However, two key technical problems still exist:the high cost and low efficiency of lignocellulose degrading enzymes, as well as the large dosage of enzymes required for lignocellulose degradation. This thesis documents the studies on three aspects aiming at improving cellulase efficiency, improvement of ethanol production and lowering ethanol production cost:1) identification of and relief from metal ion induced cellulase-catalyzed cellulose hydrolysis;2) identification of cellulase synergism;3) optimization of the cellulose hydrolysis process during lignocellulosic ethanol production. The results obtained from these studies include:
1The impact of metal ions on enzyme-catalyzed cellulose hydrolysis and the removal of metal ion-induced inhibition
Metal ions can largely impact, or even determine the activities of enzymes. During cellulase-catalyzed cellulose degradation, the presence of metal ions is inevitable. Our research investigated this metal ion impact on cellulase-catalyzed cellulose hydrolysis, in particular the impact of Fe3+on reactions catalyzed by cellulases from P. decumbens JUA10-1. We first identified the impact of metal ions on the activities of the cellulases, showing a varied degree of impact for different metal ions, out of which Fe3+leads to the highest level of inhibition. We subsequently investigated the mechanism underlying the inhibition of Fe3+on lignocellulose degradation catalyzed by cellulases from P. decumbens. The inhibitory effects are shown to be present on two different levels:Fe3+inhibits the reaction by reacting on the reducing ends of the substrate and improves the resistance to degradation; Fe3+also inhibits the catalytic abilities of cellulases by competing for enzyme binding with the substrate. Finally, we identified two approaches to abolish the inhibitory effect of Fe3+:the addition of reducing reagent DTT or chelating reagent EDTA.
2Synergism between cellulases from Trichoderma reeseil Penicillium decumbens and Aspergillus aculeatus
The P-glucosidase from A. aculeatus ZLF was purified to apparent homogeneity in this study. We further identified the synergistic effects between T. reesei cellulases or A. aculeatus cellulases and β-glucosidases. We found that both A. aculeatus cellulases show identical synergistic effects with cellulases from T. reesei. Our further investigations on the enzymatic activities of cellulase cocktails showed that the improvement of β-glucosidase activities leads to the increase of cellulose hydrolysis, while the filter paperase activity (FPA) of the cellulase cocktails stayed unchanged. The FPA was assayed by using filter paper as the substrate, which has a much simpler structure in comparison with natural substrates and more susceptible to enzymatic degradation. The FPA can therefore not represent the'true'abilities to hydrolyze cellulose for cellulases. For P. decumbens JUA10-1cellulases, although a higher level of β-glucosidase activity is present in the enzyme, supplementation of A. aculeatus ZLF cellulases can still improve the ability of the enzyme cocktails to hydrolyze cellulose. This synergistic effect is only obvious when delignined corncob residues serve as the substrate, while it is not apparent for substrates containing a higher level of lignin such as corncob residues.
3Optimization of the cellulose degradation process during lignocellulosic ethanol production
The low efficiency and high dosage requirement of cellulases have become the biggest problems during lignocellulosic ethanol production. We tried to optimize the cellulose degradation process by changing enzyme loading and substrates, in order to find solutions to these problems and optimize the lignocellulosic ethanol production process. We first used steam exploded Arundo donax Linn or delignined corncob residues as the substrate, changed the enzyme loadings, and carried out saccharification experiments at45℃. We found that both substrates and saccharification performance ceases to improve at an enzyme loading of90mg/g substrate, and the enzymes would be wasted when more enzymes were used. We further characterized the fed batch process and using cellulase cocktails for lignocellulosic ethanol production, and found both improve the production of lignocellulosic ethanol.
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44. Wang, M, and Wu, M, et al, Life-cycle energy and greenhouse gas emission impacts of different corn ethanol plant types. Environ Res Lett,2007.2(2): 024001.
45. Fang, X, and Yano, S. et al, Strain improvement of Acremonium cellulolylicus for cellulase production by mutation. J Biosci Bioeng,2009.107(3):256-261.
46. Mo, H, and Zhang, X.L, Control of gas phase for enhanced cellulose production by Penicillium decumbens in solid-state culture. Process Biochem,2004.39: 1293-1297.
47. Qu, Y.B, and Zhao, X, et al, Cellulase production from spent sulfite liquor and paper-mill waste fiber. Appl Biochem Biotechnol,1991.28(1):363-368.
48. Wei, X.M, and Zheng, K, et al, Transcription analysis of lignocellulolytic enzymes of Penicillium docwnbens 114-2 and its catabolite-repression-resistant mutant. C R Biol,2011.334:806-811.
49. Yu, B and Chen, H.Z, Effect of the ash on enzymatic hydrolysis of steam-exploded rice straw. Bioresour Technol,2010.101(23):9114-9119.
50. Rawat, R, and Tewari, L, Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3. Extremophiles,2012:1-8.
51. Shi, H, and Yin, X, et al, Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris. J Ind Microbiol Biotechnol,2012.39(2):347-357.
52. Harris, P.V, and Welner, D, et al, Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61:structure and function of a large, enigmatic family. Biochemistry,2010.49(15):3305-3316.
53. Zhou, Y, and Zhu, G, Rapid automated in-situ monitoring of total dissolved iron and total dissolved manganese in underground water by reverse-flow injection with chemiluminescence detection during the process of water treatment. Talanta, 1997.44(11):2041-2049.
54. Lowry, O.H, and Rosebrough, N.J, et al. Protein measurement with the Folin phenol reagent. J Biol Chem,1951.193(1):265-275.
55. Hideno, A, and Inoue, H, et al, Production and characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carbon source. Enzyme Microb Technol,2011.48(2): 162-168.
56. Mandels, M, and Hontz, L, et al, Enzymatic hydrolysis of waste cellulose. Biotechnol Bioeng,2004.16(11):1471-1493.
57. Ma, L, and Zhang, J, et al, Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens. Enzyme Microb Technol,2011.49(4):366-371.
58. Cheng, Y, and Song, X, et al, Genome shuffling improves production of cellulose by Penicillum decumbens JUA10. J Appl Microbiol,2009.107:1837-1846.
59. Magalhaes, P.O, and Ferraz, A, et al, Enzymatic properties of two β-glucosidases from Ceriporiopsis subvermispora produced in biopulping comditions. J Appl Microbiol,2006.101:480-486.
60. Tao, Y.M, and Zhu, X. Z, et al, Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9. J Agr Food Chem,2010.58(10):6126-6130.
61. Hoa, T, and Quyen, D.T, Cloning, Expression, Purification, and Properties of an Endoglucanase Gene (Glycosyl Hydrolase Family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris. J Microbiol Biotechnol,2011.21(10):1012-1020.
62. Clarke, A.J, and Adams, L.S, Irreversible inhibition of Schizophyllum commune cellulase by divalent transition metal ions. BBA-Protein Struct M,1987.916(2): 213-219.
63. Lassig, J.P, and Shultz, M.D, et al, Inhibition of cellobiohydrolase I from Trichoderma reesei by palladium. Arch Biochem Biophys,1995.322(1):119-126.
64. Juhasz, T, and Egyhazi, A, et al,β-glucosidase production by Trichoderma reesei. in Twenty-Sixth Symposium on Biotechnology for Fuels and Chemicals.2005: Springer.
65. Chen, M, and Zhao, L, et al, Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars. Carbonhyd Polym,2008.71(3):411-415.
66. Manonmani, H.K, and Srikantiah, K.R, Saccharification of sugar-cane bagasse with enzymes from Aspergilhis sutus and Trichoderma viride. Enzyme Microb Technol,1987.9:484-488.
67. Chahal, D.S, Solid-state fermentation with Trichoderma reesei for cellulase production. Appl Environ Microbiol,1985.49(1):205-210.
68. Hong, J, and Ye, X, et al, Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications. Langmuir,2007.23(25):12535-12540.
69. Ohgren, K, and Bura, R, et al, Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Bioresour Technol,2007. 98(13):2503-2510.
70.王体科,世界植物纤维水解概况.世界林业研究所,1993.6：51-57.
71.文新亚,李燕松等,酶解本质纤维素的预处理技术研究进展.酿酒科技,2006.146(8)：97-100.
72.张失,植物原料水解工艺学.中国林业出版社,1992.
73.刘稳,高培基,半纤维素酶分子生物学.纤维素科学与技,1998.6(1)：9-15.
74.陆中华,陈俏彪,食用菌储藏与加工技术.中国农业出版社,2004.
75. Zhang, Y.P, and Lynd, L.R, Toward an aggregated understanding of enzymatic hydrolysis of cellulose:noncomplexed cellulase systems. Biotechnol Bioeng, 2004.88(7):797-824.
76. Rosgaard, L, and Andric, P, et al, Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw. Appl Biochem Biotechnol, 2007.143(1):27-40.
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