纤维素的酶水解及超声波对其加速作用的研究
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摘要
本文主要对纤维素的酶水解机理、动力学模型、超声波的加速作用、各种因素对超声波场中纤维素酶水解的影响、纤维素酶的失活等进行了研究。
根据纤维素材料的结构和纤维素酶的水解特性，提出了结晶纤维素的晶格水解机理，非结晶纤维素的随机水解机理，所提出的机理能合理解释水解过程的各种现象。根据水解机理建立了纤维素材料的酶水解动力学模型，推导出了动力学方程的积分方程解析形式。该动力学方程描述了包括纤维素酶浓度、反应速度常数、平衡常数、原料有效浓度、密度和细度等的影响。在描述水解产生的还原糖量与时间的关系时，合并成3个参数，很容易回归。并提出了以与反应速度常数成比例的当量水解时间表示酶反应速度的方法，以解决因纤维素结构和产物抑制引起的非线性行为。分析了超声波对化学反应的效应，提出了在低超声波强度下超声波有加速酶过程中酶分子的动能和在纤维素材料表面上更新的频率，而不是空化效应。超声波有加速酶反应速度的作用，但也有加速酶失活的作用，特别是在有氧气存在的环境中，超声波更易使酶氧化而失活，因而提出采用氮气保护的方法以避免酶的失活。
通过实验研究，得到了滤纸在无超声波条件下和超声波场下的酶水解动力学数据。用本文提出的固态纤维素水解动力学方程进行拟合，也对文献报道的微晶纤维素和非结晶纤维素的酶水解实验数据进行了拟合，提出的动力学方程能很好地与实验数据吻合。证明该模型是合理的。
超声波场对纤维素的酶水解过程有明显的加速作用，通过研究发现在超声波能量为0-54mW／mL的范围内速度呈直线上升趋势。在28mW／mL的超声波场中，可提高反应速度约0.6倍。
在pH对纤维素酶水解影响的研究中，将纤维素酶简化为三种电离状态，得到酶水解当量时间与pH值的关系式。提出的关系式能很好地拟合水解时间分别为10min、40min、70min，pH4-6.5范围的实验值，并得到最优pH为4.83。在超声波场中，温度为25-50℃范围内，随着温度的升高，反应速度加快。求得在超声波场中纤维素的酶水解的活化能为57.8kJ／mol。同时也研究了
四川大学博士学位论文
酶浓度、葡萄糖浓度、滤纸浓度、原料种类对超声波场中酶水解的影响。各种
因素的影响规律与无超声波条件下水解相似，说明超声波的加速作用并未改变
反应的历程，而只改变了反应的速度常数。
对纤维素酶的失活也进行了初步实验研究，氮气环境下水解的速度比在空
气中高。说明氧促进了纤维素酶的失活，采用氮气保护进行水解是减小纤维素
酶失活的一种有效措施。
上述研究成果对深入了解纤维素酶水解机理和超声波对水解的作用具有重
要的理论和实用意义，为超声彼加速纤维素的酶水解的应用提供了依据。
The mechanism and the kinetics model of enzymatic hydrolysis of cellulose by cellulase, and the acceleration of ultrasonic energy to the hydrolysis were studied. The effect of various factors to the enzymatic hydrolysis and the deactivation of cellulase in ultrasonic field were also investigated.
According to the structure of cellulosic material (crystal cellulose and non-crystal cellulose) and the hydrolysis specialty of cellulase, a new mechanism for enzymatic hydrolysis of cellulose was proposed. The mechanism can appropriately explain various phenomena in hydrolytic process of cellulose by cellulase. On the basis of the hydrolysis mechanism, a kinetic model was suggested, and the integral equation was derived. This kinetic equation expressed the effect of cellulase concentration, reaction rate constant, equilibrium constants, cellulosic material effective concentration, density, and size and hydrolysis time on the concentration of reducing sugar produced. When the kinetic equation was used to describe the relation between concentration of produced sugar and hydrolysis time, there are only 3 parameters in the equation, which can be regressed easily. To avert some nonlinear influence caused by composition of cellulose material and product inhibition, an equivalent hydrolysis time, which is proporti
onal to reaction rate constant, was used io express enzyme reaction rate. The ultrasonic effect on chemical reaction and enzyme reaction was analyzed. The ultrasonic effect to chemical reaction is cavitation, and the ultrasonic effect on enzyme reaction is increasing kinetic energy of enzyme molecule and increasing its renewing frequency on the surface of cellulose material. Ultrasonic can accelerate reaction rale of enzyme reaction, but also can accelerate the deactivation rate of enzyme. If oxygen exits in the system, deactivation takes place more easily. Thus it was suggested to use nitrogen in the system to avoid
the deactivation.
The experimental data of enzymatic hydrolysis of filter paper obtained by us and the experimental data of avicel and non-crystal cellulose obtained by other researchers were regressed with the kinetic equation suggested by us. The kinetic equation was conformable with these experimental data. It was proved that the model is reasonable to avicel, filter paper cellulose and amorphous cellulose.
The acceleration of ultrasonic to enzymatic hydrolytic process of cellulose was obvious. The hydrolysis rate rises with increase of ultrasonic energy in 0-54mW/mL. The hydrolysis rate in the 28mW/mL ultrasonic field was 0.6 times higher than that in non-ultrasonic field.
Cellulase was simplified as 3 ionization states in the investigation of effect of pH on the enzymatic hydrolysis. A relation between equivalent time and pH was obtained. The predicted values were consistent with experiment data under 10min, 40min, 70min within pH4-6.5, and optimal pH was obtained at 4.83 through regression computation. Reaction rate was raised with increasing temperature within 25-50C. Through calculating, the activity energy of cellulose hydrolysis by cellulase in ultrasonic field was 57.8 kJ/mol. The effect of enzyme concentration, glucose concentration, filter paper concentration and material kind on the enzymatic hydrolysis in ultrasonic field was also investigated. The effect rules of various factors in ultrasonic field were similar as that in non-ultrasonic field. It was showed that ultrasonic did not change the reaction mechanism. The deactivation of cellulase was studied briefly. It was found that hydrolysis rate under nitrogen higher than that under air. It was showed that cellulase
deactivation was caused mainly by oxygen under the experimental condition.
根据纤维素材料的结构和纤维素酶的水解特性，提出了结晶纤维素的晶格水解机理，非结晶纤维素的随机水解机理，所提出的机理能合理解释水解过程的各种现象。根据水解机理建立了纤维素材料的酶水解动力学模型，推导出了动力学方程的积分方程解析形式。该动力学方程描述了包括纤维素酶浓度、反应速度常数、平衡常数、原料有效浓度、密度和细度等的影响。在描述水解产生的还原糖量与时间的关系时，合并成3个参数，很容易回归。并提出了以与反应速度常数成比例的当量水解时间表示酶反应速度的方法，以解决因纤维素结构和产物抑制引起的非线性行为。分析了超声波对化学反应的效应，提出了在低超声波强度下超声波有加速酶过程中酶分子的动能和在纤维素材料表面上更新的频率，而不是空化效应。超声波有加速酶反应速度的作用，但也有加速酶失活的作用，特别是在有氧气存在的环境中，超声波更易使酶氧化而失活，因而提出采用氮气保护的方法以避免酶的失活。
通过实验研究，得到了滤纸在无超声波条件下和超声波场下的酶水解动力学数据。用本文提出的固态纤维素水解动力学方程进行拟合，也对文献报道的微晶纤维素和非结晶纤维素的酶水解实验数据进行了拟合，提出的动力学方程能很好地与实验数据吻合。证明该模型是合理的。
超声波场对纤维素的酶水解过程有明显的加速作用，通过研究发现在超声波能量为0-54mW／mL的范围内速度呈直线上升趋势。在28mW／mL的超声波场中，可提高反应速度约0.6倍。
在pH对纤维素酶水解影响的研究中，将纤维素酶简化为三种电离状态，得到酶水解当量时间与pH值的关系式。提出的关系式能很好地拟合水解时间分别为10min、40min、70min，pH4-6.5范围的实验值，并得到最优pH为4.83。在超声波场中，温度为25-50℃范围内，随着温度的升高，反应速度加快。求得在超声波场中纤维素的酶水解的活化能为57.8kJ／mol。同时也研究了
四川大学博士学位论文
酶浓度、葡萄糖浓度、滤纸浓度、原料种类对超声波场中酶水解的影响。各种
因素的影响规律与无超声波条件下水解相似，说明超声波的加速作用并未改变
反应的历程，而只改变了反应的速度常数。
对纤维素酶的失活也进行了初步实验研究，氮气环境下水解的速度比在空
气中高。说明氧促进了纤维素酶的失活，采用氮气保护进行水解是减小纤维素
酶失活的一种有效措施。
上述研究成果对深入了解纤维素酶水解机理和超声波对水解的作用具有重
要的理论和实用意义，为超声彼加速纤维素的酶水解的应用提供了依据。
The mechanism and the kinetics model of enzymatic hydrolysis of cellulose by cellulase, and the acceleration of ultrasonic energy to the hydrolysis were studied. The effect of various factors to the enzymatic hydrolysis and the deactivation of cellulase in ultrasonic field were also investigated.
According to the structure of cellulosic material (crystal cellulose and non-crystal cellulose) and the hydrolysis specialty of cellulase, a new mechanism for enzymatic hydrolysis of cellulose was proposed. The mechanism can appropriately explain various phenomena in hydrolytic process of cellulose by cellulase. On the basis of the hydrolysis mechanism, a kinetic model was suggested, and the integral equation was derived. This kinetic equation expressed the effect of cellulase concentration, reaction rate constant, equilibrium constants, cellulosic material effective concentration, density, and size and hydrolysis time on the concentration of reducing sugar produced. When the kinetic equation was used to describe the relation between concentration of produced sugar and hydrolysis time, there are only 3 parameters in the equation, which can be regressed easily. To avert some nonlinear influence caused by composition of cellulose material and product inhibition, an equivalent hydrolysis time, which is proporti
onal to reaction rate constant, was used io express enzyme reaction rate. The ultrasonic effect on chemical reaction and enzyme reaction was analyzed. The ultrasonic effect to chemical reaction is cavitation, and the ultrasonic effect on enzyme reaction is increasing kinetic energy of enzyme molecule and increasing its renewing frequency on the surface of cellulose material. Ultrasonic can accelerate reaction rale of enzyme reaction, but also can accelerate the deactivation rate of enzyme. If oxygen exits in the system, deactivation takes place more easily. Thus it was suggested to use nitrogen in the system to avoid
the deactivation.
The experimental data of enzymatic hydrolysis of filter paper obtained by us and the experimental data of avicel and non-crystal cellulose obtained by other researchers were regressed with the kinetic equation suggested by us. The kinetic equation was conformable with these experimental data. It was proved that the model is reasonable to avicel, filter paper cellulose and amorphous cellulose.
The acceleration of ultrasonic to enzymatic hydrolytic process of cellulose was obvious. The hydrolysis rate rises with increase of ultrasonic energy in 0-54mW/mL. The hydrolysis rate in the 28mW/mL ultrasonic field was 0.6 times higher than that in non-ultrasonic field.
Cellulase was simplified as 3 ionization states in the investigation of effect of pH on the enzymatic hydrolysis. A relation between equivalent time and pH was obtained. The predicted values were consistent with experiment data under 10min, 40min, 70min within pH4-6.5, and optimal pH was obtained at 4.83 through regression computation. Reaction rate was raised with increasing temperature within 25-50C. Through calculating, the activity energy of cellulose hydrolysis by cellulase in ultrasonic field was 57.8 kJ/mol. The effect of enzyme concentration, glucose concentration, filter paper concentration and material kind on the enzymatic hydrolysis in ultrasonic field was also investigated. The effect rules of various factors in ultrasonic field were similar as that in non-ultrasonic field. It was showed that ultrasonic did not change the reaction mechanism. The deactivation of cellulase was studied briefly. It was found that hydrolysis rate under nitrogen higher than that under air. It was showed that cellulase
deactivation was caused mainly by oxygen under the experimental condition.
引文
1．张启先．纤维素和纤维素酶．微生物学通报．1976．3(1)．31-34
2. Grajek W. Production of protein by thermophilic fungi from sugar-beet pulp in solid-state fermentation. Biotechnol and Bioeng. 1988.32(2). 225-260.
3．阿尔贝·萨松著，贾谦 张玉华 李沄译．生物技术—挑战与希望．北京：科学技术文献出版社出版．1986．4．
4. Ingram L O, Gomez P F, Lai X, Moniruzzaman M, et al. Metabolic engineering of
bacteria for ethanol production. Biotechnol Bioeng. 1998.58(2-3). 204-212.
5. Cheung S, Anderson B. Ethanol production from wastewater solids. Water Environ Technol. 1996.8(5). 55-60.
6. Alvo P, Belkacemi K. Enzymatic saccharification of milled timothy (Phleum pratense L.) and alfalfa (Medicago sativa L.). Bioresourc Technol. 1997.61(3). 185-198.
7．周怡，杨守志，薛茂杰，等．由纤维素制取单细胞蛋白的反应机理模型．化工学报．1992．43(4)．395-400．
8. Venkatesh K V. Simultaneous saccharification and fermentation of cellulose to lactic acid. Bioresourc Technol. 1997.62(3). 91-98.
9．罗珺，夏黎明，林建平，等．纤维素酶水解及同时糖化和乳酸发酵过程动力学．化工学报．1998，49(2)．162-168．
10．陈卓贤，沈春明，陈国良．味精生产工艺学，北京：中国轻工业出版社，1992．82．
11．上久保正等．纤维素酶的水解．应用微生物．1984，(4)：31-35．
12. Gadgil N J, Daginawala H F, Chakrabarti T, Khanna P. Enhanced cellulase production by a mutant of trichoderma reesei. Enzyme Microb Technol. 1995. 17(10). 942-946.
13. Grethlein H E. Pretreatment For Enhanced Hydrolysis Of Cellulosic Biomass. Biotechnol Adv. 1984.2(1). 43-62.
14. Wu J, Ju L-K. Enhancing enzymatic saccharification of waste newsprint by surfactant addition. Biotechnol Prog. 1998. 14(4). 649-652.
15．冯若，李化茂．声化学及其应用．合肥：安徽科学技术出版社．1992．1-380．
16. Lin J-G, Ma Y-S. Oxidation of 2-chlorophenol in water by ultrasound/Fenton method. J Environ Eng. 2000. 126(2) 130-137.
17. Melo M L, Moreno M J S M, Pereira C S C, et al. Ultrasound assisted oxidations and reductions in steroid synthesis. Ultrason Sonochem. 1994. 1(1). 37-40.
18. Guilet R, Berlan J, Louisnard O, Schwartzentruber J. Influence of ultrasound power on the alkylation of phenylacetonitrile under solid-liquid phase transfer catalysis conditions. Ultrason Sonochem. 1998.5(1). 21-25.
19．刘云，张军．超声波催化和相转移催化合成羟基苯甲醛．化学世界．1998．39(10)．529-533．
20. Duee I, Berlan J, Casamatta G, et al. New process for the synthesis of the 2-propyl pentane nitrile under ultrasonic irradiation. Chem Eng J Biochem Eng J. 1995. 59(2). 121.
21．张明 王存德．超声波催化合成3，5-二异丙基水杨酸．中国药物化学杂志．1994．4(4)．292-294．
22. Brochette-Lemoine S, Trombotto S, Joannard D, et al. Ultrasound in carbohydrate chemistry: Sonophysical glucose oligomerization and sonocatalyzed sucrose oxidation. Ultrason Sonochem. 2000.7(4). 157-161.
23．谢冰．超声波作用下有机污染物的降解．水处理技术．2000．26(2)．114-119．
24. Price, Gareth J. Ultrasonically enhanced polymer synthesis. Ultrason Sonochem. 1996.3(3). S229-S238.
25．刘江，陈克强．超声波辐照下苯乙烯与丙烯酸乙酯共聚反应的研究．四川大学学报(工程科学版)．2000．32(2)．67-70．
26．高大维，陈满香．超声波催化糖化酶水解淀粉的初步研究．天津微生物．1993．(4)．6-9．
27．宗敏华，杜伟，李慧青，刘耘．超声辐照对有机相脂肪酶催化酯化反应的促进．华南理工大学学报(自然科学版)．2000．28(3)．101-104．
28．严慧敏，黄炜，廖兆全．超声波和TritonX 100对鸟氨酸氨基甲酰转移酶催化活性的影响．催化学报．1999．20(4)．479-480．
29．林勤保，高大维．超声波对酶反应的影响．1997．16(1)．26-28．
2. Grajek W. Production of protein by thermophilic fungi from sugar-beet pulp in solid-state fermentation. Biotechnol and Bioeng. 1988.32(2). 225-260.
3．阿尔贝·萨松著，贾谦 张玉华 李沄译．生物技术—挑战与希望．北京：科学技术文献出版社出版．1986．4．
4. Ingram L O, Gomez P F, Lai X, Moniruzzaman M, et al. Metabolic engineering of
bacteria for ethanol production. Biotechnol Bioeng. 1998.58(2-3). 204-212.
5. Cheung S, Anderson B. Ethanol production from wastewater solids. Water Environ Technol. 1996.8(5). 55-60.
6. Alvo P, Belkacemi K. Enzymatic saccharification of milled timothy (Phleum pratense L.) and alfalfa (Medicago sativa L.). Bioresourc Technol. 1997.61(3). 185-198.
7．周怡，杨守志，薛茂杰，等．由纤维素制取单细胞蛋白的反应机理模型．化工学报．1992．43(4)．395-400．
8. Venkatesh K V. Simultaneous saccharification and fermentation of cellulose to lactic acid. Bioresourc Technol. 1997.62(3). 91-98.
9．罗珺，夏黎明，林建平，等．纤维素酶水解及同时糖化和乳酸发酵过程动力学．化工学报．1998，49(2)．162-168．
10．陈卓贤，沈春明，陈国良．味精生产工艺学，北京：中国轻工业出版社，1992．82．
11．上久保正等．纤维素酶的水解．应用微生物．1984，(4)：31-35．
12. Gadgil N J, Daginawala H F, Chakrabarti T, Khanna P. Enhanced cellulase production by a mutant of trichoderma reesei. Enzyme Microb Technol. 1995. 17(10). 942-946.
13. Grethlein H E. Pretreatment For Enhanced Hydrolysis Of Cellulosic Biomass. Biotechnol Adv. 1984.2(1). 43-62.
14. Wu J, Ju L-K. Enhancing enzymatic saccharification of waste newsprint by surfactant addition. Biotechnol Prog. 1998. 14(4). 649-652.
15．冯若，李化茂．声化学及其应用．合肥：安徽科学技术出版社．1992．1-380．
16. Lin J-G, Ma Y-S. Oxidation of 2-chlorophenol in water by ultrasound/Fenton method. J Environ Eng. 2000. 126(2) 130-137.
17. Melo M L, Moreno M J S M, Pereira C S C, et al. Ultrasound assisted oxidations and reductions in steroid synthesis. Ultrason Sonochem. 1994. 1(1). 37-40.
18. Guilet R, Berlan J, Louisnard O, Schwartzentruber J. Influence of ultrasound power on the alkylation of phenylacetonitrile under solid-liquid phase transfer catalysis conditions. Ultrason Sonochem. 1998.5(1). 21-25.
19．刘云，张军．超声波催化和相转移催化合成羟基苯甲醛．化学世界．1998．39(10)．529-533．
20. Duee I, Berlan J, Casamatta G, et al. New process for the synthesis of the 2-propyl pentane nitrile under ultrasonic irradiation. Chem Eng J Biochem Eng J. 1995. 59(2). 121.
21．张明 王存德．超声波催化合成3，5-二异丙基水杨酸．中国药物化学杂志．1994．4(4)．292-294．
22. Brochette-Lemoine S, Trombotto S, Joannard D, et al. Ultrasound in carbohydrate chemistry: Sonophysical glucose oligomerization and sonocatalyzed sucrose oxidation. Ultrason Sonochem. 2000.7(4). 157-161.
23．谢冰．超声波作用下有机污染物的降解．水处理技术．2000．26(2)．114-119．
24. Price, Gareth J. Ultrasonically enhanced polymer synthesis. Ultrason Sonochem. 1996.3(3). S229-S238.
25．刘江，陈克强．超声波辐照下苯乙烯与丙烯酸乙酯共聚反应的研究．四川大学学报(工程科学版)．2000．32(2)．67-70．
26．高大维，陈满香．超声波催化糖化酶水解淀粉的初步研究．天津微生物．1993．(4)．6-9．
27．宗敏华，杜伟，李慧青，刘耘．超声辐照对有机相脂肪酶催化酯化反应的促进．华南理工大学学报(自然科学版)．2000．28(3)．101-104．
28．严慧敏，黄炜，廖兆全．超声波和TritonX 100对鸟氨酸氨基甲酰转移酶催化活性的影响．催化学报．1999．20(4)．479-480．
29．林勤保，高大维．超声波对酶反应的影响．1997．16(1)．26-28．