纤维素酶、酯酶和木聚糖酶对玉米秸秆协同作用的研究
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摘要
秸秆是一类可再生的纤维类资源,要使其高质化利用必须将其分解。目前,分解纤维类物质的主要方法有纤维素酶解和酸解等,人们研究较多是如何提高纤维素酶的活性,这是世界性的难题。因此,技术路线的优化组合问题、生产过程中成本降低的问题显得至关重要。
本文比较使用了不同前处理方法对酶解纤维素水解率的影响。首先用稀酸法、稀碱法、亚钠法对玉米秸秆进行前处理,再用纤维素酶对玉米秸秆中纤维素进行水解。结果表明：在50℃、pH为4.8,固液比为1∶30、酶浓度为4g/1、反应时间为24h的条件下,可获得较理想的酶解率。经亚钠预处理后的玉米秸秆,纤维素含量上升较多,酶解率较高,亚钠预处理后的酶解率达到39.07%,是未经处理的秸秆酶解率(9.8%)的4倍。预处理破坏了玉米秸秆的纤维素结构。
在对酶协同分解秸秆的研究中发现,两种酶协同作用分解秸秆的效果要高于单酶的效果,三种酶协同作用的效果高于两种酶的效果。三酶协同作用分解秸秆时,酶的添加顺序不同,酶分解率也不同。同时添加入纤维素酶、木聚糖酶和阿魏酸酯酶粗酶的酶分解率为79%,效果最好,高于只加纤维素酶的酶分解率65.63%。由此表明,阿魏酸酯酶、木聚糖酶和纤维素酶之间存在较大的协同作用,添加阿魏酸酯酶、木聚糖酶能够提高纤维素酶对天然木质纤维素的酶解效率。
通过研究八种金属离子对酶协同作用降解秸秆的影响发现,金属离子对酶的影响较为复杂,不同的金属离子对不同的酶影响不一样,而且同种离子在不同浓度下可表现出不同的作用。当离子浓度为1×10-3mol/L时，Cu2-、Mn2+、Ca2+、Na+、Mg2+、和Fe2+对纤维素酶起激活作用；Cu2+、K+、Ca2+、Na+、Fe2+对木聚糖酶起激活作用：Cu2+、Ca2+、Zn2+对阿魏酸酯酶粗酶起激活作用。对三酶协同作用的激活作用最强的是Cu2+、Ca2+和Na+。加Cu2+、Ca2+和Na+的三酶协同作用的酶解率为82.61%,高于不加金属离子的79%。
Stalk is a renewable resource. The mainly methods on hydrolysis of cellulose from stalk are using cellulase and acid. It is a hot potato how can improve the rate of enzymatic hydrolysis on cellulose in the world. It becomes important to combinatorial optimization and operating cost reduction.
Three different pretreatment methods were tested to study the effect on hydrolysis of cellulose from corn stalk. The dilute-acid、dilute-alkali and sodium sulfite anhydrous pretreatment of corn stalk were carried out, The pretreatment of obtained sample was conducted for cellulose enzymolys is saccharification. The results showed that the optimum condition for enzymatic hydrolysis is at temperature of 50℃, pH of 4.8, ratio of solid to liquid 1:30, enzyme concentration of 4 g/L, and the hydrolysis time of 24 h. Under the optimized condition, the rate of enzymatic hydrolysis on corn stalk pretreatment using sodium sulfite anhydrous was 39.07%, which is four multiple higher than that of corn stalk hydrolysis without pretreatment. Pretreatment destroyed the fibrous structure of corn stalk significantly. Relative good rate of enzymatic hydrolysis on corn stalk pretreatment using sodium sulfite anhydrous can be achieved。
The study showed that the rate of enzymatic hydrolysis on synergism of cellulase、FAE and xylanase was higher than two enzymes, the rate of two enzymes was higher than ones. The rate of enzymatic hydrolysis was different with different orders of adding enzymes when synergistic reaction,the optimum order was adding them at one time,and the rate of enzymatic hydrolysis on synergism was 79%, which is higher than that of cellulase(65.63%). It is concluded that FAE, xylanase and cellulase have great synergistic effect, FAE、xylanase can help cellulase hydrolyze natural lignocellulose and enhance hydrolytic efficiency.
The effects of eight kinds of metallic ions on cellulase、FAE and xylanase activity were studied. The results were as following:The effects of metallic ions on enzyme activity were complicacy. The enzyme activity with different mental ions was different. When the concentration of mental ions was 1×10-3mol/L,cellulase activity improved by Cu2+, Mn2+, Ca2+, Na+, Mg2+, Fe2+, xylanase activity improved by Cu2+, K+, Ca2+, Na+, Fe2+, FAE activity improved by Cu2+, Ca2+, Zn2+,Cu2+, Ca2+ andNa+ had the strongest activation effect on synergism of cellulase、esterase and xylanase.The rate of enzymatic hydrolysis on addition of metallic ions was 82.99%, it is higher than that of whithout metallic ions(79%).
本文比较使用了不同前处理方法对酶解纤维素水解率的影响。首先用稀酸法、稀碱法、亚钠法对玉米秸秆进行前处理,再用纤维素酶对玉米秸秆中纤维素进行水解。结果表明：在50℃、pH为4.8,固液比为1∶30、酶浓度为4g/1、反应时间为24h的条件下,可获得较理想的酶解率。经亚钠预处理后的玉米秸秆,纤维素含量上升较多,酶解率较高,亚钠预处理后的酶解率达到39.07%,是未经处理的秸秆酶解率(9.8%)的4倍。预处理破坏了玉米秸秆的纤维素结构。
在对酶协同分解秸秆的研究中发现,两种酶协同作用分解秸秆的效果要高于单酶的效果,三种酶协同作用的效果高于两种酶的效果。三酶协同作用分解秸秆时,酶的添加顺序不同,酶分解率也不同。同时添加入纤维素酶、木聚糖酶和阿魏酸酯酶粗酶的酶分解率为79%,效果最好,高于只加纤维素酶的酶分解率65.63%。由此表明,阿魏酸酯酶、木聚糖酶和纤维素酶之间存在较大的协同作用,添加阿魏酸酯酶、木聚糖酶能够提高纤维素酶对天然木质纤维素的酶解效率。
通过研究八种金属离子对酶协同作用降解秸秆的影响发现,金属离子对酶的影响较为复杂,不同的金属离子对不同的酶影响不一样,而且同种离子在不同浓度下可表现出不同的作用。当离子浓度为1×10-3mol/L时，Cu2-、Mn2+、Ca2+、Na+、Mg2+、和Fe2+对纤维素酶起激活作用；Cu2+、K+、Ca2+、Na+、Fe2+对木聚糖酶起激活作用：Cu2+、Ca2+、Zn2+对阿魏酸酯酶粗酶起激活作用。对三酶协同作用的激活作用最强的是Cu2+、Ca2+和Na+。加Cu2+、Ca2+和Na+的三酶协同作用的酶解率为82.61%,高于不加金属离子的79%。
Stalk is a renewable resource. The mainly methods on hydrolysis of cellulose from stalk are using cellulase and acid. It is a hot potato how can improve the rate of enzymatic hydrolysis on cellulose in the world. It becomes important to combinatorial optimization and operating cost reduction.
Three different pretreatment methods were tested to study the effect on hydrolysis of cellulose from corn stalk. The dilute-acid、dilute-alkali and sodium sulfite anhydrous pretreatment of corn stalk were carried out, The pretreatment of obtained sample was conducted for cellulose enzymolys is saccharification. The results showed that the optimum condition for enzymatic hydrolysis is at temperature of 50℃, pH of 4.8, ratio of solid to liquid 1:30, enzyme concentration of 4 g/L, and the hydrolysis time of 24 h. Under the optimized condition, the rate of enzymatic hydrolysis on corn stalk pretreatment using sodium sulfite anhydrous was 39.07%, which is four multiple higher than that of corn stalk hydrolysis without pretreatment. Pretreatment destroyed the fibrous structure of corn stalk significantly. Relative good rate of enzymatic hydrolysis on corn stalk pretreatment using sodium sulfite anhydrous can be achieved。
The study showed that the rate of enzymatic hydrolysis on synergism of cellulase、FAE and xylanase was higher than two enzymes, the rate of two enzymes was higher than ones. The rate of enzymatic hydrolysis was different with different orders of adding enzymes when synergistic reaction,the optimum order was adding them at one time,and the rate of enzymatic hydrolysis on synergism was 79%, which is higher than that of cellulase(65.63%). It is concluded that FAE, xylanase and cellulase have great synergistic effect, FAE、xylanase can help cellulase hydrolyze natural lignocellulose and enhance hydrolytic efficiency.
The effects of eight kinds of metallic ions on cellulase、FAE and xylanase activity were studied. The results were as following:The effects of metallic ions on enzyme activity were complicacy. The enzyme activity with different mental ions was different. When the concentration of mental ions was 1×10-3mol/L,cellulase activity improved by Cu2+, Mn2+, Ca2+, Na+, Mg2+, Fe2+, xylanase activity improved by Cu2+, K+, Ca2+, Na+, Fe2+, FAE activity improved by Cu2+, Ca2+, Zn2+,Cu2+, Ca2+ andNa+ had the strongest activation effect on synergism of cellulase、esterase and xylanase.The rate of enzymatic hydrolysis on addition of metallic ions was 82.99%, it is higher than that of whithout metallic ions(79%).
引文
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[2]蒋挺大.木质素[M].北京：化学工业出版社,2001.
[3]Chosdu R, Hilmy N, Erizal Erlinda TB, et al. Radiation and chemical pretreatment of cellulosic waste[J].Radiat PhysChem,1993,42:695-698.
[4]杨涛.离子液体预处理玉米秸秆及纤维素酶水解研究[D].天津：天津大学化工学院,2008：1.
[5]杜风光,冯文生.燃料乙醇发展现状和前景展望[J].现代化工,2006,26(1)：6-9.
[6]Sun Y, Cheng J.Hydrolysis of lignocellulosic materials for ethanol production:a review[J].Bioresour Technol,2002,83:1-11.
[7]孙亚东,蒋建新,孙冉,等.纤维乙醇及渗透汽化原位分离技术研究进展[J].中国能源,2008,30(9)：17-24.
[8]刘洪斌.燃料乙醇非粮化-我国发展纤维乙醇的挑战与对策[J].生物加工过程,2008,6(1)：7-11.
[9]Choteborsk P'Palmarola.Adrados B, Gable M, et al.Processing of wheat bran to sugar solution [J].Journal of Food Engineering,2004,61(4):561-565.
[10]孙健,陈砺,王红林,等.纤维素原料生产燃料酒精的技术现状[J].可再生能源,2003,112(6)：5-9.
[11]章冬霞,亓伟,孙秀云,等.木质纤维素酶水解技术的研究[J].吉林化工学院学报,2009,26(1)： 1-5.
[12]周建,罗学刚,苏林.纤维素酶法水解的研究现状及展望[J].化工科技,2006,14(2)：51-56.
[13]阮同琦,赵祥颖,刘建军.木聚糖酶及其应用研究进展[J].山东食品发酵,2008(1)：42-45.
[14]陈洪章,李佐虎.影响纤维素酶解的因素和纤维素酶被吸附性能的研究[J].化学反应工程与工艺,2000,16(1)：30-35.
[15]张丽萍,董超,王迎春,等.几种离子对纤维素酶活力的影响[J].河北省科学院学报,2000,17(4)：235-238.
[16]赵玉蓉,金宏,陈清华,等.金属离子对纤维素酶及木聚糖酶活性影响的研究[J].饲料博览,2005(1)：1-3.
[17]Chang, V.S.Holtzapple, M.T.Fundamental factors affecting biomass enzymatic reactivity [J].Applied Biochemistry and Biotechnology,2000,84:5-37.
[18]Yoshikuni Teramoto,Seung Hwan Lee, Takashi Endo.Pretreatment ofwoody and herbaceous biomass for enzymatic saccharification using sulfuric acid-free ethanol cooking[J].Bioresource Technol,2008,99(18):8856-8863
[19]文新亚,李燕松,张志鹏,等.酶解木质纤维素的预处理技术研究进展[J].酿酒科技,2006,(8)：97-100.
[20]李松晔,刘晓非,庄旭品,等.棉浆粕纤维素的超声波处理[J].应用化学,2003,20(11)：1030-1034.
[21]Palmqvist E, Hahn-Hagerdal B.Fermentation of lignoeellulosie hydrolysates:Inhibitors and menehanisms of inhibition[J].Bioresource Technology,2000,74(1):25-33.
[22]Esteghlalian A, Hashimoto A G, Fenske J J, et al. Modeling and optimization of the dilute sulfuric add pretreatme nt of COFU stover, poplar and switehgrass[J].Bioresource Technology,1997,59: 129-136.
[23]McMillan J D. Pretreatment of lignocellulosic biomass. In:Himmet M E. Enzymatic conversion of bionmass fro fuels production [J].Amercian Chemical Society,1992,292-324.
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