固定化纤维二糖酶的研究

黑曲霉（Aspergillus niger LORRE 012）的孢子中富含纤维二糖酶,将这些孢子用海藻酸钙凝胶包埋后,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定,半衰期为38 d,耐热性和适宜的pH范围均比固定化前有所增加,其K_m和V_max值分别为6.01 mmol/L和7.06 mmol/(min·L)。利用固定化纤维二糖酶重复分批酶解10 g/L的纤维二糖,连续10批的酶解得率均可保持在97%以上;采用连续酶解工艺,当稀释率为0.4 h^-1,酶解得率可达98.5%。玉米芯经稀酸预处理后,其纤维残渣用里氏木霉（Trichoderma reesei）纤维素酶降解,酶解得率为69.5%;通过固定化纤维二糖酶的进一步作用,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除,酶解得率提高到84.2%,还原糖中葡萄糖的比例由53.6%升至89.5%,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。     

裂褶菌纤维二糖脱氢酶吸附纤维素性质

纤维二糖脱氢酶（ＣＤＨ）可以吸附棉花、微晶纤维素和酸处理纤维素,４ｍｉｎ便都达到平衡。与纤维素酶明显不同,该酶的Ｓｃａｔｃｈａｒｄ吸附曲线都是一条直线,为典型的单结合位点模型（ｏｎｅ－ｂｉｎｄｉｎｇ－ｓｉｔｅ ｍｏｄｅｌ）。观察到ｐＨ值、温度、乙二醇和ＮａＣｌ对ＣＤＨ吸附微昌纤维素有影响,并进行了讨论。     

纤维二糖抑制外切纤维素酶水解作用机理的分析

纤维二糖是纤维素分子的结构单元, 也是纤维素酶水解纤维素时的主要产物. 它能强烈抑制纤维素酶的水解作用, 但并不影响酶对纤维素的吸附. 红外、荧光、圆二色谱分析表明, 纤维二糖可结合在外切纤维素酶活性部位附近的色氨酸残基上形成“位阻效应”, 从而阻止纤维素分子链进入活性中心. 同时, 结合纤维二糖后, 外切纤维素酶分子构象发生了较大变化, 致使吸附纤维素后不能导致微纤维的分离, 为“无效吸附”.     

绿色木霉JFY-14产纤维素酶的产酶条件研究

利用实验室现有的纤维素酶高产菌株制备纤维素酶。考察了JFY-14菌株产酶培养过程中pH值、培养时间、氮源等条件的影响。得到了最佳产纤维素酶条件:培养时间为72~75 h,初始pH值为4.5~5.0以及最佳培养氮源为1%的硫酸铵。     

纤维二糖差向异构酶在毕赤酵母中的表达及酶学性质研究

首先将来源于Caldicellulosiruptor saccharolyticus的纤维二糖差向异构酶基因CsCEm进行密码子优化,然后进行全基因合成,再将其引入到载体pPIC9K中,构建重组质粒pPIC9K-CsCEm并转化入毕赤酵母GS115,得到酵母工程菌株.经微孔板筛选、摇瓶筛选得到酶活最高的重组工程茵GS115-4-19.该菌株经甲醇诱导144 h后,摇瓶发酵液上清酶活达到0.42 U/mL.酶学性质研究结果表明:该酶的最适pH为7.5,且在pH 6.0 ～8.0范围内相对酶活都在80％以上;在pH 4～9的缓冲液中放置24 h后仍保持原酶活力的80％以上;最适温度为80℃,在60℃～80℃保温30 min后,相对酶活在80％以上.动力学研究结果表明该酶对底物乳糖的Km和Vmax分别为(120.27±9.96) mmol/L和(1.035±0.05) mmol/L/min.纤维二糖差向异构酶在毕赤酵母中的成功表达为生物酶法合成乳果糖提供了重要参考.     

纤维二糖在纤维素生物降解中调控作用的探讨*

对纤维二糖在真菌纤维素酶类合成中的诱导和阻遏作用机制、水解活性的抑制作用等的研究进展做了简要评述。根据对纤维素酶分子的纤维结合结构域的研究结果,提出了外切纤维素酶的主要功能是破坏纤维素的结晶结构,为β-1,4糖苷键的水解提供条件的论点。提出了经济有效转化纤维性材料的新策略。     

蔗糖酶和GOD的共固定化研究

本文研究了用吸附交联技术共固定化蔗糖酶和葡萄糖氧化酶（ＧＯＤ）的方法,考查了共固定化酶的动力学性质。试验结果表明：与溶液酶相比较,固定化蔗糖酶和ＧＯＤ的响应滞迟期分别为３分钟和２分钟,４态响应时间增加６分钟和４分钟,Ｋｍ值增大,ｐＨ─活力曲线变宽,最适ｐＨ值分别增大０．７和０．６４,最适温度则降低７．３℃和１６℃。以活性氧化铝作载体,戊二醛作交联剂制备的共固定化蔗糖酶和ＧＯＤ,其蛋白质固定化率为９２．９％,分解葡萄糖的总速度为４４１．６ＩＵ,当蔗糖浓度在０．２％以内时其反应速度与蔗糖浓度呈正相关（ｒ＝０．９９６）,使用半衰期１６２３次,在４℃下保存１２０天活力残存为８３．７％。     

裂褶菌产纤维二糖脱氢酶条件优化及部分酶学性质研究

研究了裂褶菌Ｓｃｈｉｚｏｐｈｙｌｌｕｍ ｃｏｍｍｕｎｅＡＳ ５．３９１产纤维二糖胶氢酶的条件。该酶是诱导酶棉花是最好的诱导底物。培养基中加入琥珀酸纳缓冲液和土温８０利于酶的合成。而木素相关物质藜芦醇或愈创木酚对酶的生成没有影响。该酶在ｐＨ３．５～１０．５和４５℃以下保持稳定,其最适作用温度为３０℃,最适ｐＨ为４．５。Ｚｎ＾２＋和Ａｇ＾＋对该酶活性有很大的抑制作用,而叠氮化钠和氰化钾对酶活力没有影响     

纤维素酶固定化的研究进展

纤维素酶成本过高是纤维糖化工艺中的主要障碍,因此,各国学者都在研究和探索降低纤维素酶成本及提高其酶解效率的有效方法,酶的固定化技术便有很有希望的方法之一。本文综述了近２０年国内外应用不同性质的载体研究纤维素酶固定化的若干结果和进展。     

纤维剩余物酶解工艺研究

富含单宁的塔拉豆荚经水解制备没食子酸,其剩余物中富含纤维素。本研究探讨了酶解各因素对塔拉纤维剩余物还原糖产率的影响,在单因素实验的基础上,进行料液比、酶解温度、p H和酶解时间四因素L16(4)5正交优化实验,其优化工艺条件为料液比1∶6 g·m L-1,酶解温度55℃,p H6,酶解时间48 h。在此条件下,酶解塔拉纤维剩余物还原糖产率均值为43.95%。     

Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate

The inhibition effect of cellobiose on the initial stage of hydrolysis when cellobiohydrolase Cel 7A and endoglucanases Cel 7B, Cel 5A, and Cel 12A from Trichoderma reesei were acting on bacterial cellulose and amorphous cellulose that were [(3)H]- labeled at the reducing end was quantified. The apparent competitive inhibition constant (K(i)) for Cel 7A on [(3)H]-bacterial cellulose was found to be 1.6 +/- 0.5 mM, 100-fold higher than that for Cel 7A acting on low-molecular-weight model substrates. The hydrolysis of [(3)H]-amorphous cellulose by endoglucanases was even less affected by cellobiose inhibition with apparent K(i) values of 11 +/- 3 mM and 34 +/- 6 mM for Cel 7B and Cel 5A, respectively. Contrary to the case for the other enzymes studied, the release of radioactive label by Cel 12A was stimulated by cellobiose, possibly due to a more pronounced transglycosylating activity. Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme-substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzyme-substrate complex is nonproductive.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

微球载体固定化纤维素酶的反应动力学模型研究

建立了固定化纤维素酶的反应动力学模型,该模型以米氏方程为基础并考虑了产物竞争性抑制的影响。在此模型的基础上进行模拟,系统研究了产物竞争性抑制、内扩散限制、溶液中的宏观底物浓度、载体大小等因素对球形载体内部的底物、产物浓度分布和效率因子的影响。产物竞争性抑制的存在将增加载体颗粒内的底物浓度,对效率因子的影响较小。底物内扩散系数或者反应体系中底物浓度增大时,载体颗粒内的底物浓度和效率因子都将增大。载体粒径增大,载体颗粒内的底物浓度和效率因子均减小。     

2种纤维素酶水解效果比较及酶蛋白组分分析

比较了自产纤维素酶和商品纤维素酶的水解效果,并采用超滤、层析、SDS-PAGE相结合的方法分析2种纤维素酶蛋白组分的差异。里氏木霉以纸浆为C源合成的自产纤维素酶的水解得率高于商品纤维素酶,自产纤维素酶水解48h的得率为66.24%,商品纤维素酶的得率为52.19%。自产纤维素酶中存在着Cel6A酶组分和XYNⅡ酶组分,而商品纤维素酶中没有检测到这2种酶组分。自产纤维素酶和商品纤维素酶的Cel1A酶组分和Cel7A酶组分间存在着分布和含量上的差异。自产纤维素酶在相对分子质量(2.5～3.5)×104范围内存在着几条蛋白条带,而商品纤维素酶则是在相对分子质量3.5×104附近存在着几条蛋白条带。     

Some kinetic properties of the β-d-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β-d-glucosidase (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β-d-glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. d-Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. d-Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while d-fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β-d-glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

碳纳米管固定化纤维素酶的最佳工艺研究

纤维素酶在环保、医药、食品等领域都具有广泛的应用前景,但由于纤维素酶的生产成本较高,生物活性较低,使得纤维素酶的应用受到了限制。为了寻找一种固定化纤维素酶的方法,使酶可以重复多次使用,首次以多壁碳纳米管为载体固定化纤维素酶,研究功能化的多壁碳纳米管固定化纤维素酶的固定化条件,采用正交试验对酶固定化中的主要条件进行优化,并通过傅里叶变换红外光谱仪对多壁碳纳米管(multiwalled carbon nanotube,MWCNTs)、纤维素酶及固定化纤维素酶的结构进行表征。结果表明,固定化纤维素酶的最佳工艺条件为:酶浓度5 mg·mL^-1,温度40℃,pH 5.0,固定化时间3 h;通过傅里叶变换红外光谱证实纤维素酶成功固定到多壁碳纳米管上。     

Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion

The slow down in enzymatic hydrolysis of cellulose with conversion has often been attributed to declining reactivity of the substrate as the more easily reacted material is thought to be consumed preferentially. To better understand the cause of this phenomenon, the enzymatic reaction of the nearly pure cellulose in Avicel was interrupted over the course of nearly complete hydrolysis. Then, the solids were treated with proteinase to degrade the cellulase enzymes remaining on the solid surface, followed by proteinase inhibitors to inactive the proteinase and successive washing with water, 1.0 M NaCl solution, and water. Next, fresh cellulase and buffer were added to the solids to restart hydrolysis. The rate of cellulose hydrolysis, expressed as a percent of substrate remaining at that time, was approximately constant over a wide range of conversions for restart experiments but declined continually with conversion for uninterrupted hydrolysis. Furthermore, the cellulose hydrolysis rate per adsorbed enzyme was approximately constant for the restart procedure but declined with conversion when enzymes were left to react. Thus, the drop off in reaction rate for uninterrupted cellulose digestion by enzymes could not be attributed to changes in substrate reactivity, suggesting that other effects such as enzymes getting "stuck" or otherwise slowing down may be responsible.     

Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm^?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate V_max was enhanced by sonication relative to controls, but the value of the saturation constant K_m was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm^?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm^?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013