外切-β-葡聚糖酶基因重组里氏木霉的筛选及其产酶性能

外切-b-葡聚糖酶是纤维素酶的重要组分之一,提高该组分的活力是增强纤维素酶协同降解性能、降低纤维素水解成本的关键。分别采用微晶纤维素琼脂平板法和滤纸崩解法,对已有的基因重组转化子进行筛选试验,获得了6个优良转化子,其滤纸崩解速率和微晶纤维素琼脂平板上的生长速率都较大。进一步在摇瓶条件下进行复筛试验,获得了外切-β-葡聚糖酶（C₁）高产转化子Trichoderma reesei ZU-101,液体培养48 h,其C₁酶活力可达18.24 U·ml^-1,是出发菌株的2.16倍;分析结果表明：重组转化子的纤维素酶体系中内切-b-葡聚糖酶和纤维二糖酶的活力与出发菌株相比变化不大,但由于外切-b-葡聚糖酶活力得到了大幅度提高,纤维素酶的总活力（滤纸酶活力FPA）也提高了61.9%。采用纤维素酶对碱预处理玉米秸秆进行酶解试验,当酶用量为20 FPIU·（g底物）^-1,水解48 h,重组转化子T.reesei ZU-101纤维素酶的酶解得率高达94.4%。本文的研究结果在可再生纤维素资源的生物转化与利用方面具有广阔的应用前景。     

玉米秸秆中不同木质素脱除方法对纤维素酶吸附及酶解效果的比较

利用不同预处理方法获得的玉米秸秆底物研究木质素脱除对纤维素酶吸附量及酶解效率的影响。相比于其他处理方法,2%(质量分数)NaOH处理的底物具有最高的木质素脱除率(85%),最高的底物可及性[4.7 mg·(g 葡聚糖) ^-1]及酶解效率(18.9%)。通过对不同处理获得的底物进行Langmuir吸附等温曲线模拟,获得了最大吸附量(W_max)与吸附平衡常数(K),且木质纤维素酶水解效率与纤维素酶吸附量具有很好的线性关系(R²>0.8),表明脱除木质素能很好地提高底物可及性与酶解效率。然而,提高NaOH浓度(3%,4%)进一步脱除木质素时,底物可及性与碳水化合物转化为单糖的效率反而明显下降。因此,适当脱除木质素而提高底物对纤维素酶的可及性将有助于获得更有效的酶水解效果。     

离子及表面活性剂对甜高粱秆渣酶解的影响

为了提高纤维素酶水解经高温液态水处理后的甜高粱秆渣的效率,探讨了多种阴离子、阳离子以及吐温80(Tween 80)对纤维素酶活力的影响,并初步探讨了Tween 80影响甜高粱秆渣酶解的机制。酶激活试验表明,Br^-、I^-、NO₃^-、Ca²⁺、Mg²⁺和Co²⁺对纤维素酶有激活作用,但对甜高粱秆渣的水解效率提高不明显。添加Tween 80发现,随着浓度的增加,它对纤维素酶的抑制作用增强,而Tween 80添加量为0.175 ml·(g甜高粱秆渣)^-1时,甜高粱秆渣的酶解效率由16.6%提高到37.9%。吸附试验表明,甜高粱秆渣对纤维素酶和Tween 80的吸附达到一定限度后不再上升,Tween 80能显著降低甜高粱秆渣对纤维素酶的吸附。红外光谱分析发现,木质素对Tween 80的吸附要强于它对纤维素酶的吸附。     

玉米芯残渣的碱性亚硫酸盐预处理及其反应动力学模型

玉米芯提取木糖后残留了大量富含纤维素和木质素的废弃物。针对玉米芯残渣（corncob residues,CCR）中木质素含量高和半纤维素含量很低的特点,采用碱性亚硫酸盐法进行预处理。研究了预处理pH、液固比、温度、亚硫酸盐用量等条件对纤维素保留率、木质素去除率、底物酶解效率以及预处理液中木质素磺酸钠含量的影响规律。结果表明,当亚硫酸钠用量为10%（质量）、氢氧化钠为5%（质量）、液固比为6：1、160℃预处理1 h时,可去除86.1%的木质素、保留82.4%的纤维素,底物的72 h酶解率达85.1%[酶载量为5 FPU·（g葡聚糖）^-1],预处理液中木质素磺酸钠的收率为31.5 g·（100 g CCR）^-1。为了指导放大试验和工程应用,提出了一个能准确预测底物木质素含量的参数--木质素因子（lignin factor,LF）,在此基础上成功建立了脱木质素反应动力学经验公式以及底物酶解效率的预测方程,预测值与实测值误差在10%之内。     

甘露聚糖酶促进纤维素酶水解转化马尾松聚糖至可发酵单糖

由于形态结构和化学组成等原因,针叶材原料利用纤维素酶水解法转化聚糖至可发酵单糖一直存在着难水解、总糖转化率低的问题。采用硫酸盐(KP)蒸煮联合氧脱木素和机械打浆对马尾松木片进行预处理,基于针叶材原料中含有较多的甘露聚糖的特点,研究在纤维素酶水解预处理后的浆料过程中添加甘露聚糖酶对酶解效率的影响。研究结果显示,添加甘露聚糖酶对纤维素酶水解具有促进作用,其作用效果因原料预处理方法和程度的不同而不同。KP蒸煮后再经氧脱木素或打浆处理,则甘露聚糖酶对纤维素酶水解的促进作用比单独KP蒸煮效果更加明显。当KP蒸煮至样品木素含量相对于原料为4.1%时,再对原料进行氧脱木素,将木素含量降至相对于原料为1.9%时,分别用10 FPU/g和15FPU/g的复合纤维素酶CTec2对预处理后的样品进行酶解,酶解总糖得率分别为66.0%和83.6%;添加5U/g甘露聚糖酶,上述样品酶解总糖得率分别提高至70.6%和89.2%;相对于原料中聚糖含量,上述条件下酶解总糖转化率分别为53.9%和68.1%。纤维素酶水解至3小时左右再添加甘露聚糖酶,其促进纤维素酶水解效果更好。     

玉米芯氨水预处理及酶解工艺研究

为有效提高木质纤维素酶解转化率,文中以玉米芯为研究对象,在常压中温下采用氨水浸泡工艺处理原料,考察了预处理条件对木质素脱除率和纤维素、半纤维素酶解转化率的影响规律。确定了最适预处理条件:氨水质量分数为15%、固液质量体积比为1∶6 g/mL、反应温度为60℃和预处理时间为12 h。该条件下纤维素、半纤维素回收率和木质素脱除率分别为94.5%,86.7%和48.1%;在每g葡聚糖加入30 FPU纤维素酶和60 CBUβ-葡萄糖苷酶条件下,酶解24 h后纤维素和半纤维素酶解转化率分别可达83.0%和81.6%。     

玉米芯的2步法预处理及其酶解条件优化研究

生物燃料将成为主要新能源之一,以玉米芯为原料,碱氧和稀酸为处理剂对其进行2步法预处理,使原料中纤维素相对含量增加,以提供转化乙醇的纤维素原料。采用扫描电镜表征2步法预处理玉米芯,其表面形成疏松、沟纹和孔洞形态,这有利于酶解。采用瑞氏木霉生产的纤维素酶水解该预处理玉米芯,利用正交实验得到酶水解优化条件为,酶用量75 FPU g 1,底物质量浓度60 g L 1,pH值4.8,反应温度50℃,还原糖得率可达69.3%。为提高纤维素酶中β-葡聚糖酶的酶活效率,并减少产物葡萄糖对β-葡聚糖酶的抑制作用,进一步优化β-葡聚糖酶加量。结果表明,当β-葡聚糖酶加量达6.5 CBU时,还原糖得率显著提高到78.2%。这表明该预处理玉米芯是有效降解的玉米芯原料,适于提高还原糖得率。     

氢氧化钠预处理对甘蔗渣酶解和发酵性能的影响

采用氢氧化钠预处理甘蔗渣,通过单因素和正交试验考察了不同预处理条件对甘蔗渣酶解和发酵性能的影响,并进一步分析了比表面积和木质素含量对酶解性能的影响。结果表明：预处理温度、氢氧化钠质量分数及预处理时间对酶解和发酵效率影响较为显著,最佳的预处理条件为：温度85℃、时间11 h、NaOH质量分数4.5%,在此优化条件下预处理的甘蔗渣,含纤维素56.46%,与原料相比提高了46.16%;半纤维素20.30%、Klason木质素5.79%,与原料相比分别降低了15.77%和72.87%,酶解36 h的还原糖得率为0.69 g/g（以甘蔗渣质量计）。经过氢氧化钠预处理后的甘蔗渣比表面积显著增加（由原料的0.07 m²/g最大可增加到1.07 m²/g）,木质素显著降低,有利于提高酶解和发酵效率。当比表面积超过0.30 m²/g时,酶解初始速率和酶解效率达到平衡;当木质素低于11%时,酶解效率达到平衡。     

NaOH/AQ预处理对玉米秸秆脱木质素及酶解性能的影响

对玉米秸秆进行氢氧化钠/蒽醌（NaOH/AQ）去木质化预处理,考察了预处理温度、时间和NaOH用量对玉米秸秆脱木质素程度的影响,并探讨了脱木质素程度对提高预处理后物料酶解性能的影响。L₉（3⁴）正交试验得出较适宜预处理工艺条件为：温度160℃,时间60 min,NaOH用量（以绝干原料质量计）2.8%;其他条件为AQ用量0.05%,固液比1：5（g：mL）,此时木质素脱除率为75%,酶解后聚糖转化率达到73.79%。随着物料脱木质素程度的提高,其酶解效率相应增加;当木质素脱除率达到一定程度后,预处理后的聚糖转化率达到最大值,继续提高木质素脱除率,聚糖转化率反而降低。响应面优化的酶水解工艺条件为纤维素酶用量30 FPU/g,β-葡萄糖苷酶10 IU/g,反应时间72 h,温度50℃,底物质量分数2.5%,此时还原糖得率为85.62%。对酶解液进行HPLC分析,酶解液中的葡萄糖质量浓度为14.83 g/L,木糖质量浓度为4.83 g/L。XRD分析显示,预处理前后纤维素的晶型没有变化,而结晶度由31.40%提高至46.91%,表明物料中木质素和半纤维素发生了不同程度的溶出。     

碳源对平菇选择性降解稻草木素及其后续酶解性能的影响

前期研究发现,平菇预处理稻草表现出比较好的脱木素选择性和纤维素酶水解效果。为了使预处理后的稻草在纤维素酶水解过程中得到更多的单糖,通过向平菇预处理稻草固体培养基中添加麸皮、玉米皮和木聚糖,研究外加碳源对平菇降解稻草木素的选择性及后续酶水解效果的影响。结果表明,添加适量的麸皮和木聚糖可提高平菇处理稻草的脱木素选择性。麸皮和木聚糖添加量为稻草粉的10%时,脱木素选择系数分别由对照的1.86增加至2.58和2.03;预处理样品酶解后原料中总糖的46.8%和45.9%转化为可发酵单糖,分别比对照的35.5%提高了30%和28%。添加玉米皮对平菇预处理酶解效果影响相对较小,添加量为5%时,预处理样品酶解后总糖转化率仅比对照提高15%,增加添加量酶解总糖转化率反而低于对照样品。     

Long‐term lime pretreatment of poplar wood

Long‐term lime pretreatment has proven to increase digestibility of many herbaceous lignocellulose sources; but until this work, its effects had not been evaluated on wood, whose lignin content is higher, and therefore, more recalcitrant to enzymatic hydrolysis. In this study, the mild conditions of long‐term lime pretreatment (1‐atm pressure, temperatures ranging from 25 to 75°C, and reaction times between 1 and 12 weeks, with and without air) were systematically applied to poplar wood available in two batches with different lignin contents. These batches were designated as low‐lignin biomass (LLB) with lignin content of 21.4% and high‐lignin biomass (HLB) with lignin content of 29.1%. Full factorial designs resulted in 79 samples of pretreated poplar that were analyzed for lignin and carbohydrates pretreatment yields, and enzymatic digestibility (15 FPU/g glucan in raw biomass cellulose loading). After aerated lime pretreatment at 65°C for 4 weeks, and subsequent enzymatic hydrolysis, an overall yield of 0.76 g glucan + xylan recovered per gram glucan + xylan in raw biomass was obtained. This is equivalent to an increased poplar wood digestibility of 7.5‐fold compared with untreated biomass. Different batches of the feedstock resulted in different lignin and carbohydrates pretreatment yields; however, overall yields of carbohydrates (combining pretreatment and enzymatic hydrolysis) were similar. © 2010 American Institute of Chemical Engineers AIChE J, 2011     

醋酸预水解对稻草化学成分及酶水解性能的影响

研究了醋酸预处理对稻草主要化学成分及酶水解糖化效率的影响。在160℃下以不同的醋酸用量(0～4%)对稻草进行处理,预处理后稻草的Klason木质素含量基本保持不变,约60%的酸溶木质素被脱除;灰分含量(质量分数)约下降30%,灰分中SiO2则几乎全部保留在预处理浆料中。预处理醋酸用量的增加对酸溶木质素和灰分含量的变化均无显著影响。预处理后高聚糖的降解程度随醋酸用量的增加而上升,其中半纤维素的降解程度尤为显著,阿拉伯聚糖、半乳聚糖大量溶出。对经醋酸预处理稻草的酶水解研究表明,预处理中醋酸用量的增加无助于酶水解液中还原糖得率的提高。稻草于160℃下经不添加醋酸的自水解预处理后,其酶解还原糖得率均高于经醋酸预处理的稻草,当纤维素酶用量为40 FPU/g(对底物)时,稻草中高聚糖的酶水解转化效率最高,葡聚糖、木聚糖的转化率分别为67.8%和45.3%,总糖转化率为58.8%。     

绿液预处理对棉秆化学成分及酶水解糖化的影响

绿液的主要成分是Na2CO3和Na2S,可在硫酸盐浆厂碱回收系统中循环产生。将新疆棉秆在不同温度、硫化度和用碱量下经绿液预处理后,在不同酶用量下进行酶水解,并测定酶水解液中总糖得率以评价预处理效果。结果表明,原料预处理得率随用碱量的增加而下降。适当地提高蒸煮温度和用碱量,可以增加木质素脱除量,有利于后续酶水解的进行,但过于剧烈的预处理条件将导致碳水化合物的过度降解,因而降低酶水解糖的转化率。另外,由于新疆棉秆细小,棉秆皮占全秆比例较大,原料总糖含量较低,导致最终酶水解总糖得率偏低。在温度120?C,用碱量16%,硫化度32%条件下绿液预处理,酶水解糖转化率达到最大值,当酶水解酶用量为20 FPU/g(对浆料)时,葡聚糖、木聚糖和总糖的转化率分别为74.0%、61.3%和68.5%。     

皇竹草稀硫酸预处理研究

研究了中低温稀酸预处理对皇竹草化学组成变化、纤维素酶水解得率与总糖得率的影响,并采用扫描电镜(SEM)对皇竹草纤维结构变化进行了分析.结果表明,随着硫酸浓度的增大、温度的升高和时间的延长,半纤维素含量大幅度降低,且预处理后纤维素酶水解得率也逐渐增大.较好的预处理条件为100 g皇竹草原料,在固液比1:5(g:mL)条件下,用质量分数4.0%硫酸在温度110 ℃下,经过8 h预处理后,纤维素保留率为87.48%,半纤维素水解率为93.68%,所得固体渣经纤维素酶水解72 h后得率为86.3%(纤维素酶用量40 FPIU/g,以纤维素质量计),100 g原料可得到总糖量为54.53 g.预处理后皇竹草纤维表面和细胞壁受到破坏,表面积增大,有利于纤维素酶水解作用的进行.     

Pilot‐plant production of xylo‐oligosaccharides from corncob by steaming,enzymatic hydrolysis and nanofiltration

This paper reports a pilot‐plant production process for xylo‐oligosaccharides (mainly xylobiose and xylotriose) from corncob meal by steaming treatment followed by enzymatic hydrolysis and nanofiltration. The effects of corncob meal pretreatment, steaming temperature and time were investigated in order to obtain maximum extraction of xylan and to minimize the autohydrolysis of xylan into xylose. The enzymatic reaction was carried out using Aspergillus niger AN‐1.15 endo‐xylanase at 55 °C and the optimum enzymatic hydrolysis time was 5 h. The conventional downstream processing for purification of xylo‐oligosaccharides was incorporated with nanofiltration technology, giving benefits of energy saving and removal of monosaccharides. The final product from 40 kg of corncob meal was 10 dm³ of xylo‐oligosaccharide syrup (800 g dm^?3 total sugar), containing 74.5% xylobiose and xylotriose. Copyright © 2004 Society of Chemical Industry     

稀碱-Fenton试剂预处理对云南苦竹酶水解得率的影响

利用响应面法对稀碱-Fenton反应预处理竹粉的条件进行优化,确定最佳的Fenton预处理条件为:1 g 稀碱预处理后竹粉底物加入质量分数30 %的 H₂O₂ 溶液3.4 mL,Fe²⁺浓度15.8 mmol/L,反应时间12 h,获得的 72 h 酶水解得率为49.98%。与原料和经2%NaOH 预处理后的样品相比,经2%NaOH-Fenton 预处理后的样品中纤维素含量升高,半纤维素和木质素含量降低,72 h酶水解得率为48.24%,分别提高了47.79和37.44个百分点。当纤维素酶和β-葡萄糖苷酶的用量分别为32 FPIU/g和16 IU/g(以纤维素质量计)时,72 h 酶水解得率为76.64%,比单独使用纤维素酶时的酶水解得率提高了22.80%。     

碳酸钠―蒸汽爆破预处理对麦草化学成分及酶水解效率的影响

研究了碳酸钠―蒸汽爆破预处理对麦草化学成分及后续酶水解的影响。结果表明,预处理麦草浆料中木质素含量随预处理中碳酸钠用量的增大而下降,木聚糖和阿拉伯聚糖含量随碳酸钠用量的增大而上升。当碳酸钠用量增加到8%以后,继续增加碳酸钠用量,预处理麦草浆料中的葡聚糖、木聚糖、阿拉伯聚糖和木质素含量基本保持稳定。酶用量较低时,不添加碳酸钠的预处理麦草浆料酶水解葡聚糖得率最大。除不添加碳酸钠的预处理外,酶水解葡聚糖和总糖得率随木质素含量下降而提高。提高酶用量后木质素含量对预处理麦草浆料酶水解葡聚糖得率的影响变缓。     

Enzymatic hydrolysis from carbohydrates of barley straw pretreated by ionic liquids

BACKGROUND: Lignocellulosic biomass offers many potential advantages in comparison with the traditionally used sugars or starchy biomass since it is very widely available and does not compete with food and feed production. The abundance and high carbohydrates content of barley straw make it a good candidate for bioethanol production in Europe. Since biomass must be pretreated before enzymatic hydrolysis to improve the digestibility of both the cellulose and the hemicellulose biomass, the use of ionic liquids (IL) has been proposed as an environment‐friendly pretreatment of biomass. RESULTS: Different pretreatment conditions were investigated to determine the effects of the experimental conditions (temperature and time) on the enzymatic digestibility of pretreated material. The pretreatment of barley straw with 1‐ethyl‐3‐methyl imidazolium acetate treatment resulted in up to a 9‐fold increase in the cellulose conversion and a 13‐fold increase in the xylan conversion when compared with the untreated barley straw. CONCLUSION: Ionic liquid pretreatment of barley straw at 110°C for 30 min, followed by enzymatic hydrolysis, leads to a sugar yield of 53.5 g per 100 g raw material. It is then ready available for conversion into ethanol and is equivalent to more than 86% from potential sugars. The increase in saccharification was possible due to rupture of the lignin–hemicellulose linkages by treatment with 1‐ethyl‐3‐methyl imidazolium acetate. © 2012 Society of Chemical Industry     

Wet oxidation pretreatment of lignocellulosic residues of sugarcane,rice, cassava and peanuts for ethanol production

Wet oxidation (WO) pretreatment of sugarcane bagasse, rice hulls, cassava stalks and peanut shells was investigated. WO was performed at 195 °C for 10 min, with 2 g kg^?1 of Na₂CO₃ and under either 3 or 12 bar of oxygen. Oxygen pressure and the type of raw material used had a major effect on the fractionation of the materials, formation of sugars and by‐products, and cellulose enzymatic convertibility. Cellulose content in the solid fraction increased after pretreatment of all materials, except rice hulls. The greatest increase, from 361 g kg^?1 to almost 600 g kg^?1, occurred for bagasse. The solubilisation of individual components was different for each material. Bagasse xylan was solubilised to a large extent, and 45.2% of it was recovered as xylose and xylo‐oligosaccharides in the liquid fraction. In the prehydrolysates of rice hulls around 40% of the original glucan was recovered as gluco‐oligosaccharides, due to hydrolysis of starch contained in grain remains. The formation of by‐products was modest for all the materials, but increased with increasing oxygen pressure. The highest yield of acetic acid (34–36 g kg^?1 of raw material) and furfural (0.7–1.8 g kg^?1) occurred for bagasse. The pretreatment enhanced the enzymatic convertibility of cellulose giving the best result (670.2 g kg^?1) for bagasse pretreated at the highest oxygen pressure. However, for the other materials the pretreatment conditions were not effective in achieving cellulose conversions above 450 g kg^?1. Some enzymatic conversion of xylan was observed. Copyright © 2007 Society of Chemical Industry