Lignin Interaction with Cellulase during Enzymatic Hydrolysis

The conversion of lignocellulosic biomass into biofuels or biochemicals typically involves a pretreatment process followed by the enzyme-catalyzed hydrolysis of cellulose and hemicellulose components to fermentable sugars. Many factors can contribute to the recalcitrance of biomass, e.g., the lignin content and structure, crystallinity of cellulose, degree of fiber polymerization, and hemicellulose content, among others. However, nonproductive binding between cellulase and lignin is the factor with the greatest impact on enzymatic hydrolysis. To reduce the nonproductive adsorption of enzymes on lignin and improve the efficiency of enzymatic hydrolysis, this review comprehensively summarized the progress that has been made in understanding the interactions between lignin and enzymes. Firstly, the effects of pretreatment techniques on lignin content and enzymatic hydrolysis were reviewed. The effects of lignin content and functional groups on enzymatic hydrolysis were then summarized. Methods for the preparation and characterization of lignin films were assessed. Finally, the methods applied to characterize the interactions between lignin and cellulase were reviewed, and methods for decreasing the nonproductive binding of enzymes to lignin were discussed. This review provides an overview of the current understanding of how lignin hinders the enzymatic hydrolysis of lignocellulosic biomass, and provides a theoretical basis for the development of more economical and effective methods and additives to reduce the interaction of lignin and enzymes to improve the efficiency of enzymatic hydrolysis.     

高温蒸煮协同纤维素酶改性竹笋膳食纤维

以竹笋膳食纤维(bamboo shoot dietary fiber,BSDF)为研究对象,分别采用纤维素酶酶解(ET)、高温蒸煮(HT)、高温蒸煮协同纤维素酶(ET-HT)处理BSDF,分析其结构和理化性质(持水力、膨胀力、持油力、色泽)的变化。结果表明,改性后BSDF的粒径均显著减小(P <0. 05),ET-HT40组BSDF的粒径((423±23. 7) nm)最小,改性处理后的BSDF的电位均显著下降(P <0. 05)。ET-HT处理后BSDF呈片状结构,ET-HT组BSDF的L*值(54. 26±0. 64)和b*值(18. 41±0. 29)最小,a*值(9. 63±0. 17)最大。ET-HT20组BSDF的持水力((5. 29±0. 17) g/g)和膨胀力((13. 22±0. 12) mL/g)最大,ET-HT40组BSDF的持油力((8. 35±0. 03) g/g)最大。热重分析表明ET-HT处理BSDF的热稳定性最强。红外光谱表明ET、HT和ET-HT改性后BSDF的主要官能团结构未发生改变。综上,ET-HT较单独ET和HT更有效地改善了BSDF的理化性质,是提升BSDF品质的有效方式。     

浏阳豆豉发酵中高产酶活菌株的分离鉴定及酶活性分析

从浏阳豆豉发酵过程中分离产高酶活菌株，通过形态观察结合分子生物学技术进行鉴定，并对其产蛋白酶、脂肪酶及纤维素酶的活性进行分析。结果表明，分离得到3株菌（编号为000、5132、621）均被鉴定为溜曲霉菌（Aspergillus tamarri）。3株菌的蛋白酶、脂肪酶及纤维素酶的活性测定结果表明，菌株621蛋白酶活性最强，为（207.98±3.20）U/mL；菌株5132的纤维素酶活性最强，为（3.40±1.40）U/mL；菌株000的脂肪酶活性最高，为（90.7±0.64）U/mL。     

N~＋注入诱变选育纤维素酶高产菌株及发酵产酶营养因子优化研究

应用低能氮离子（N＋）注入技术对纤维素酶产生菌里氏木霉（Trichoderma reesei）进行诱变选育,在能量为10 keV,注量为150×10^14和200×10^14N＋/cm^2的条件下分别筛选得到3株纤维素酶高产菌株,连续5代遗传稳定性实验结果表明,所得到的高产菌株遗传稳定性较好,羧甲基纤维素酶活力均提高到3.300 IU/mL以上,较出发菌株（2.698 IU/mL）提高了20.0%以上。采用Plackett-Burman实验设计法和旋转中心组合设计法系统地研究高产菌株150-1-1发酵营养因子组成,得到了纤维素酶产量随葡萄糖、麸皮和微晶纤维素等营养因子的变化规律及相应的响应面分析图。实验结果表明,葡萄糖、麸皮和微晶纤维素浓度与纤维素酶活存在显著的相关性,当葡萄糖浓度为4.9 g/L,麸皮浓度为23.0 g/L,微晶纤维素浓度为7.7 g/L时,150-1-1纤维素酶滤纸酶活力达到2.439 IU/mL,较优化前（2.000 IU/mL）提高了22.0%。     

高产纤维素酶突变株的筛选及其产酶条件优化

通过常压室温等离子体技术诱变里氏木霉RUT-C30,筛选高产纤维素酶突变株,并对其产酶进行优化,提高纤维素酶的产量。筛选得到高产纤维素酶突变株后,进行全基因组测序分析突变型,并对产酶培养基和培养条件进行优化。结果表明:经过筛选获得高产纤维素酶突变株JNDY-13,其摇瓶发酵最高滤纸酶活可达2.21 IU/mL,为出发菌株的2.21倍,优化后JNDY-13在5 L罐中流加发酵所产最高滤纸酶活为5.40 IU/mL;测序结果显示JNDY-13基因组中共有752个突变发生,其中半乳糖激酶基因中被插入的18个碱基可能是突变株纤维素酶活力增加的原因。     

Effect of LHW,HCl, and NaOH pretreatment on enzymatic hydrolysis of sugarcane bagasse: sugar recovery and fractal-like kinetics

Conversion of lignocellulose to sugars involves two main processes, namely pretreatment and enzymatic hydrolysis. Lignocellulose pretreatment leads to the degradation of enzymatic recalcitrance of substrate for achieving efficient saccharification. In this study, liquid hot water (LHW), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were as reagents used to pretreat sugarcane bagasse (SB). Results showed that LHW, HCl, and NaOH pretreatment could solubilize 95.3%, 94.7% xylan and 88.7% lignin, respectively. Enzymatic hydrolysis of pretreated SB showed that the maximum glucose (26.0?g/L) and xylose (12.7?g/L) concentration were produced by NaOH pretreatment, and slightly more glucose and less xylose were produced after HCl pretreatment compared to LHW pretreatment. Addition of Tween 80 or xylanase could significantly improve both glucose and xylose production. At 48?h, the glucose increase for LHW, HC1 and NaOH pretreatment was 38.3%, 26.4% and 8.0%, respectively, and the xylose increase for them was 35.0%, 24.9% and 1.7%, respectively. Fractal-like kinetics showed that the value of rate constant increased after the addition of Tween 80 or xylanase, and the efficiency of enzymatic hydrolysis mainly depended on rate constant other than fractal dimension of substrate. Totally, substrate accessibility was dominated for efficient of lignocellulose to sugar compared to enzyme loading. The application of fractal-like theory on the heterogeneous enzymatic hydrolysis of lignocellulose was quite successful.     

枯草芽孢杆菌纤维素酶基因整合载体的构建

目的:以枯草芽孢杆菌(Bacillus subtilis)为宿主,构建纤维素酶基因整合表达载体,获得能够表达纤维素酶并且降解纤维素的工程菌。方法:通过聚合酶链式反应(polymerase chain reaction,PCR)从B.subtilis LN基因组中克隆同源片段M1、M2基因片段,以质粒pGEM-T为载体,将同源片段M1、M2、启动子P43和葡萄糖苷酶基因CelKg连接在一起构建整合载体pGEM-Kmpgmt,并采用双交换同源重组的方式将其转化进入B.subtilis LN基因组中。结果:通过PCR和双酶切验证整合载体构建完成,并成功整合到野生型B.subtilis LN中,获得重组菌B.subtilis Kpg。刚果红染色结果显示重组菌对羧甲基纤维素钠有降解作用。改良培养基37℃条件下摇瓶培养,重组菌B.subtilis Kpg生长至18 h时上清液中纤维素酶活力比野生型B.subtilis LN提高了115%。     

酶预处理对纤维打浆性能的影响

采用纤维素酶对漂白阔叶木浆进行预处理,研究了酶预处理工艺对纤维形态和打浆能耗的影响,并进一步分析浆料通过PFI磨打浆后的纤维形态变化,为酶预处理漂白阔叶木浆制备纤维素微纤丝(CMF)提供理论指导。结果表明,酶预处理并没有明显改变纤维形态,但经PFI磨打浆后的纤维更易被切断和分丝帚化,纤维润胀程度得以提高,且当酶用量8 U/g,打浆度达到50°SR和68°SR时,浆料的扭结纤维含量相比未经酶预处理的对照样分别减少了17. 2个百分点和16. 2个百分点,细小纤维含量分别增加了20. 8个百分点和17. 6个百分点;此外,酶预处理能显著降低磨浆能耗。当酶用量8 U/g时,打浆度达到50°SR和68°SR时,打浆能耗相比未添加酶的对照样分别节省了50%和33. 3%。     

蒲公英总黄酮复合酶酶法提取工艺及抗氧化活性研究

在考察加酶量、pH值、料液比、乙醇浓度、酶解温度和酶解时间对蒲公英总黄酮得率影响的基础上,进行四因素三水平响应面试验,优化得到蒲公英总黄酮复合酶酶法的最佳提取条件为料液比122(g/mL)、pH 4.5、乙醇浓度52%、酶解温度51℃,该条件下蒲公英总黄酮的得率为15.09mg/g。体外功能性试验显示,蒲公英总黄酮提取液的抗氧化活性及对自由基的清除能力均较强。     

Technological Implications of Modifying the Extent of Cell Wall-Proanthocyanidin Interactions Using Enzymes

The transference and reactivity of proanthocyanidins is an important issue that affects the technological processing of some fruits, such as grapes and apples. These processes are affected by proanthocyanidins bound to cell wall polysaccharides, which are present in high concentrations during the processing of the fruits. Therefore, the effective extraction of proanthocyanidins from fruits to their juices or derived products will depend on the ability to manage these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the role of pure hydrolytic enzymes (polygalacturonase and cellulose) and a commercial enzyme containing these two activities on the extent of proanthocyanidin-cell wall interactions. The results showed that the modification promoted by enzymes reduced the amount of proanthocyanidins adsorbed to cell walls since they contributed to the degradation and release of the cell wall polysaccharides, which diffused into the model solution. Some of these released polysaccharides also presented some reactivity towards the proanthocyanidins present in a model solution.