Enzymatic hydrolysis and simultaneous saccharification and fermentation of

山西农业科学 2023，51（12）：1426-1434Journal of Shanxi Agricultural SciencesCFS 预处理对不同秸秆原料酶解和理化结构的影响田鑫，王雨萌，徐师苗，汪强杰，胡轲，张海波，程红艳（山西农业大学 资源环境学院，山西 太谷 030801）摘要：高铁酸钾复合液（CFS ）是制备高铁酸钾的剩余滤液，其含有大量碱（OH -）和氧化剂（ClO -和Fe 6+），具有破坏木质纤维素顽固结构、提升酶解效率的潜力。

为实现秸秆的资源化利用与高铁酸钾制备废液的再利用，以山西储量丰富的玉米秸秆（CS ）、高粱秸秆（SS ）和谷子秸秆（MS ）为原料，采用CFS 进行预处理，对比3种秸秆的酶解糖化率，分析秸秆的理化结构变化。

结果表明，CFS 预处理中碱和氧化剂共同参与了3种秸秆的降解，促进了酶解糖化率；在最佳预处理时间24 h 下，CS 、SS 和MS 的还原糖产量分别较对照提高252.77%、236.39%、216.66%，其中CS 的酶解效率最高；组分分析表明，CFS 处理能有效去除3种秸秆中木质素成分，增加纤维素相对含量，进而有利于纤维素酶的可及性；结构分析显示，CFS 处理后，3种秸秆的理化结构发生了不同程度变化，粗糙度增加，官能团发生断裂，纤维结晶度升高，热稳定性变差。

在3种秸秆中，CS 结构变化最明显，更有利于被生物转化。

综上，CFS 预处理可改变作物秸秆的理化结构，破坏其致密结构，促进后续酶解效率，是一种理想的预处理技术。

关键词：高铁酸钾复合液（CFS ）；预处理；作物秸秆；还原糖产量；理化结构中图分类号：S141.4 文献标识码：A 文章编号：1002‒2481（2023）12‒1426‒09Effects of CFS Pretreatment on Enzymatic Hydrolysis and PhysicochemicalStructure of Different Straw MaterialsTIAN Xin ，WANG Yumeng ，XU Shimiao ，WANG Qiangjie ，HU Ke ，ZHANG Haibo ，CHENG Hongyan（College of Resources and Environment ，Shanxi Agricultural University ，Taigu 030801，China ）Abstract ： Composite ferrate solution(CFS) is the residual filtrate for preparing potassium ferrate. It contains a lot of alkali (OH -) and oxidant(ClO - and Fe 6+), which has the potential to destroy the recalcitrant structure of lignocellulose and improve the efficiency of enzymatic hydrolysis. In order to realize the utilization of straw resources and reuse of preparation waste liquid of potassium ferrate, in this paper, corn straw(CS), sorghum straw(SS), and millet straw(MS), which are abundant in Shanxi province, were pretreated with CFS, the enzymolysis and saccharification rates of the three kinds of straw were compared, and the change of physicochemical structure of the straw was analyzed. The results showed that the alkali and oxidant in the pretreatment of CFS were involved in the degradation of three kinds of straw, which promoted the enzymatic hydrolysis rate and saccharification rate. Under the optimal pretreatment time of 24 h, the reducing sugar yield of CS, SS, and MS was increased by 252.77%, 236.39%, and 216.66% compared with that of the control, respectively, and the enzymatic hydrolysis efficiency of CS was the highest. Component analysis showed that CFS treatment could effectively remove lignin in three kinds of straw and increase the relative content of cellulose, which was beneficial to the accessibility of cellulase. Structural analysis showed that after CFS treatment, the physicochemical structure of the three kinds of straw changed in different degrees, roughness increased, functional group fractured, fiber crystallinity increased, and thermal stability decreased. Among the three kinds of straw, CS had the most obvious structural change and was more conducive to biotransformation. In conclusion, CFS pretreatment could change the physicochemical structure of crop straws, destroy the dense structure and promote the efficiency of subsequent enzymatic hydrolysis, so it was an ideal pretreatment technology.Key words ：composite ferrate solution(CFS); pretreatment; crop straw; reducing sugar yield; physicochemical structuredoidoi:10.3969/j.issn.1002-2481.2023.12.11收稿日期：2023-01-04基金项目：山西省高等学校科技创新项目（2020L0137）；山西农业大学科技创新基金项目 （2018YJ39）；山西省优秀博士来晋工作奖励基金（SXYBKY201803）；国家自然科学基金（52100149）；山西省水利科学技术研究与推广项目（2022GM034）作者简介：田　鑫（1997-），女，山西汾阳人，在读硕士，研究方向：农业环境保护与废弃物资源化利用。

生物质能工业我国能源短缺，随着经济的迅速发展和对环保标准要求的逐步提高，迫切需要开发新的、清洁的可替代能源。

在众多可能替代化石燃料的能源中，生物质以其可再生、产量巨大、可储存等优点而引人注目。

而且生物质能是唯一一种可以转换为清洁燃料的可再生能源，其利用技术和化石燃料的利用方式具有很大的兼容性，因此以生物质作为原料不但可以弥补化石燃料的不足，缓解过分依赖大量进口石油的被动局面，实现我国能源安全战略，而且达到保护生态环境的目的。

对于我国这样一个幅员辽阔的农业大国来说，单就农作物秸秆而言，年产量高达7亿多吨，相当于3.5亿吨标准煤。

但目前，如此巨大的秸秆资源非但没有得到有效利用，反而由于就地焚烧已成为我国一大社会公害。

因此，在我国开发利用秸秆生产燃料乙醇和裂解油既具有现实意义，又可推动我国甚至世界范围内以秸秆等农作物废弃物为代表的生物质生产液体燃料更上层楼。

虽然秸秆和木材同属于木质纤维素，都有纤维素、半纤维素和木质素组成（4：3：3），然而在结构和化学组成却有较大的差异，因而秸秆和木材的转化特性不相同。

在秸秆中各种组分的转化特性也不同的，其转化反应特性和转化产品也随着秸秆组分结构的不同而变化。

例如，秸秆生物转化过程主要利用的是秸秆中的纤维素，对木质素和半纤维素生物转化效率低，难于适应工业化的要求。

而秸秆快速热解得到的液体产物中含有大量的酸类（如乙酸）产品，木材热解则以醇类和酮类产品为主。

这表明，秸秆中纤维素、半纤维素和灰分影响了热解过程产生液体产物的品位。

为解决在秸秆转化过程中采用单一的生物或热转化方式存在的问题，应将生物转化技术和快速热解技术有机结合起来，避免在秸秆原料转化液体燃料研究上，套用或沿用木材的技术，传统的生物转化、热化学转化过程把秸秆作为性质“单一组分”的原料，致使其转化的技术经济关久攻不破，因此，为秸秆高效转化的根本出路在于其生物量的全利用，新的高效转化过程应该建立在秸秆组分分离后的分级定向转化以及转化过程间的集成优化原则之上。

新能源的发现英语作文Title: Discoveries in New Energy Sources。

In the quest for sustainable energy solutions, scientists and researchers worldwide have been tirelessly exploring new avenues to harness clean and renewable energy sources. This pursuit has led to remarkable discoveries and innovations that hold the potential to revolutionize the way we power our world. In this essay, we will delve into some of the most significant advancements in the field of new energy sources and their implications for the future.One groundbreaking discovery in recent years is the development of perovskite solar cells. Perovskite materials have emerged as a promising alternative to traditional silicon-based solar cells due to their high efficiency and low production costs. Researchers have achieved impressive progress in improving the performance and stability of perovskite solar cells, paving the way for their commercialization on a large scale. These advancements holdthe promise of making solar energy more accessible and affordable for communities around the globe.Another area of intense exploration is the field of hydrogen energy. Hydrogen has long been recognized as a clean and abundant fuel source, but technologicalchallenges have hindered its widespread adoption. However, recent breakthroughs in hydrogen production and storage technologies have reignited interest in this versatile energy carrier. Innovations such as electrolysis, photoelectrochemical water splitting, and solid-state hydrogen storage materials have made significant strides in making hydrogen a viable option for powering transportation, industry, and residential applications. The development of hydrogen fuel cells, which convert hydrogen gas into electricity with zero emissions, holds particular promisefor decarbonizing sectors such as transportation and manufacturing.Furthermore, the exploration of advanced battery technologies has opened up new possibilities for energy storage and grid integration. Lithium-ion batteries havedominated the market for portable electronics and electric vehicles, but concerns about their limited capacity, safety issues, and reliance on scarce resources have spurredefforts to develop alternative battery chemistries. Researchers are investigating various options, including solid-state batteries, sodium-ion batteries, and flow batteries, which offer higher energy density, faster charging capabilities, and improved safety profiles. These next-generation batteries have the potential torevolutionize energy storage, enabling greater integrationof renewable energy sources into the grid and facilitating the transition to a more sustainable energy system.In addition to these technological innovations, thereis growing interest in harnessing the power of bioenergy as a renewable energy source. Bioenergy encompasses a range of technologies that utilize organic materials such as biomass, biogas, and biofuels to generate heat, electricity, and transportation fuels. Advances in biofuel production processes, such as enzymatic hydrolysis and microbial fermentation, have made it possible to convert a wide range of feedstocks, including agricultural residues, algae, andmunicipal solid waste, into biofuels with high energy content and low carbon emissions. Bioenergy offers the dual benefits of reducing greenhouse gas emissions and providing an alternative revenue stream for farmers and rural communities.In conclusion, the discovery and development of new energy sources represent a critical step towards achieving a sustainable and resilient energy future. From perovskite solar cells to hydrogen fuel cells to advanced battery technologies, these innovations hold the promise of powering our world while mitigating the impacts of climate change. As we continue to invest in research and development in this field, it is essential to prioritize collaboration, innovation, and investment to accelerate the transition to a clean energy economy. By harnessing the power of science and technology, we can build a brighter and more sustainable future for generations to come.。

利用一株凝结芽孢杆菌发酵酸解玉米秸秆生产乳酸倪志华;张玉明【摘要】该研究拟考察凝结芽孢杆菌(B acilluscoagulans)利用玉米秸秆生物炼制乳酸的效果.以凝结芽孢杆菌C G M C C N o.7635为菌种,利用2％硫酸预处理后的玉米秸秆为碳源、20 g/L酵母粉为氮源,添加20 FPU/g纤维素酶后开展糖化发酵生产乳酸实验.结果表明,发酵65 h后可获得乳酸含量为(38.38±1.03)g/L,其中L-乳酸光学纯度为(99.23±0.22)％.进一步使用补料发酵工艺,添加经预处理的玉米秸秆,可最终获得乳酸含量为(82.56±1.28)g/L.建立的玉米秸秆生物炼制乳酸工艺操作简单、产物浓度高,具有工业应用潜力.【期刊名称】《中国酿造》【年(卷),期】2019(038)007【总页数】4页(P44-47)【关键词】乳酸;玉米秸秆;生物炼制;木质纤维素;凝结芽孢杆菌【作者】倪志华;张玉明【作者单位】河北大学生命科学学院,河北保定 071002;河北大学生命科学学院,河北保定 071002【正文语种】中文【中图分类】TQ921.3乳酸是一种重要的有机酸，以乳酸为单体合成的聚乳酸生物塑料被认为是石化塑料的最佳替代品之一，利用可再生生物质生产乳酸可实现资源绿色循环应用[1]。

我国是农业大国，玉米秸秆资源丰富，利用玉米秸秆生物炼制乳酸，不但可以降低原料成本、实现农业废弃物绿色利用，而且可以解决秸秆丢弃、焚烧造成的环境污染问题[2-3]。

微生物是生物炼制的主要参与者，根霉菌和乳酸细菌是研究最多的乳酸生产菌。

刘占英等[4]对一株可直接降解纤维素生产乳酸的粪肠球菌（Enterococcus faecalis）进行紫外诱变育种，该菌株可利用玉米秸秆获得0.63 g/L乳酸。

李鑫等[5]以米根霉（Rhizopus oryzae）为菌种，生物转化经汽爆和碱处理后玉米秸秆酶解液（含有葡萄糖100 g/L），最终获得69.15 g/L乳酸。

液氨预浸预处理甜菜渣提高纤维素酶解率-生物工程专业毕业论文剂降解了48．95％，纤维素含量提高了85．96％，破坏半纤维素和木质素之间的连接键。

3、液氨预浸预处理使得甜菜渣孔隙度增大，表面致密结构受到破坏，酶解的有效比表面积增大，增加了纤维素酶与底物的酶解可及性；无定形纤维素受到破坏，结晶度升高，纤维素结晶区暴露。

4、液氨预浸预处理破坏甜菜渣细胞壁，使得细胞间隙扩大，细胞大小发生改变；相比未处理甜菜渣，细胞壁所产生的蓝色自发荧光均减弱，表明显著去除木质素和半纤维素；破坏纤维结构，纤维表面变得粗糙并出现大量缝隙。

关键词：甜菜渣；液氨预浸；酶解率；化学组成；物理结构；微观形态III万方数据Enhanced Enzymatic Hydrolysis of SugarBeet Pulp by Aqueous Ammonia PretreatmentABSTRACTCellulosic ethanol，with the advantages of renewable resource，‘cleanliness and safety，is the mainstream direction of new energy development and has been obtained extensive attention from countries and enterprises around the world，Sugar beet pulp，byproducts derived from sugar beet industry，is deemed as the potential feedstocks for the cellulosic ethanol production．However the low enzymatic hydrolysis of cellulose severely restricted the development of cellulosic ethanol production．Hencevarious pretreatments were established and used in ethanol production．In this study，aqueous ammonia pretreatment of sugar beet pulp at low temperature were established．The cellulose conversions of sugar beet pulp werethe chemical composition，physical analyzed．The effects of the pretreatrnent onstructure，and cell wall morphology were discussed．The effects hydrochloric acid pretreatment，aqueous ammonia pretreatment, ammonium oxalate pretreatment and pectinase pretreatment on cellulose conversions of sugar beet pulp were compared．The optimal process of aqueous ammonia pretreatment of sugar beet pulp at low temperature was established．The effects of万方数据the pretreatment on the chemical composition，physical structure were discussed．And the changes of cell wall morphology were characterized by fluorescence microscopy，scanning electron microscopy，confocal laser scanning microscope in different level．The main conclusions ale as follows：1．Aqueous ammonia pretreatment at low temperature was established．The cellulose conversion of sugar beet pulp could reach to 63．72％，which was 82．47％higher than that of raw material，under pretreatment condition of 1 0％aqueous ammonia，at 80。

摘要安徽省拥有丰富的竹笋资源。

竹笋作为我国一种传统食材，含有多种营养物质和活性物质，并具有较高的食用价值和保健功能。

为充分利用竹笋资源，本论文对三种竹笋的基本成分进行测定，研究了竹笋壳中多酚类物质的提取工艺，竹笋膳食纤维的制备工艺，同时探讨了三种竹笋食品的加工和检测方法。

为进一步开发竹笋资源提供了一定的理论依据。

试验测定了雷竹笋、燕竹笋、毛竹笋三种竹笋中基本成分的含量：含水率约为84.24%-91.36%，灰分含量约为5.93%-10.35%，粗纤维含量约为1.27%-3.37%，Vc含量约为5.8mg/g-9.2mg/g,，总糖含量（干样，以下同）约为10.8%-22.5%，还原糖含量约为8.53%-10.5%，蛋白质含量约为 1.1%-3.78%，粗脂肪含量约为0.98%-1.89%，黄酮含量约为0.42%-0.91%。

以燕竹笋壳为原料，运用超声波辅助提取技术对其中多酚类物质进行提取，以其多酚提取率作为指标，考察超声功率、提取时间、乙醇浓度、料液比对多酚提取率的影响，并利用响应面法对燕竹笋壳多酚提取工艺进行分析，得到最佳工艺参数为：超声功率200W，提取时间40.2min，乙醇浓度42%，料液比1:50.5。

在此条件下，燕竹笋壳多酚的理论提取得率为 5.50%，实际平均提取得率为5.57%，理论预测值与实际值相对误差为1.27%。

以燕竹笋粉为原料分别采用化学法、酶解法、发酵法制备竹笋膳食纤维，以IDF和SDF的含量以及溶胀性、持水力等指标作为考察依据，得到结论：发酵法得到的膳食纤维中SDF的含量明显高于化学法和酶解法。

以燕竹笋为原料，采用霉菌发酵法制备膳食纤维，以其中SDF含量作为指标，考察接菌比例、发酵温度、发酵时间和发酵基质初始pH对SDF含量的影响，并利用正交分析法对膳食纤维制备工艺进行分析，得到最佳工艺参数为：接菌比例绿色木霉：黑曲霉=2:1，发酵基质pH为8，在28℃条件下震荡培养48h。

木质纤维素糖化发酵工艺研究进展前言目前，世界乙醇生产主要以淀粉类(粮食作物为主，如玉米、木薯等)和糖类（如甘蔗、甜菜等）[1-2]作为发酵原料．采用微生物法发酵生产乙醇技术成熟，但是高昂的原料成本使粮食发酵生产乙醇的工业应用受到限制，同时存在与人争粮或与粮争地等弊端，并且导致粮食价格持续走高，因此寻找新的原料势在必行．所以现在科学家把目光投向成本更为低廉、来源更广泛的木质纤维素原料[3]．它不仅包括秸秆等农业废弃物，城市固体废弃物、办公废纸、杂草、锯末等以及市政废水中的固体部分[4].地球上每年植物光合作用的生物量可达2 000亿 t，其中大部分为木质纤维素类．它的主要成分是纤维素、木质素、半纤维素．在植物组织中木质素与半纤维素以共价键形式结合，并将纤维素分子包埋其中，形成一种坚固的天然屏障，使一般微生物很难进入使其降解。

木质纤维素原料生产燃料乙醇的过程主要包括预处理、糖化、发酵等，其预处理是生物转化的关键步骤，影响整个纤维素酒精生产过程．因此高效、便捷的预处理技术是木质纤维素原料生产燃料乙醇的关键所在．一、分步糖化和发酵（SHF）前处理后的木质纤维素经水解糖化生成葡萄糖，然后在另一反应器中进行发酵转化为乙醇，这种糖化发酵工艺被称为分步糖化和发酵。

其主要优点是糖化和发酵都能在各自最优条件下进行——纤维素酶水解糖化所需的最适温度在 45～5℃，而大多数发酵产乙醇的微生物最适温度在 28～37 ℃[5]。

缺点是糖化产物葡萄糖和纤维二糖的积累会抑制纤维素酶的活力，最终导致产率的降低。

研究发现，纤维二糖的浓度达到 6 g/L 时，纤维素酶的活力就将降低 60%，葡萄糖对纤维素酶的抑制作用则没有那么明显，但是，它会对β-葡糖苷酶（一种关键的纤维素水解酶）产生强烈的抑制，葡萄糖浓度达到3 g/L时，β-葡糖苷酶的活力就将降低75%。

此外，水解用的纤维素酶（主要来自于真菌）不仅组分相对单一而且价格昂贵，当其活力受到抑制时，就得增加用量，最终导致使用成本的提高。

玉米蛋白酶解产物对a■淀粉酶活性抑制研究战旭梅，祁兴普,赖梦宇，刘萍(江苏农牧科技职业学院，江苏泰州225300)摘要：以玉米蛋白粉为原料，采用碱性蛋白酶、复合风味蛋白酶、胰蛋白酶对玉米蛋白进行酶解，研究不同酶解条件下得到的酶解产物对！淀粉酶活性的抑制作用&通过单因素和响应面方法对酶解条件进行优化。

结果表明：在玉米浓缩蛋白粉稀释料液比1：10(g/mL)、酶解时间3.0h、酶添加量4000U/g、pH8-0、酶解温度45.5C的条件下，酶解液对！-淀粉酶抑制率达到15.01%&关键词：玉米蛋白；酶解;响应面试验;!淀粉酶抑制率Stiidy on tlie inhinition of corr protein hydrolysates to a-amylast activity ZHAN Xu-met,QI Xing-pu,LAI Merg-yu$LIU Ping$(Jiangst Agri-animal Hustandry Vocational College,Taizhou225300,Jiangsu,China) Abstrad:C on protein meal was hydrolyzed by alkaline protease,tavourzyma and typsin,and the in­hibition of a-Cmylase activity by the hydrolysates under di/eynt conditions was studied-The conditions of enzymatic hydrolysis were optimized by single factor and response surface methods.The results showed that con protein concentrate at a dilui material-to-liquid ratio of1：10(gmL):the enzymatic hydrolysis iomewas3.0h,iheenyymeaddoioon was4000Ugg,pH8.30,and iheenyymaio hydeoeysosiempeeaC ture was45.5C-Under the conditions,the inhibition rate of the enzyme hydrolysate on a-Cmylase cached15.01%-Key wo S s：con protein;enzyme hydrolysis;response surface test;a-Cmylase inhibition rate中图分类号：TS201.2文献标志码：A文章编号：1008-9578(2021)05-0142-06玉米蛋白粉是玉米湿法生产淀粉的主要副产物之一，含有60%以上的蛋白质，约68%的蛋白质为醇溶蛋白，难溶于水［1］）利用酶法对玉米蛋白进行水解生产的玉米肽不仅具有高效性%安全性等特点'2（,并且具有纯天然%易被人体消化吸收、抗氧化、抗疲劳、促进乙醇代谢等优良特性［3］，可以开发成运动饮料、醒酒饮料等功能性食品。

生物技术进展 2023 年 第 13 卷 第 6 期 934 ~ 939Current Biotechnology ISSN 2095‑2341研究论文ArticlespH 渐变条件下多酶分步连续酶解工艺制备鲟鱼硫酸软骨素与胶原蛋白肽王珍瑜1 ， 陆文超1 ， 钟康荣2 ， 关永健2 ， 汪真2 ， 陈超1 *1.清华大学化学系，北京 100084；2.贵州千鲟生物科技有限公司，贵州 黔东南苗族侗族自治州 556300摘要：pH 渐变条件下采用碱性蛋白酶、木瓜蛋白酶和菠萝蛋白酶分步连续酶解鲟鱼软骨，同时制备鲟鱼硫酸软骨素和胶原蛋白肽。

分别通过温度、酶解时间、酶用量3个单因素实验，研究其对连续酶解效果的影响。

综合分析得出最佳条件：碱性蛋白酶酶解温度为45 ℃，酶解时间为2 h ，酶用量为100 U ·g -1底物；木瓜蛋白酶的酶解温度为50 ℃，酶解时间为2 h ，酶用量为50 U ·g -1底物；菠萝蛋白酶的酶解温度为50 ℃，酶解时间为2 h ，酶用量为30 U ·g -1底物。

该工艺条件下制备的鲟鱼硫酸软骨素提取率为85%，纯度93%，胶原蛋白肽的提取率为82%，纯度为90%。

研究建立的pH 渐变条件下多酶分步连续酶解工艺，产率与纯度较国内现有生产方法均有提高，且碱液使用量减少，避免了有机溶剂的引入，从而可以降低生产成本，减轻环境污染负担。

关键词：酶解工艺；硫酸软骨素；胶原蛋白肽DOI ：10.19586/j.2095­2341.2023.0119中图分类号：Q556， TQ925+.2 文献标志码：APreparation of Chondroitin Sulfate and Collagen Peptide of Sturgeon by Multi -enzyme Step -by -step Enzymatic Hydrolysis Under the Condition of pH GradientWANG Zhenyu 1 ， LU Wenchao 1 ， ZHONG Kangrong 2 ， GUAN Yongjian 2 ， WANG Zhen 2 ， CHEN Chao 1 *1.Department of Chemistry ， Tsinghua University ， Beijing 100084， China ；2.Guizhou Qiansturgeon Biotechnology Co.， Ltd ， Guizhou Qiandongnan Miao and Dong Autonomous Prefecture 556300， ChinaAbstract ：Alkaline protease ， papain and bromelain were used to hydrolyze cartilage of sturgeon step by step under pH gradient conditions to prepare sturgeon chondroitin sulfate and collagen peptide simultaneous. The effects of enzymatic hydrolysis tempera­ture ， enzymatic hydrolysis time ， and enzyme dosage on the efficiency of enzymatic hydrolysis were investigated. Based on com­prehensive analysis ， the optimal temperature for alkaline protease hydrolysis was 45 ℃， hydrolysis time was 2 hours ， and enzyme dosage was 100 U ·g -1 cartilage ； the optimal temperature for papain hydrolysis was 50 ℃， hydrolysis time was 2 hours ， and en­zyme dosage was 50 U ·g -1 cartilage ； the optimal temperature for bromelain hydrolysis was 50 ℃， hydrolysis time was 2 hours ， and enzyme dosage was 30 U ·g -1 cartilage. Sturgeon chondroitin sulfate was extracted at a rate of 85% with a purity of 93%， while collagen peptide was extracted at a rate of 82% with a purity of 90%. In comparison to the currently used domestic production techniques ， the multi -enzyme step -by -step enzymatic hydrolysis process established in this study under pH gradient conditions could improve yield and purity ， reduce the amount of alkali ， and avoid the introduction of organic solvents ， thereby lowering the production cost and lessening the burden of environmental pollution.Key words ：enzymatic hydrolysis process ； chondroitin sulfate ； collagen peptide收稿日期：2023­09­25； 接受日期：2023­10­24基金项目：清华大学国强研究院项目。

Toward an Aggregated Understanding of Enzymatic Hydrolysis of Cellulose:Noncomplexed Cellulase SystemsYi-Heng Percival Zhang,1Lee R.Lynd 1,21Thayer School of Engineering,Dartmouth College,Hanover,New Hampshire 03755;e-mail:percival.zhang @,Lee.R.Lynd @ 2Department of Biological Sciences,Dartmouth College,Hanover,New Hampshire 03755Received 2June 2004;accepted 29July 2004Published online 10November 2004in Wiley InterScience ().DOI:10.1002/bit.20282Abstract:Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase compo-nents.Topics considered include properties of cellulose,adsorption,cellulose hydrolysis,and quantitative models.A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables in-cluded.We suggest that it is timely to revisit and reinvig-orate functional modeling of cellulose hydrolysis,and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.B 2004Wiley Periodicals,Inc.Keywords:adsorption;cellulose;cellulase;hydrolysis;ki-netic modelINTRODUCTIONThe potential importance of cellulose hydrolysis in the con-text of conversion of plant biomass to fuels and chemicals is widely recognized (Lynd et al.,1991,1999;Himmel et al.,1999),and cellulose hydrolysis also represents one of the largest material flows in the global carbon cycle (Falkowski et al.,2000).The quantity of scientific information on components of cellulose-hydrolyzing enzyme system has expanded dramatically in recent years.Over the 12-year period from 1991to 2003,for example,the number of known glycosyl hydrolases gene sequences has increased from f 300to >10,000,and the number of cellulase crystal structures has increased from several to f 230(H.Henrissat,mun.).Also during this period,exten-sive structurally based classification schemes have been introduced for both catalytic and cellulose-binding mod-ules,and have led to new insights and hypotheses with respect to the evolution of cellulase systems (Henrissat,1991;Henrissat and Bairoch,1993,1996),updated fre-quently at http//:rs.mrs.fr/CAZY.In order for the large volume of available information on cellulase components to be brought to bear on the primary applications that motivate interest in cellulose hydrolysis,e.g.,conversion of renewably produced biomass to fuels and commodity chemicals,it is necessary to incorporate this information into an understanding of cellulase systems comprised of multiple components with distinct modes of action.The situation is further complicated because the action of cellulase enzyme systems is impacted by substrate properties in addition to concentration—such as degree of polymerization,crystallinity,accessible area,the presence of lignin—which depend on the particular substrate being investigated and change as the reaction proceeds.In the course of seeking an ‘‘aggregated’’understanding of enzy-matic hydrolysis of cellulose that incorporates informa-tion about cellulase components and substrate features in addition to concentration,quantitative models are tremen-dously valuable.Of particular importance,measured pa-rameters for cellulase components and substrates could in principle be incorporated into models used to predict the behavior of multicomponent cellulase enzyme parison of such predictions to experimental measure-ments is the most systematic and rigorous means available by which to test whether understanding of cellulase compo-nents and their interactions is sufficient to explain a given observation.In addition,once a quantitative model is vali-dated,it can be used to rapidly formulate new hypotheses of significance in both fundamental and applied contexts.B 2004Wiley Periodicals,Inc.Correspondence to:Y.-H.P.Zhang or L.R.LyndContract grant sponsors:Department of Energy and National Institute of Standards and TechnologyContract grant numbers:DE-FG02-02ER15350and 60NANB1D0064REVIEWThis article reviews available information on enzymatic hydrolysis by noncomplexed cellulase systems;that is,sys-tems based on components that act discretely rather than as stable complexes(Lamed et al.,1983;Tomme et al., 1995a).A considerable portion of this review is spent on the properties of cellulose in light of the central role such properties play in mechanistically based quantitative models of cellulose hydrolysis.In particular,the following section considers crystallinity,degree of polymerization, accessibility,preparation and properties of model sub-strates,and pretreated lignocellulosic materials.The section Cellulase Adsorption is devoted to adsorption leading to the formation of cellulose–cellulase complexes,including ad-sorption models,reversibility,and enzyme mobility,as well as inferred accessibility of cellulose from cellulase adsorp-tion.Thereafter,mechanistic understanding of cellulose hydrolysis by noncomplexed systems is addressed in Cellulose Hydrolysis,with attention given to concep-tual understanding of cellulose hydrolysis,features of the widely studied Trichoderma reesei cellulase system,docu-mentation and understanding of synergism among cellu-lase components,and a summary of current mechanistic understanding.The section Quantitative Models presents a classification scheme and summarizes features of mod-els reported in the literature.The final section offers con-cluding perspectives and outlines outstanding challenges associated with understanding and modeling noncomplexed cellulase systems.Since our primary focus is on the function of cellulases rather than their structure,we use the older, functionally defined nomenclature rather than the newer nomenclature based on amino-acid sequence and molecu-lar structure.CELLULOSEAll cellulose is produced biosynthetically.While cellulose production by photosynthetic higher plants and algae is thought to be by far the most important in terms of global carbon flows,cellulose production by nonphotosynthetic organisms(certain bacteria,marine invertebrates,fungi, slime molds and amoebae)has also been documented (Coughlan,1985;Jarvis,2003;Lynd et al.,2002;Tomme et al.,1995a).Cellulose is a linear condensation polymer consisting of D-anhydroglucopyranose joined together by h-1,4-glycosidic bonds.Anhydrocellobiose is the repeating unit of cellulose,since adjacent anhydroglucose mole-cules are rotated180j with respect to their neighbors (Fig.1a).This rotation causes cellulose to be highly sym-metrical,since each side of the chain has an equal number of hydroxyl groups.Coupling of adjacent cellulose mol-ecules by hydrogen bonds and van der Waal’s forces re-sults in a parallel alignment and a crystalline structure. The extensive hydrogen bonds of interchain(2per anhy-droglucopyranose)and intrachain(2f3per anhydrogluco-pyranose)produces straight,stable supramolecular fibers of great tensile strength(Gardner and Blackwell,1974a,b;Krassig,1993;Nevell and Zeronian,1985).In contrast, starch contains amylose and amylopectin connected by a-1,4and to some extent a-1,6glucosidic bonds,forming a tightly coiled helical structure maintained by interchain hydrogen bonds(Buleon et al.,1998;Calvert,1997).Na-tive cellulose,referred to as cellulose I,has two distinct crystallite forms,I a,which is dominant in bacterial and algal cellulose,and I h,which is dominant in higher plants (Atalla and Vanderhart,1984).Native cellulose(cellulose I) can be converted to other crystalline forms(II–IV)by var-ious treatments(Klein and Snodgrass,1993;Krassig,1993; O’Sullivan,1997).Cellulose exist as sheets of glucopyranose rings lying in a plane with successive sheets stacked on top of each other to form a three-dimensional particle.Because of this ar-rangement,the surface of a cellulose particle has distinct ‘‘faces’’that interact with the aqueous environment and cellulase enzymes.The six carbons in the glucopyranose ring and internal h-glucosidic bonds lie in the ab plane or ‘‘110’’face,whereas the ac plane or11¯0face consists of the edges of rings(see Fig.1b).Additional faces present reducing and nonreducing ends,respectively.The repeating unit of the110face is the cellobiose lattice,which mea-sures1.04nm along the axis of the cellulose molecule and 0.54nm in the perpendicular direction.About100cellu-lose glucans are aggregated into elementary fibrils with a crystalline width of4–5nm(O’Sullivan,1997),and bunches of elementary fibrils are embedded in a matrix of hemicellulose with a thickness of7–30nm.The lignifica-tion process occurs late in the process of synthesizing nat-ural fibers,so lignin is located primarily on the exterior of microfibrils where it covalently bonds to hemicellulose (Fig.1c;Klein and Snodgrass,1993).The relationship between structural features of cellu-lose and rates of enzymatic hydrolysis has been the subject of extensive study and several reviews(Converse,1993; Cowling and Kirk,1976;Lynd et al.,2002;Mansfield et al., 1999;McMillian,1994),but is still incompletely under-stood.Structural features of cellulose commonly considered as rate-impacting factors include crystallinity index,degree of polymerization,and accessible area.Crystallinity Index(CrI)Crystallinity has often been thought of as providing an indication of substrate reactivity,and is prominently fea-tured in the model of Wood(1975)as well as other models. The crystallinity of dried cellulose samples can be quan-titatively measured from the wide-range X-ray diffraction pattern(Krassig,1993).In the case of cellulose-I,the crys-tallinity index(CrI)is calculated using the formula:CrI¼1Àh am=h cr¼1Àh am=ðh totÀh amÞð1Þbased on the ratio of the height of crystalline cellulose in the002reflection at2u=22.5j(h cr)to the height of amorphous cellulose(h am),and h tot=h cr+h am.Cotton798BIOTECHNOLOGY AND BIOENGINEERING,VOL.88,NO.7,DECEMBER30,2004(Hoshino et al.,1997;Lee et al.,1982;Sinitsyn et al.,1991), bacterial cellulose from Aacetobacter xylinum(Boisset et al.,1999;Gilkes et al.,1992;Valjamae et al.,1999),and cellulose from the alga Valonia ventricosa(Boisset et al.,1999;Fierobe et al.,2002)provide examples of highly crystalline cellulose,while phosphoric acid swollen cellu-lose and ball-milled cellulose are regarded as amorphous cellulose(Hoshino et al.,1997;Lee et al.,1982;Ooshima Figure1.a:Structure of cellulose featuring repeating h1,4-linked anhydrocellobiose units.b:Cellulose I crystal.The axes of the repeating unit (cellobiose)are:a=0.817nm,b=1.04nm,and c=0.786nm.The faces of the glucopyranose rings are parallel to the ab plane(110face)of the crystal (Mosier et al.,1999).c:Organization of lignocellulose origanization into elementary fibrils and microfibrils(Klein and Snodgrass,1993).ZHANG AND LYND:NONCOMPLEXED CELLULASE SYSTEMS799et al.,1983).Common model substrates derived from bleached commercial wood pulps,such as Avicel(Wood and Bhat,1988;Wood,1988),filter paper(Henrissat et al., 1985),and Solka Floc(Bertrain and Dale,1985;Fan et al., 1980;Lee et al.,1982;Sinitsyn et al.,1991)are regarded as a blend of amorphous and crystalline forms(Gilkes et al., 1991).Typical values of CrI for various model cellulosic substrates are presented in Table I.The CrI value of cel-lulose increases after a period of water swelling due to re-crystallization(Fan et al.,1980;Lee et al.,1983;Fengel and Wegener,1984),and the variations in drying condition prior to measurement of CrI may cause differences between substrates arising from the method of substrate preparation rather than properties of the substrate per se(Lenze et al., 1990;Weimer et al.,1995).The presence of residual cells and proteins can also result in artifacts in the CrI assay (Converse,1993).Cellulose hydrolysis rates mediated by fungal cellulases are typically3–30times faster for amorphous cellulose as compared to high crystalline cellulose(Lynd et al.,2002; Table III).This observation led investigators in the1980s to postulate a model for cellulose structure consisting of amorphous and crystalline fractions(Fan et al.,1980,1981; Lee et al.,1983).If this hypothesis were correct,it would be expected that crystallinity should increase over the course of cellulose hydrolysis as a result of preferential reaction of amorphous cellulose(Betrabet and Paralikar, 1977;Ooshima et al.,1983).However,several studies have found that crystallinity does not increase during enzymatic hydrolysis(Lenze et al.,1990;Ohmine et al.,1983;Puls and Wood,1991;Schurz et al.,1985;Sinitsyn et al.,1989).Con-sidering both the uncertainty of methodologies for mea-suring CrI as well as conflicting results on the change of CrI during hydrolysis,it is difficult to conclude at this time that CrI is a key determinant of the rate of enzymatic hy-drolysis(Lynd et al.,2002;Mansfield et al.,1999). Future studies aimed at developing and applying im-proved methods would be useful to more definitively re-solve the role of CrI in impacting hydrolysis.In interpreting crystallinity data,and indeed data for all cellulose physical properties,care must be taken to distinguish correlation from cause and effect.For example,several treatments that decrease crystallinity also increase surface area,and it has been suggested that the increased hydrolysis rates observed with substrates arising from such treatments may be due to increasing adsorptive capacity rather than substrate reac-tivity(Caulfield and Moore,1974;Howell and Stuck,1975; Lee and Fan,1982).Comparing the hydrolysis rates on various sources of model cellulosic substrates,Fierobe et al. (2002)concluded that accessibility of cellulose is a more important factor than crystallinity index in determining the hydrolysis rate.Degree of PolymerizationThe degree of polymerization(DP)of cellulosic substrates determines the relative abundance of terminal and interior h-glucosidic bonds,and of substrates for exo-acting and endo-acting enzymes,respectively.DP may be defined in terms of the number average DP(DP N),weight average DP (DP W),or DP inferred from viscosity(DP V):DP N¼M nMW glu¼P N i M i P N i=MW gluð2ÞDP W¼M WMW glu¼P N i M2i P N i=MW gluð3ÞDP V¼M VMW glu¼P N i D P N i=MW gluð4Þwhere N i is the number of moles of a given fraction i having molar mass M i,M N is the number-average molecular weight,M w is the weight-average molecular weight,M V is the viscosity-average molecular weight,MW glu is the molecular weight of anhydroglucose(162g/mol),and D is viscosity.Measurement of DP begins with dissolution of cellulose using a technique that does not alter chain length. Several such methods appear satisfactory,including:1) metal complex solutions such as Cuam solution(Klemm et al.,1998)and cupriethylenediamine(Klemen-Leyer et al., 1992,1994,1996);2)forming cellulose derivatives by reacting with organic solvents(Ng and Zeikus,1980)or inorganic acids such as nitric acid(Whitaker,1957);and3) ionic solutions such as N,N-dimethylacetamide(DMAc)/ LiCl(Striegel,1997).After dissolution,DP N can be mea-sured by membrane or vapor pressure osmometry,cry-oscopy,ebullioscopy,determination of reducing end con-centration,or electron microscopy(Krassig,1993).DP W can be measured based on light scattering,sedimentation equilibrium,and X-ray small angle scattering,and DP V is measured based on viscosity.The viscosity of dissolved cellulose or cellulose derivatives has been found to equal:D¼K m M aþ1ið5ÞTable I.Summary of some physical properties of model cellulosicsubstrates.Substrate1CrI2SSA2(m2/g)DP N2F RE(%)Avicel0.5–0.6203000.33BC0.76–0.9520020000.05PASC0–0.04240100 1.0Cotton0.81–0.95na.1000–30000.1–0.033Filter Paper–0.45na.7500.13Wood pulp0.5–0.761–55500–15000.06–0.21BC,bacterial cellulose;PASC,phosphoric acid swollen cellulose;CrIdenotes crystallinity index;SSA denotes specific surface area by BET;DP N denotes the number-average degree of polymerization;F RE denotesthe fraction of reducing ends.2References in text.800BIOTECHNOLOGY AND BIOENGINEERING,VOL.88,NO.7,DECEMBER30,2004in which K m =constant,with the value of a for cellulose and cellulose derivatives in most cases ranging from 0.75to 1(Krassig,1993).Therefore,DP V can be written as:DP V ¼PN i M 1:75À2i P N i=MW glu ð6ÞSince cellulose is polydisperse,DP W z DP V >DP N .The DP N values are adequate in dealing with cellulose hydrol-ysis,and DP W and DP V frequently show a good correlation to polymer properties (Klemm et al.,1998;Krassig,1993).The distribution of DPs among a population of cellulose molecules can be measured by size exclusion chromatog-raphy (Yau et al.,1979).The reciprocal of DP corresponds to the fraction of reducing ends relative to all glucan units present (F NR ,unitless).Cellulose solubility decreases drastically with increasing DP due to intermolecular hydrogen bonds.Cellodextrins with DP from 2–6are soluble in water (Klemm et al.,1998;Miller,1963;Pereira et al.,1988),while cellodextrins from 7–13or longer are somewhat soluble in hot water (Zhang and Lynd,2003;Schmid et al.,1988).A glucan of DP =30already represents the polymer ‘‘cellulose’’in its structure and properties (Klemm et al.,1998).The DP of cellulosic substrates varies greatly,from <100to >15,000,depending on substrate origin and preparation,as shown in Figure 2.The DP of wood after pulping is reduced to 500–1,500(Bertrain and Dale,1985;Klein and Snodgrass,1993;Lee et al.,1982;Swatloski et al.,2002).After partial acid hydrolysis,the DP of Avicel is further decreased to 130–800(Hoshino et al.,1997;Ng and Zeikus,1980;Ross-Murphy,1985;Steiner et al.,1988;Wood,1985),depending on hydrolysis conditions (Dong et al.,1998)and the DP of the original substrate (Wood,1988).Similarly,the DP of natural cotton can be as high as 15,000,but is reduced to 1,000–3,000or less in the preparation of cotton linters involving treatment to accomplish dewaxing and whitening (Kleman-Leyer et al.,1992,1996;Okazaki and Moo-Young,1978;Ryu and Lee,1982),and filter paper made from cotton pulp has a DP of 500–1,000or higher (Nisizawa,1973;Kongruang et al.,2004).Bacterial cel-lulose (BC)has an average DP of 2,000–3,000(Hestrin,1963;Fierobe et al.,2002;Valjamae et al.,1999),while bacterial microcrystalline cellulose (BMCC)prepared by treatment of BC with acids ranges from 130–1,300,de-pending on hydrolysis conditions (Valjamae et al.,1999).The DP of phosphoric-acid swollen cellulose (PASC)ranges from 30to more than 1,000(Fan et al.,1980;Krassig,1985;Petre et al.,1981;Wood and McCrae,1972),depending on the DP of the starting substrate (Wood,1988;Hoshino et al.,1997),as well as the phosphoric acid incubation time and temperature (Krassig,1993).The change in DP over the course of hydrolysis for cellulosic substrates is determined by the relative propor-tion of exo-and endo-acting activities and cellulose proper-ties.Exoglucanases act on chain ends,and thus decrease DP only incrementally (Kleman-Leyer et al.,1992,1996;Srisodsuk et al.,1998).Endoglucanases act on interior portions of the chain and thus rapidly decrease DP (Kleman-Leyer et al.,1992,1994;Selby,1961;Srisodsuk et al.,1998;Whitaker,1957;Wood and McCrae,1978).Exoglucanase has been found to have a marked preference for substrates with lower DP (Wood,1975),as would be expected given the greater availability of chain ends with decreasing DP.It is well known that endoglucanase activity leads to an increase in chain ends without resulting in appreciable solubilization (Irwin et al.,1993;Kruus et al.,1995;Re-verbel-Leroy et al.,1997).We know of no indication in the literature that the rate of chain end creation by endogluca-nase is impacted by substrate DP.AccessibilityCellulase enzymes must bind to the surface of substrate particles before hydrolysis of insoluble cellulose can take place.The 3D structure of such particles (including micro-structure)in combination with the size and shape of the cellulase enzyme(s)under consideration determine whether h -glucosidic bonds are or are not accessible to enzymatic attack.Cellulosic particles have both external and internal surfaces.In general,the internal surface area of cellulose is 1–2orders higher than the external surface area (Chang et al.,1981),but this is not always the case,for example,in the case of bacterial cellulose.The internal surface area can be measured by small angle X-ray scattering (SAXS),mer-cury porosimetry,water vapor sorption,and size exclusion (Grethlein,1985;Neuman and Walker,1992;Stone etal.,Figure 2.Typical DP values of cellulose and soluble cellodextrins.NC,natural cotton;NW,natural wood;P,pulp;CT,cotton linter;FP,filter paper.ZHANG AND LYND:NONCOMPLEXED CELLULASE SYSTEMS 8011969).The internal surface area of porous cellulose particles depends on the capillary structure and includes intrapar-ticulate pores(1–10nm)as well as interparticulate voids (>5A m)(Marshall and Sixsmith,1974).Grethlein(1985) found linear correlations between the initial hydrolysis rate of pretreated biomass and the pore size accessible to a molecule with a diameter of51A˚,similar to the size of T.reesei cellulase components.But the surface exposed to dextran cannot distinguish the specific active cellulose sur-face area at which enzymatic hydrolysis occurs from the surface area which is not a site for enzymatic attack(Chanzy et al.,1984;Gilkes et al.,1992;Lehtio et al.,2003),re-sulting in potential overestimation of effective cellulase-accessible area.Techniques for measuring internal surface generally do not estimate external area(Converse,1993). External surface area is closely related to shape and par-ticle size,and can be estimated by microscopic observation (Gilkes et al.,1992;Henrissat et al.,1988;Reinikainen et al., 1995b;Weimer et al.,1990;White and Brown,1981).For example,the external surface area of BMCC is f115m2/g (Gilkes et al.,1992)whereas that of Avicel is f0.3m2/g (Weimer et al.,1990).Increasing cellulase adsorption and cellulose reactivity with decreasing particle size has been reported(Kim et al.,1992;Mandels et al.,1971).However, this may be due to causes other than increased external area, perhaps decreasing mass transfer resistance,since external surface is thought to be a small fraction of overall surface area for most substrates.The gross cellulose accessibility is generally measured by the sorption of nitrogen,argon or water vapor,dimensional change or weight gain by swelling in water or organic liquids,and exchange of H to D atoms with D2H.The most widely used procedure for specific surface area(SSA)is the Brunauer-Emmett-Teller(BET)method using nitrogen adsorption.Due to variations in the experimental condi-tions such as adsorption time,vacuum time and vacuum pressure(Marshall and Sixsmith,1974),sample prepara-tion(Grethlein,1985;Lee et al.,1983),and sample origin and features(Marshall and Sixsmith,1974;Weimer et al., 1990),a wide range of gross area values have been reported in the literature even for the same substrate.The specific area of Avicel PH102increases from5.4m2/g surface area to18m2/g after a long time of water swelling,because the capillary structure of air-dried cellulose from the water-swollen state collapses,resulting in drastic changes in phys-ical parameters(Grethlein,1985;Lee et al.,1983).To keep substrate capillary structure as it exists in the hydrated state,it is recommended that SSA be measured using solvent-dried samples(Grethlein,1985;Lee et al.,1983). The typical SSA of BMCC,Avicel,and wet pulp are f200m2/g BMCC(Bothwell et al.,1997),1.8–22m2/g Avicel(Fan et al.,1980;Lee et al.,1983;Marshall and Sixsmith,1974),and55–61m2/g pulp(Fan et al.,1980; Kyriacou et al.,1988).The specific surface area of PASC from Solka Floc increases from19.5to239m2/g when phosphoric acid concentration increases from75%to85% (Lee et al.,1982).Because a nitrogen molecule is much smaller than cellulase,it has access to pores and cavities on the fiber surface that cellulase cannot enter.Therefore, there is limited basis to infer that SSA measured using the BET method is a key determinant of enzymatic hydrolysis rate(Mansfield et al.,1999).Preparation and Properties of Model Substrates Wood pulp is made from wood using several steps,in-cluding shredding,delignification,bleaching,and washing (Klemm et al.,1998).For example,Solka Floc is made from SO2-bleached spruce pulp by ball milling(Ghose, 1969).Avicel,also called hydrocellulose and microcrystal-line cellulose,is prepared from cellulosic fibers(wood pulp) by partial acid hydrolysis and then spray drying of the washed pulp slurry,but microcystalline cellulose(Avicel) still contains a substantial amount(f30–50%)of amor-phous cellulose(Krassig,1993).Bacterial cellulose(BC) is prepared from the pellicle produced by Acetobacter xylinum(ATCC23769)(Hestrin,1963)or from Nata de Coco(Daiwa Fine Produces,Singapore;Boisset et al., 2000).Bacterial microcrystalline cellulose(BMCC)is pre-pared from BC by partial acid hydrolysis to remove amor-phous cellulose(Valjamae et al.,1999).Cotton cellulose is made from natural cotton after removing impurities such as wax,pectin,and colored matter(Corbett,1963). Whatman No.1filter paper is made from cotton pulp (Dong et al.,1998).Homogenous amorphous cellulose can be made from various pure cellulose powders,e.g.,Avicel, cotton linters,by swelling treatments such as phosphoric acid,alkali,DMSO,DMAc/LiCl.Phosphoric acid swol-len cellulose(PASC)is most commonly made by swelling cellulose powder using concentrated phosphoric acid,re-sulting in decreased crystallinity(Wood,1988).Typical values for CrI,DP,gross surface area values(SSA by BET), and fraction of reducing ends(F NR,reciprocal of DP)for model cellulosic substrates are presented in Table I. Characteristics of Pretreated LignocelluloseNatural cellulose molecules occur in elementary fibrils closely associated with hemicellulose and other structural polysaccharides as well as lignin(Fig.1c).Such ligno-cellulose typically contains cellulose(35–50wt.%),hemi-cellulose(20–35wt.%),and lignin(5–30wt.%)(Chang et al.,1981;Klein and Snodgrass,1993;Lynd et al.,2002; Mansfield et al.,1999).A detailed consideration of en-zymatic hydrolysis of native lignocellulose may be found elsewhere(Hatfield et al.,1999).Since enzymatic hydro-lysis of native lignocellulose usually results in solubiliza-tion of V20%of the originally present glucan,some form of pretreatment to increase amenability to enzymatic hy-drolysis is included in most process concepts for biological conversion of lignocellulose.Pretreatment,under appro-priate conditions,retains nearly all of the cellulose pres-ent in the original material and allows close to theoretical yields upon enzymatic hydrolysis.Proposed pretreatment802BIOTECHNOLOGY AND BIOENGINEERING,VOL.88,NO.7,DECEMBER30,2004processes include dilute acid,steam explosion at high solid concentration,‘‘hydrothermal’’process,‘‘organosolv’’pro-cesses involving organic acid solvents in an aqueous phase, ammonia fiber explosion(AFEX),strong alkali process (Lynd et al.,2002),as well as mechanical treatments such as hammer and ball milling(Millett et al.,1976;Sun and Cheng,2002).Comparative features of these processes as well as consideration of substrate factors impacting the hy-drolysis rate are reviewed elsewhere(Chang et al.,1981; Converse,1993;Cowling and Kirk,1976;Dale,1985;Hsu, 1996;Ladisch et al.,1983;Mansfield et al.,1999;McMillian 1994;Lynd,1996;Sun and Cheng,2002;Weil et al.,1994; Wood and Saddler,1988).Hydrolysis of lignocellulosic biomass is more compli-cated than that of pure cellulose due to the presence of nonglucan components such as lignin and hemicellulose. Lignin removal and/or redistribution are thought to have a significant effect on observed rates of enzymatic hydrolysis (Chernoglazov et al.,1988;Converse,1993;Lynd et al., 2002).Lignin has been implicated as a competitive cel-lulase adsorbent which reduces the amount of cellulase available to catalyze cellulose hydrolysis(Bernardez et al., 1993;Ooshima et al.,1990;Sutcliffe and Saddler,1986). In addition,it has been suggested that residual lignin blocks the progress of cellulase down the cellulose chain(Eriksson et al.,2002;Mansfield et al.,1999).The measured crystallinity index of lignocellulose is impacted by the presence of lignin and hemicellulose.Thus, care must be taken in comparing CrI values for lignocellu-losic substrates to values for cellulosic substrates,and also in comparing the CrI of lignocellulosic substrates before and after pretreatment.Reported CrI values for pretreated materials are generally in the range of0.4–0.7(Chang and Holtzapple,2000;Gharpuray et al.,1983;Koullas et al., 1992;Sinitsyn et al.,1989,1991).Pretreatment by either dilute-acid or steam explosion under conditions that are quite effective in enhancing hydrolysis has been found to increase the composite CrI of lignocellulose(Deschamps et al.,1996;Kim et al.,2003;Knappert et al.,1980; Meunier-Goddik et al.,1999).Consistent with this,a negative correlation between hydrolysis rate and CrI has been shown in experiments that involved chemical pre-treatments followed by ball milling(Chang and Holtzapple, 2000;Gharpuray et al.,1983;Knappert et al.,1980;Koullas et al.,1992;Sinitsyn et al.,1989,1991),and also ex-periments that examined various pretreatment conditions (Chang and Holtzapple,2000).In contrast to the trend ob-served for other pretreatment processes,AFEX pretreat-ment has been reported to result in a decrease in CrI (Gollapalli et al.,2002).Several investigators have impli-cated accessible surface area as an important factor in determining the effectiveness of pretreatment(Gharpuray et al.,1983;Grethlein,1985;Grethlein and Converse,1991; Sinitsyn et al.,1991).A significant difficulty in interpreting the effects of pretreatment at a mechanistic level is that exposure of substrates to conditions that cause one poten-tial determinant of reactivity to change usually bring about changes in other such potential determinants.For example, Sinitsyn et al.(1991)found a strong negative correlation between CrI and accessible surface area accompanying several pretreatment processes.We suspect that the impact of increased surface area accompanying pretreatment may in many cases be more important than changes in CrI,al-though further work will be needed to establish this point and the relative significance of these and other factors may well be different for different processes.DP values of lignocellulosic substrates such as ba-gasse,wheat straw,and Eucalyptus regnans pretreated using steam explosion,supercritical CO2,alkali,and ozone mostly fall in the range of600–1,100,although values as high as3,000have been recorded for Pinus radiata chips (Puri,1984;Sinitsyn et al.,1991).During dilute acid-catalyzed cellulose hydrolysis,the DP of cellulosic ma-terials decreases rapidly initially and achieves a nearly constant value thereafter called the level-off DP(LODP) (Klemm et al.,1998;Krassig,1993;Wood,1988).LODP values in the range of100–300have been measured,de-pending on the substrate and conditions such as temperature and acid concentration(Krassig,1993;Wood,1988).This LODP value may limit the rates of hydrolysis that can oc-cur with dilute acid pretreated lignocellulose,although this has not been investigated experimentally.Different con-clusions about the importance of DP in determining hydrolysis rates of pretreated cellulosic biomass have been drawn,with Sinitsyn et al.(1991)concluding that DP is relatively unimportant,but Puri(1984)concluding that it is quite important.CELLULASE ADSORPTIONAdsorptionCellulase adsorption is rapid compared to the time re-quired for hydrolysis,with many studies finding that ad-sorption reaches steady-state within half an hour(Lynd et al.,2002).The most common description of cellulase adsorption is the Langmuir isotherm(Eq.[7]),derived as-suming that adsorption can be described by a single ad-sorption equilibrium constant and a specified adsorption capacity.The Langmuir isotherm may be represented as:E a¼W max K P E f1þK P E fð7Þin which E a is adsorbed cellulase(mg or A mol cellulase/L), W max is the maximum cellulase adsorption=A max*S(mg or A mol cellulase/L),A max is the maximum cellulase adsorp-tion per g cellulose(mg or A mol cellulase/g cellulose),S is cellulose concentration(g cellulose/L),E f is free cellulase (mg or A mol cellulase/L),and K P is the dissociation constant (K P¼E a f)in terms of L/g cellulose.The distribution coef-ficient or partition coefficient,R,is defined as:R¼K P W maxð8ÞZHANG AND LYND:NONCOMPLEXED CELLULASE SYSTEMS803。

L-阿拉伯糖研究进展黄淳【摘要】L- arabinose is a new functional and low -caloric sugar. In nature, L -arabinose indwells in the corn bran, beetroot, arabic gum, etc. L- arabinose plays an important role in the modulating of blood sugar and blood fat. The preparation methods of L - arabinose are summarized and the nature of L - arabinose areintrodured in this paper. The article also gives some outlook for the development trend of L -arabinose.%L-阿拉伯糖是一种新兴的低热量功能性糖，在自然界中，广泛存在于玉米皮、甜菜根、阿拉伯胶等中。

L-阿拉伯糖在血糖、血脂的调节方面有广阔的前景。

本文综述了L-阿拉伯糖的制备方法，并介绍了L-阿拉伯糖的性质，展望了L-阿拉伯糖生产的发展趋势。

【期刊名称】《河南化工》【年(卷),期】2011(000)023【总页数】4页(P21-24)【关键词】L-阿拉伯糖;制备;提取;合成【作者】黄淳【作者单位】青岛科技大学化工学院,山东青岛266042【正文语种】中文【中图分类】TS245L－阿拉伯糖，是从一种叫阿拉伯树分泌的胶体中经复杂的化学和物理方法分离提取出来的一种右旋单糖，分子式C5H10O5。

作为一种低热量的甜味剂，L－阿拉伯糖已被美国食品药品监督局和日本厚生省批准列入健康食品添加剂。

2008年5月，我国卫生部将L－阿拉伯糖批准为新资源食品。

自然界中，L－阿拉伯糖广泛存在于水果、稻子、麦子等粗粮皮壳、落叶松木、玉米皮、甜菜根、阿拉伯胶中。

Enzymatic Hydrolysis ProcessIntroductionEnzymatic hydrolysis is a process that involves the breakdown of complex organic compounds using enzymes. This process plays a crucial role in various industries, including biofuels, food, pharmaceuticals, and agriculture. Enzymes act as catalysts, accelerating the rate of chemical reactions and enabling the transformation of substrates into desired products. In this article, we will explore the different aspects of enzymatic hydrolysis, including its significance, applications, factors affecting the process, and future prospects.Importance of Enzymatic HydrolysisEnzymatic hydrolysis is a highly significant process due to its wide range of applications. Some key reasons for the importance of this process are:1.Biofuel Production: Enzymes are extensively utilized in theproduction of biofuels, such as ethanol and biodiesel. Enzymatichydrolysis of lignocellulosic biomass, such as agriculturalresidues and energy crops, breaks down complex carbohydrates intosimple sugars, which can then be fermented to produce biofuels.2.Food Industry: Enzymatic hydrolysis is employed in the foodindustry to enhance the nutritional properties of various foodproducts. For example, proteases are widely used to hydrolyzeproteins into amino acids, improving their digestibility.3.Pharmaceuticals: Enzymatic hydrolysis plays a critical role inthe pharmaceutical industry, particularly in drug formulation.Enzymes are used to break down active pharmaceutical ingredientsinto biologically active compounds, facilitating their absorptionand effectiveness.4.Waste Treatment: Enzymatic hydrolysis is utilized in thetreatment of various types of waste, including sewage andagricultural waste. Enzymes aid in the breakdown of organic matter,reducing the environmental impact and facilitating the productionof valuable by-products.Enzymatic Hydrolysis ProcessThe enzymatic hydrolysis process involves a series of steps, including substrate preparation, enzyme selection, enzymatic reaction, and product recovery.1.Substrate Preparation: The substrate used in enzymatic hydrolysisneeds to be properly prepared to ensure efficient enzyme-substrate interaction. This often involves pretreatment methods such as size reduction, chemical or physical treatment, and removal ofinhibitors.2.Enzyme Selection: The choice of enzyme is crucial as itdetermines the specificity and efficiency of the hydrolysisprocess. Different enzymes are suitable for different substrates,and factors such as pH, temperature, and enzyme concentration need to be considered.3.Enzymatic Reaction: The hydrolysis reaction typically occurs in acontrolled environment, with optimum pH and temperature conditions.The enzyme is added to the substrate, and the reaction is allowedto proceed for a specific period. During this time, the enzymebreaks down the substrate into smaller molecules.4.Product Recovery: After the enzymatic hydrolysis, the desiredproducts need to be separated from the reaction mixture. This mayinvolve techniques such as filtration, centrifugation, orchromatography, depending on the nature of the products andimpurities present.Factors Affecting Enzymatic HydrolysisSeveral factors can influence the efficiency of enzymatic hydrolysis. These factors need to be carefully considered and optimized to achieve the desired results. Some key factors include:1.pH: Enzymatic reactions are highly sensitive to pH. Differentenzymes have different pH optima, and maintaining the appropriatepH level ensures optimal enzyme activity.2.Temperature: Temperature significantly affects enzyme activity.Each enzyme has an optimum temperature range within which itfunctions best. Deviating from this range can either decrease the reaction rate or denature the enzyme.3.Enzyme Concentration: The concentration of enzymes in thereaction mixture influences the rate and efficiency of hydrolysis.Higher enzyme concentrations usually result in faster reactionrates, up to a certain limit.4.Substrate Concentration: The concentration of the substrate alsoaffects the hydrolysis process. Extremely high or low substrateconcentrations can inhibit enzyme activity and reduce overallefficiency.5.Inhibitors and Activators: Inhibitors can significantly affectenzymatic hydrolysis by interfering with the enzyme-substrateinteraction. Activators, on the other hand, can enhance enzymeactivity. Identifying and managing these factors is crucial foroptimum results.Future ProspectsEnzymatic hydrolysis continues to be an area of active research and development. Scientists are continually exploring new enzymes, optimizing reaction conditions, and developing innovative techniques to improve the efficiency and cost-effectiveness of the process. In addition, advancements in genetic engineering and enzyme immobilization techniques hold promising potential for further enhancing enzymatic hydrolysis processes.ConclusionEnzymatic hydrolysis is a vital process with significant applications in various industries. Understanding the factors influencing the process and optimizing reaction conditions are crucial for achieving efficient and cost-effective hydrolysis. Ongoing research efforts and technological advancements continue to drive progress in this field, opening doors to new possibilities and innovations in the future.。

CHEMICAL INDUSTRY AND ENGINEERING PROGRESS 2018年第37卷第1期·276·化 工 进展木薯酒精渣的预处理及补料同步糖化发酵制取乙醇岳军，徐友海，王继艳，胡世洋，惠继星，金刚（中国石油吉林石化公司研究院，吉林 吉林 132021）摘要：木薯酒精渣的处置是制约木薯燃料乙醇大规模产业化的问题之一。

本文立足于探索木薯酒精渣利用途径，分析了木薯酒精渣的主要成分，对比了氨水、氢氧化钠、氨水组合稀硫酸3种预处理方式对于木薯酒精渣纤维素和木素含量及纤维素酶水解效率的影响，分析了处理前后木薯酒精渣的表面结构及纤维素结晶度，并以氨水处理后的木薯酒精渣为底物，进行了同步糖化发酵。

结果表明，3种预处理方法中组合预处理能更好地增加纤维素含量和提高纤维素酶水解效率，与未处理原料相比，组合预处理后纤维素含量增加了111.26%，木素下降了35.05%，酶水解72h 纤维素转化率从42.10%增加到61.71%。

氨水预处理后，原料的木素含量降低，处理后木薯酒精渣的表面变得更加粗糙，纤维素结晶度有所增加，以氨水处理后的木薯酒精渣为底物进行分批补料同步糖化发酵，当初始底物浓度为100.0g/L ，分别在20h 、40h 、60h 进行补料至最终底物浓度为400.0g/L 时，发酵120h 乙醇浓度达到51.0g/L 。

关键词：燃料乙醇；木薯酒精渣；同步糖化发酵；预处理；纤维素结晶度中图分类号：S216.2 文献标志码：A 文章编号：1000–6613（2018）01–0276–07 DOI ：10.16085/j.issn.1000-6613.2017-0752Pretreatment of cassava alcohol residues and ethanol production by simultaneous saccharification and fermentationYUE Jun ，XU Youhai ，WANG Jiyan ，HU Shiyang ，HUI Jixing ，JIN Gang（Research Institute of Jilin Petrochemical Co.，Ltd.，PetroChina ，Jilin 132021，Jilin ，China ）Abstract: Processing cassava alcohol residues （CAR ）is one of the bottlenecks of the large scale application of the cassava based alcohol production. This study focused on the ethanol production using CAR as raw material by pretreatment of CAR and simultaneous saccharification and fermentation （SSF ）. The chemical compositions of cassava alcohol residues were analyzed. Pretreatment was conducted by different methods such as aqueous ammonia pretreatment ，sodium hydroxide pretreatment and the combined pretreatment with aqueous ammonia and dilute sulfuric acid. The effect of different pretreatment methods on enzymatic digestibility of CAR were studied. The structural feature of CAR was analyzed by X-ray diffraction （XRD ）and scanning electron microscope （SEM ）. The fed-batch method was combined with simultaneous saccharification and fermentation （SSF ）to enhance ethanol concentration further and reduce enzyme loading. The results showed that among the three pretreatment methods the combined pretreatment was more effective on increasing cellulose and digestibility of cellulose. Cellulose increased 111.26% and lignin decreased 35.05% after being pretreated by the combined pretreatment. Compared with no pretreated cassava alcohol residues ，cellulose conversion rate increased from 42.10% to 61.71% at 72h of enzyme hydrolysis when the第一作者及通讯作者：岳军（1984—），男，硕士，工程师。

玫瑰鲜花醋的试制朱绍华;杨光福【摘要】试验以重瓣红玫瑰鲜花为原料,通过糖渍、酶解、萃取工艺,在单因素试验结果的基础上,采用正交试验对玫瑰鲜花醋的工艺条件进行了优化.结果表明:玫瑰鲜花醋工艺配料组合为醋液萃取总酸3.5%,玫瑰鲜花与醋液比1∶12,白砂糖添加量18%,食盐添加量1.0%时,感官评价最佳;玫瑰鲜花醋经取得CNAS实验室认可机构检测,产品符合GB 2719-2003 及GB/T 18187-2000 标准的要求.%Take the fresh flower of Rose rugosa cv.Plena as the raw material,the process conditions of rose vinegar are optimized by orthogonal experiment on the basis of single-factor experiment with the process of sugaring,enzymatic hydrolysis and extraction.The optimal parameters are achieved as follows:the ingredients preparation should be 3.5% total acidity of extracted vinegar;the ratio of fresh rose flower and vinegar is 1∶12 ;the additive amount of sugar is 18%;the additive amount of salt is 1.0%,the sensory evaluation is the best.Rose vinegar is accredited by CNAS laboratory.This product meets the qualifications of GB 2719-2003 and GB/T 18187-2000.【期刊名称】《中国调味品》【年(卷),期】2018(043)007【总页数】5页(P121-125)【关键词】重瓣红玫瑰;玫瑰花青素;玫瑰鲜花醋【作者】朱绍华;杨光福【作者单位】昆明拓东调味食品有限公司,昆明 650228;云南省餐饮与美食行业协会,昆明 650221【正文语种】中文【中图分类】TS264.221 概述玫瑰花（Rosa rugosa Thunb.）是蔷薇科（Rosaceae）蔷薇属（Rosa L.）多年生常绿或落叶性灌木。

木质纤维素的酶水解Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. En zymatic hydrolysis that conv erts lig no cellulosic biomass to ferme ntable sugars may be the most complex step in this process due to substrate-related and en zyme-related effects and their in teracti ons. Although en zymatic hydrolysis offers the pote ntial for higher yields, higher selectivity, lower en ergy costs and milder operat ing con diti ons than chemical processes, the mecha nism of en zymatic hydrolysis and the relati on ship between the substrate structure and function of various glycosyl hydrolase comp onents is not well un derstood. Con seque ntly, limited success has bee n realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and en zyme features that in flue nee performa nee, to better un dersta nd fun dame ntal strategies to adva nce en zymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view in cludi ng characteristics of cellulose (e.g., crystalli ni ty, degree of polymerizati on and accessible surface area) and soluble and in soluble biomass comp onents (e.g., oligomeric xyla n and lig nin) released in pretreatme nt, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability and activity of in dividual en zymes and their syn ergistic effects in dec on struct ing complex lig no cellulosic biomass. Adva need tech no logies to discover and characterize novel enzymes and to improve enzyme characteristics by mutage nesis, post-tra nslatio nal modificati on and over-expressi on of selected en zymes and modificati ons in lig no cellulosic biomass are also discussed.基于酶水解技术基础上的纤维素乙醇生产技术是20世纪80年代生物质技术的主要研究领域，自从20世纪70年代能源危机”之后，美国能源部一直积极支持规模以上乙醇生产，并建立独立部门用于管理和支持这项工作。