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Lesson One 细胞器的结构和功能Actin：肌动蛋白，是微丝的结构蛋白, 以两种形式存在, 即单体和多聚体。
basal body:：基体，真核细胞的纤毛或鞭毛基底部由微管及其相关蛋白质构成的短筒状结构，是纤毛和鞭毛的微管组织中心。
centriole：中心粒，动物、某些藻类和菌类细胞中的圆筒状细胞器，位于间期细胞核附近或有丝分裂细胞的纺锤体极区中心。
chemotaxis：趋化性，即由介质中化学物质的浓度差异形成的刺激所引起的趋向性。
chloroplast：叶绿体，绿色植物细胞内进行光合作用的结构，是一种质体。
chromosome：染色体，实质是脱氧核甘酸，为细胞核内由核蛋白组成、能用碱性染料染色、有结构的线状体，是遗传物质基因的载体。
cilia：纤毛，从一些原核细胞和真核细胞表面伸出的、能运动的突起。
cytoplasm：胞质，由细胞质基质、内膜系统、细胞骨架和包涵物组成。
cytoskeleton：细胞骨架，真核细胞中与保持细胞形态结构和细胞运动有关的纤维网络。
包括微管、微丝和中间丝。
dynein：动力蛋白，即纤毛中的一种具有ATP酶活性的巨大的蛋白质复合体。
endoplasmic reticulum：内质网，指细胞质中一系列囊腔和细管，彼此相通，形成一个隔离于细胞质基质的管道系统。
flagella：鞭毛，在某些细菌菌体上具有细长而弯曲的丝状物，是细菌的运动器官。
Golgi complex：高尔基复合体，由许多扁平的囊泡构成的以分泌为主要功能的细胞器。
lysosome：溶酶体，真核细胞中一种膜包围的异质的消化性细胞器。
是细胞内大分子降解的主要场所。
microfilament：微丝，由肌动蛋白分子螺旋状聚合成的纤丝，又称肌动蛋白丝，是细胞骨架的主要成分之一。
microtubule：微管，由微管蛋白原丝组成的不分支的中空管状结构，是细胞骨架成分，与细胞支持和运动有关。
mitochondrion：线粒体，真核细胞中由双层高度特化的单位膜围成的细胞器。
研究揭示nmn对肾的好处，nmn对男性的功效与作用不止如此研究揭示nmn对肾的好处，nmn对男性的功效与作用不止如此！随着年龄的增长，身体所有器官的衰退和功能障碍——肾脏也不例外。
日本W+NMN25000黑金版开展了一项研究，他们监测了老龄小鼠的肾脏细胞中细胞的变化，测试了补充W+NMN可以恢复肾脏功能随着年龄的增加，肾小球会出现变化，肾小球是从血液中过滤掉废物的重要细胞单元。
衰老会引起肾小球功能下降，废物过滤率也逐渐下降，终导致肾功能丧失。
在本研究中，研究者探索了NMN对衰老过程中肾脏功能的恢复作用。
NMN是烟酰胺腺嘌呤二核苷酸（NAD+）的前体，NAD+对细胞能量产生和DNA完整性的维持具有非常重要的作用。
NAD+在体内的水平随着动物和人的年龄而下降，从而引起器官功能下降。
NMN is a precursor of nicotinamide adenine dinucleotide (NAD+), which plays a very important role in cell energy production and maintenance of DNA integrity. Levels of NAD+ in the body decline with age in animals and humans, causing organ function to decline.研究人员比较了8周龄和96周龄的老年小鼠肾脏中的基因和蛋白质水平。
共筛选出108差异基因，这些基因调控的蛋白质富集于氧化磷酸化途径、sirtuin信号传导、脂肪酸β-氧化途径和胆汁酸合成途径。
该研究一共筛选到7208种蛋白质。
为了更好的筛选差异蛋白，他们将该研究成果与其他研究组的两个数据集进行比较，最终得到27种差异蛋白，有19种随衰老而增加的蛋白（上调表达蛋白），有8种随年龄增加而减少的蛋白（下调表达蛋白）。
说明这27种蛋白可能是衰老的生物标记蛋白。
细胞生物学习题第一章绪论二、是非判断1.细胞是生命体的结构与生命活动的基本单位。
2.细胞生物学研究的总趋势是细胞生物学与分子生物学相互渗透和交融，即分子细胞生物学。
3.世界上第一个观察到活细胞有机体的是RobertHooke。
4.细胞生物学是20世纪80年代以后逐渐形成和发展起来的。
5.细胞生物学研究内容可分为细胞结构功能和细胞重要生命活动两大部分。
6.细胞的显微结构是指在电子显微镜下所观察到的结构。
7.细胞生物学形成发展的前身是细胞学。
8.20世纪60年代DNA双螺旋结构的阐明，标志着分子生物学的诞生。
三、填空1.细胞生物学是在、和三个不同层次上研究细胞的和的科学。
2.第一位观察并命名“cell”的科学家是，而真正观察到活细胞有机体的科学家是3.和于1953年正式提出了DNA分子的双螺旋结构模型。
4.现代生物学的三大基石是：1838～1839年和的；1859年的；1866年的5.由于德国病理学家Virchow于1858年提出的观点，才使得细胞学说最终完善。
6.细胞生物学的发展历史大致可划分为、、、和分子细胞生物学几个时期。
7.19世纪自然科学的三大发现是指、和四、选择1.细胞学发展的经典时期主要是指（C）。
A.1665年前后B.1838～1839C.19世纪最后25年D.20世纪以后2.分子细胞生物学是在20世纪（A）逐渐形成和发展起来的。
A.50年代B.60年代C.70年代D.80年代3.细胞学说主要是由（B）提出的。
A.WilonandHertwigB.SchleidenandSchwannC.SingerandNicolonD.Ro bertHookeandLeeuwenHoek4.细胞生物学的形成和发展与以下（B）方法技术密切相关。
A.石蜡切片技术B.电子显微镜技术。
C.光学显微镜技术D.细胞培养技术5.当前细胞生物学研究的热门领域是（A）。
A.细胞信号转导B.细胞增殖C.细胞起源D.细胞核的结构五、简答1.生命科学的发展可大致划分为哪几个阶段？2.细胞生物学的发展可划分为哪几个阶段？3.细胞学说的内容包括哪些？有何重要意义？4.试列举国内外你所了解的有关细胞生物学方面的教科书及学术期刊。
细胞生物学中-英-法文名词表第一章细胞cell (cellule)细胞生物学cell biology (biologie cellulaire)原核细胞prokaryocyte (prokaryote)真核细胞eukaryocyte (eukaryocyte)生殖细胞germ cell (cellules germinales)体细胞somatic cell (cellule somatique )癌细胞cancer cell (cellule du cancer)干细胞stem cell (cellules souches)细胞治疗cell therapy (thérapie cellulaire)组织工程tissue engineering (ingénierie tissulaire)第二章构建单元building block (élément fondamental, élément de base)单糖monosaccharide (monosaccharide)脂肪酸fatty acid (acide gras)氨基酸amino acid (acide aminé)核苷酸nucleotide (nucleotide)环腺苷酸adenosine 3’,5’-monophosphate, cAMP(l'adénosine 3 ',5 ' –monophosphate)环鸟苷酸guanosine 3’,5’-monophosphate, cGMP(guanosine 3 ',5 ' –monophosphate)多糖polysaccharide (polysaccharide)糖原glycogen (glycogène)脂质lipid (lipide)三酰甘油triacylglycerol (triacylglycérol)蛋白质protein (protéine)肽peptide (peptide)多肽链polypeptide chain (chaîne polypeptide)α螺旋α helix (α hélice)β折叠β pleated sheet (β pliage)翻译后修饰post-translational modification(modification post-traductionnelle)核酸nucleic acid (acides nucléiques)核糖核酸ribonucleic acid, RNA (acide ribonucléique, ARN)脱氧核糖核酸deoxyribonucleic acid, DNA (acide désoxyribonucléique, ADN) （DNA）双螺旋结构double helix structure (structure en double hélice)信使核糖核酸messenger ribonucleic acid, mRNA(acide ribonucléique messager, ARN messager ouARNm)转运核糖核酸transfer ribonucleic acid, tRNA(acide ribonucléique de transfert, ARNt) 核糖体核糖核酸ribosome ribonucleic acid, rRNA(acide ribonucléique ribosomique, ARNr) 质膜plasma membrane (membrane plasmique )细胞质cytoplasm (cytoplasme)细胞器organelle (organite)细胞骨架cytoskeleton (cytosquelette)胞质溶胶cytosol (cytosol)细胞核nucleus (noyau)染色质chromatin (chromatine)染色体chromosome (chromosome)核仁nucleolus (nucleoli)第三章激光扫描共聚焦显微镜laser scanning confocal microscope(microscope confocal à balayage laser)电子显微镜electron microscope (microscope électronique)细胞化学技术cytochemistry (cytochimie)免疫细胞化学技术immunocytochemistry (immunocytochimie) 流式细胞术flow cytometry (cytométrie de flux)细胞培养cell culture (la culture cellulaire)第四章细胞核nucleus (noyau)核被膜nuclear envelope (envelope nucléaire)核膜nuclear membrane (membrane nucléaire)核孔nuclear pore (pores nucléaires)核孔复合体nuclear pore complex (pores nucléaires complexes) 核纤层nuclear lamina (lamina nucléaire)染色质chromatin (chromatine)染色体chromosome (chromosome)异染色质heterochromatin (hétérochromatine)常染色质euchromatin (euchromatine)核型karyotype (caryotype)基因gene (gène)基因表达gene expression (expression génique)外显子exon (exon)内含子intron (intron)基因组genome (genome)复制起始点replication origin (origine de replication)着丝粒centromere (centromère)端粒telomere (telomère)组蛋白histon (histone)非组蛋白non-histon (non-histone)核小体nucleosome (nucléosome)组蛋白修饰histon modification (histone modification)DNA复制DNA replication (réplication de l'ADN)半保留复制semiconservative replication (réplication semiconservative )复制叉replication fork (fourche de replication)前导链leading strand ( brin précoce)后随链lagging strand (brin tardif)DNA修复DNA repair (réparation de l'ADN)转录transcription (transcription)非编码RNA non-coding RNA (ARN non codant)微小RNA microRNA (microARN)小干扰RNA small interfering RNA (petits ARN interférents)核仁组织者nucleolus organizer (nucléole organiseur)核仁nucleolus (nucléole)第五章核糖体ribosome (ribosome)游离核糖体free ribosome (ribosomes libres)膜结合核糖体membrane-bound ribosome (ribosomes associés aux membranes) 密码子codon (codon)mRNA (ARNm)tRNA (ARNt)rRNA (ARNr)泛素－蛋白酶体系统ubiquitin-proteasome system(systèm de l'ubiquitine-protéasome)泛素ubiquitin (ubiquitine)蛋白酶体proteasome (protéasome)泛素化ubiquitination (l'ubiquitination)内质网endoplasmic reticulum，ER (réticulum endoplasmique)糙面内质网rough endoplasmic reticulum，RER(réticulum endoplasmique rugueux)光面内质网smooth endoplasmic reticulum，SER(réticulum endoplasmique lisse)蛋白质糖基化protein glycosylation (glycosylation des protéines)蛋白质折叠protein folding (repliement des protéines )内质网应激ER stress未折叠蛋白反应unfolded protein response高尔基体Golgi apparatus (appareil de Golgi)蛋白质分选protein sorting (tri des protéines)溶酶体lysosome (lysosome)异体吞噬泡heterophagic vacuole (vacuole heterophagique)自体吞噬泡autophagic vacuole (vacuole autophagique)吞噬作用phagocytosis (phagocytose)吞饮作用pinocytosis (pinocytosis)自体吞噬autophagy (autophagie)过氧化物酶体peroxisome (peroxysome)线粒体mitochondria (mitochondrie)电子传递链electron-transport chain (la chaîne de transport d'électrons)呼吸链respiratory chain (chaîne respiratoire)（线粒体）基粒elementary particle (particule élémentaire)化学渗透偶联chemiosmotic coupling (couplage chimiosmotique)质子动力势proton-motive force (la force proton motrice)氧化磷酸化oxidative phosphorylation (la phosphorylation oxydative )线粒体DNA mitochondrial DNA, mtDNA (ADN mitochondrial, ADNmt) 细胞骨架cytoskeleton (cytosquelette)微管microtubule (microtubule)微管蛋白tubulin (tubuline)马达蛋白motor protein (protéines du moteur)微管组织中心microtubule organizing center, MTOC(le centre organisateur des microtubule)中心体centrosome (centrosome )中心粒centriole (centriole)细胞质微管cytoplasmic microtubule (microtubule cytoplasmique)纺锤体微管spindle microtubule ( microtubules du fuseau)微丝microfilament (microfilaments)肌动蛋白actin (actine)微绒毛microivilli (microivilli)中间丝intermediate filament (filament intermédiaire)角蛋白keratin (kératine)第六章质膜plasma membrane (membrane cytoplasmique)脂筏lipid raft (radeaux lipidiques)第七章细胞连接cell junction (jonctions cellulaires)细胞黏附cell adhesion (adhérence cellulaire )细胞外基质extracellular matrix (matrice extracellulaire)紧密连接tight junction (jonctions serrées ou jonctions étanches)锚定连接anchoring junction (jonction d'ancrage)黏合带adhesion belt (adhésion en ceinture)v http://biologie.univ-mrs.fr/upload/p204/adherence2.pdf黏合斑adhesion plaque (plaque adherence)桥粒desmosome (desmosome)半桥粒hemidesmosome (hemidesmosome)间隙连接gap junction (jonction gap)细胞黏附分子cell adhesion molecules (molécules d'adhérence cellulaire)钙黏素Cadherin (cadhérine)上皮－间质转变epithelial-mesenchymal transition(transition épithéliale-mésenchymateuse)选择素selectin (sélectine)免疫球蛋白超家族黏附分子the immunoglobulin superfamily cell adhesion molecules(les molécules d'adhérence cellulaire de la superfamille des immunoglobulines) 整合素integrin (intégrine)胶原collagen (collagène)纤粘连蛋白fibronectin (fibronectine)层粘连蛋白laminin (laminine)糖胺聚糖glycosaminoglycan (glycosaminoglycanes)蛋白聚糖proteoglycan (protéoglycanes)基膜basal lamina (lamina basale)失巢凋亡anoikis (anoïkis)第八章单纯扩散simple diffusion (simple diffusion)膜运输蛋白membrane transport protein (protéine de transport de la membrane)易化扩散facilitated diffusion (diffusion facilitée)电化学梯度 electrochemical gradient (gradient électrochimique)转运体transporter (transporteur)偶联转运体 coupled transporter (transporteur couplé)钠钾泵 sodium potassium pump (pompe sodium-potassium)通道蛋白 channel protein (protéine de canal)离子通道ion channel (canal ionique)水通道water channel (canal d'eau)电压门控通道voltage-gated channel (canal voltage-dépendant)递质门控通道transmitter-gated channel (canal transmetteur-dépendant)乙酰胆碱受体acetylcholinergic receptor (récepteur acétylcholinergique)水孔蛋白aquaporin (aquaporine)第九章蛋白质分选 protein sorting (tri des protéines)蛋白质分选信号protein sorting signals (signaux de tri de protéines)信号肽 signal peptide (peptide signal)信号斑Signal spot (tache de signal)门控运输gated transport (transports fermée)穿膜运输transmembrane transport (transport transmembranaire)小泡运输vesicular transport (transport vésiculaire)共翻译转运Cotranslational translocation (translocation cotraductionnelle)信号识别颗粒signal recognition particle(particle de reconnaissance de signaux)有被小泡coated vesicle (vésicule couché)内体endosome (endosome)受体介导的胞吞receptor-mediated endocytosis固有分泌途径constitutive secretory pathway (voie de sécrétion constitutive) 受调分泌途径regulated secretory pathway (voie de sécrétion régulée)第十章细胞通讯cell communication (communication cellulaire)信号分子signaling molecule (molécule de signalisation)配体ligand (ligand)（细胞）信号转导(cell) signalling (la transduction du signal cellulaire)膜受体membrane receptor (récepteur membranaire)细胞内受体（核受体）intracellular receptor(récepteur intracellulaire, récepteur nuclaire)细胞内信号转导蛋白intracellular signaling proteins(protéines de signalisation intracellulaires)分子开关molecular switch (commutateur moléculaire)小分子信使small messenger molecule (petite molécule messagère)G蛋白偶联受体G protein-coupled receptor (récepteur couplé à la G-protéine) 酶偶联受体enzyme-linked receptor (récepteurs liés à une enzyme)受体酪氨酸激酶receptor tyrosine kinase (récepteur tyrosine kinase)第十一章转录调控蛋白transcription regulator (régulateur transcriptionnelle)基因调节蛋白gene regulatory protein (protéine de régulation génique)转录因子transcription factor (facteur de transcription)组成性基因表达constitutive gene expression (l'expression du gène constitutif) 核糖开关riboswitch微小RNA micro RNA (micro ARN)RNA干扰RNA interference, RNAi (l'interférence ARN)组合调控combinatorial regulation (réglementation combinatoire)细胞记忆cell memory (mémoire cellulaire)第十二章细胞增殖cell proliferation (prolifération cellulaire)细胞周期cell cycle (cycle cellulaire)限制点restriction point (point de restriction)有丝分裂mitosis (mitose)细胞核分裂karyokinesis (karyokinèse)细胞质分裂cytokinesis (cytokinèse)减数分裂meiosis (méiose)动粒kinetochore (kinétochore)纺锤体(mitotic) spindle (fuseau mitotique)周期蛋白cyclin (cycline)周期蛋白依赖性激酶cyclin-dependent kinase, Cdk (la kinase cyclin-dépendante) 周期蛋白依赖性激酶抑制物cyclin-dependent kinase inhibitor, CKI(inhibitrices de Cdk)检查点check point (points de contrôle)第十三章细胞分化cell differentiation (la différenciation cellulaire)分化潜能differentiation potential (potentiel de différenciation)全能性totipotency (totipotence)胚胎干细胞embryonic stem cell, ES cell (les cellules souches embryonnaires)去分化dedifferentiation (dédifférenciation)（干细胞）自我更新(stem cell) self-renewal(auto-renouvellement (de cellules souches))组织干细胞（成体干细胞）tissue stem cells, adult stem cells(les cellules souches de tissu)奢侈基因luxury gene (gène de luxe)基因的差异性表达differential expression of genes(gènes exprimés de manière différentielle)第十四章细胞凋亡apoptosis (apoptose)坏死necrosis (nécrose)程序性坏死programmed necrosis (nécrose programmée)程序性细胞死亡programmed cell death (la mort cellulaire programmée)胱冬肽酶caspases (caspases)死亡受体death receptors (récepteurs de mort)。
欧洲药典EP8.02.6.1⽆菌检验sterility中英⽂翻译2.6.1. STERILITY2.6.1 ⽆菌检查法The test is applied to substances, preparations or articles which, according to the Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test.本检查⽅法适⽤于按照药典要求应当⽆菌的原料、制剂或其他物质。
但是，如果按照本⽆菌检查法的结果符合要求，仅表明在该检查条件下未发现微⽣物污染。
PRECAUTIONS AGAINST MICROBIAL CONTAMINATION微⽣物污染防范The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any micro-organisms which are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.⽆菌检测试验应在⽆菌的条件下进⾏。
Biochemical Engineering Journal 65 (2012) 70–81Contents lists available at SciVerse ScienceDirectBiochemical EngineeringJournalj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b ejReviewReview on production and medical applications of -polylysineSwet Chand Shukla a ,Amit Singh b ,Anand Kumar Pandey c ,Abha Mishra a ,∗aSchool of Biochemical Engineering,Institute of Technology,Banaras Hindu University,Varanasi 221005,India bDepartment of Pharmacology,Institute of Medical Sciences,Banaras Hindu University,Varanasi 221005,India cSchool of Biomedical Engineering,Institute of Technology,Banaras Hindu University,Varanasi 221005,Indiaa r t i c l ei n f oArticle history:Received 3May 2011Received in revised form 28March 2012Accepted 2April 2012Available online 11 April 2012Keywords:-PolylysineHomopolyamideS.albulus Lysinopolymerus Conjugate Drug carrier Targetinga b s t r a c t-Polylysine (-PL)is a homopolyamide linked by the peptide bond between the carboxylic and epsilon amino group of adjacent lysine molecules.It is naturally occurring biodegradable and nontoxic towards human.This review article gives an insight about the various -PL producing strains,their screening procedures,mechanism of synthesis,characterization,and its application in the medical field.The poly cationic nature of -PL at physiological pH makes it as one of the potential candidates in the field of drug delivery.Most of the biomedical applications till date use synthetic ␣-PLL as a raw material.However,it is believed that naturally occurring -PL would be an ideal substitute.© 2012 Elsevier B.V. All rights reserved.Contents 1.Introduction ..........................................................................................................................................712.Origin and distribution of -PL ......................................................................................................................713.Mechanism of synthesis .............................................................................................................................714.Biosynthesis and molecular genetics ................................................................................................................715.Microbial production of -polylysine ................................................................................................................726.Screening and detection of -PL production in microbial system...................................................................................737.Purification and characterization of -PL ............................................................................................................738.Conformation of -PL ................................................................................................................................749.Application of polylysine in medicine ...............................................................................................................749.1.Polylysine as a drug carrier ...................................................................................................................749.2.Polylysine as nanoparticles...................................................................................................................759.3.Polylysine as a gene carrier...................................................................................................................759.4.Polylysine as liposomes ......................................................................................................................769.5.Polylysine as interferon inducer .............................................................................................................769.6.Polylysine as lipase inhibitor .................................................................................................................779.7.Polylysine as hydrogel ........................................................................................................................779.8.Polylysine as coating material................................................................................................................779.9.Other applications ............................................................................................................................7810.Conclusion ..........................................................................................................................................78References ...........................................................................................................................................78Abbreviations:Pls,polylysine synthetase;NaSCN,sodium thiocynate;FTIR,Fourier transform infrared spectroscopy;NMR,nuclear magnetic resonance spectroscopy;MION,monocrystalline iron oxide nanoparticle;NPs,nanoparticles;IgM,immunoglobulin M.∗Corresponding author.Tel.:+919451887940.E-mail address:abham.bce@itbhu.ac.in (A.Mishra).1369-703X/$–see front matter © 2012 Elsevier B.V. All rights reserved./10.1016/j.bej.2012.04.001S.C.Shukla et al./Biochemical Engineering Journal 65 (2012) 70–81711.Introduction-Polylysine (-PL)is a basic polyamide that consists of 25–30residues of l -lysine with an -amino group-␣-carboxyl group link-age (Fig.1).Polyamide can be grouped into two categories,one in which the polyamide consists of only one type of amino acid linked by amide bonds called homopolyamide and the other which consists of different amino acids in their chain called proteins [1].Furthermore,proteins are biosynthesized under the direction of DNA,while the biosynthesis of homopolyamides is catalyzed by peptide synthetases.Therefore,the antibiotics that are inhibitors of translation such as chloramphenicol,do not affect the biosyn-thesis of polyamides.Proteins in general exhibit exact length,whereas homopolyamides show a remarkable variation in molec-ular weight.Amide linkages in proteins are only formed between ␣-amino and ␣-carboxylic groups (␣-amide linkages),whereas amide bonds in homopolyamide involve other side chain functions such as -and ␥-carboxylic with -amino groups [1].Particularly,chemically synthesized polylysine were found to have linkages between ␣-carboxyl and ␣-amino group.Many workers investi-gated various applications of ␣-PL in the drug delivery system.However,␣-PL was reported to be toxic to human beings,and there-fore,research has now been diverted towards finding naturally occurring polymers [2,3].-PL is an unusual naturally occurring homopolyamide having linkages between the -amino group and ␣-carboxylic group,and it shows high water solubility and sta-bility.No degradation is observed even when the -PL solution is boiled at 100◦C for 30min or autoclaved at 120◦C for 20min [4].-PL was discovered as an extracellular material of Streptomyces albulus ssp.Lysinopolymerus strain 346during screening for Dra-gendorff’s positive substances [5–7].Mutation studies were made by nitrosoguanidine treatment on wild type Lysinopolymerus strain 346to enhance the -PL production.As a result of mutation,S-(2-aminoethyl)-l -cysteine and glycine resistant mutant were isolated,with four times higher amounts of -PL than the wild type [8].-PL is a cationic surface active agent due to its positively charged amino group in water,and hence they were shown to have a wide antimi-crobial activity against yeast,fungi,Gram positive,Gram negative bacterial species [4,9].The excreted polymer is absorbed to the cell surfaces by its cationic property,leading to the striping of outer membrane and by this mechanism the growth of microbes sensi-tive to -PL is inhibited.-PL degrading enzyme plays an important role in self-protection of -PL producing microbes [9].Due to its excellent antimicrobial activity,heat stability and lack of toxicity,it is being used as a food preservative [10,11].Naturally occurring -PL is water soluble,biodegradable,edible and nontoxic toward humans and the environment.Therefore,-PL and its derivatives have been of interest in the recent few years in food,medicine and electronics industries.Derivatives of -PL are also available which offers a wide range of unique applications such as emul-sifying agent,dietary agent,biodegradable fibers,highly water absorbable hydrogels,drug carriers,anticancer agent enhancer,biochip coatings,etc.Polylysine exhibits variety of secondary struc-tures such as random coil,␣-helix,or -sheet conformations in aqueous solution.Moreover,transitions between conformations can be easily achieved using,salt concentration,alcohol con-tent,pH or temperature as an environmental stimulus.There is aH NH*CH 2CH 2CH 2CH 2CH NH 2CO*OHnFig.1.Chemical structure of epsilon polylysine.growing interest in using -PL and its derivatives as biomaterials and extensive research has been done leading to a large number of publications [4,12–15].The present review focuses on various pro-cess parameters for maximal yield of polymer by microbial system more specifically by actinomycetes,probable biosynthetic route and its application,especially in pharmaceutical industries.2.Origin and distribution of -PLNot much is known about the -PL producing microbial species existing in the environment.It is observed that -PL producers mainly belong to two groups of bacteria’s:Streptomycetaceae and Ergot fungi .Besides Streptomyces albulus ,a number of other -PL producing species belonging to Streptomyces,Kitasatospora and an Ergot fungi,Epichole species have been isolated [16].Recently,two Streptomyces species (USE-11and USE-51)have been isolated using two stage culture method [17].3.Mechanism of synthesis-Polylysine (-PL)is a homopolymer characterized by a pep-tide bond between ␣-carboxyl and -amino groups of l -lysine molecules.Biosynthetic study of -PL was carried out in a cell-free system by using a sensitive radioisotopic -PL assay method,suggested that the biosynthesis of -PL is a non ribosomal peptide synthesis and is catalyzed by membrane bound enzymes.In vitro ,-PL synthesis was found to be dependent on ATP and was not affected by ribonuclease,kanamycin or chloramphenicol [18].In a peptide biosynthesis,amino acids are activated either by adeny-lation or phosphorylation of carboxyl group.Adenylation occurs in translation and in the nonribosomal synthesis of a variety of unusual peptides [19,20];Phosphorylation has been suggested for the biosynthesis of glutathione [21].In the former,ATP is con-verted to AMP and pyrophosphate by adenylation,and in the latter,phosphorylation leads to ADP and phosphate as the final prod-ucts.The synthesis of -PL,a homopolypeptide of the basic amino acid l -lysine,is similar to that of poly-(␥-d -glutamate)in terms of adenylation of the substrate amino acid [18].Through the exper-imental observations,the probable mechanism of synthesis was suggested by Kawai et al.showed that in the first step of -PL biosynthesis l -lysine is adenylated at its own carboxyl groups with an ATP-PPi exchange reaction.The active site of a sulfhydryl group of an enzyme forms active aminoacyl thioester intermediates,lead-ing to condensation of activated l -lysine monomer.This is the characteristic feature of nonribosomal peptide synthetase enzyme [22–24].-PL producing strain of Streptomyces albulus was found to pro-duce -PL synthetase (Pls).A gene isolated from the strain was identified as a membrane protein with adenylation and thiolation domains which are characteristic features of the nonribosomal pep-tide synthetases (NRPSs).-PL synthetase has six transmembrane domains surrounding three tandem soluble domains without any thioesterase and condensation domain.This tandem domain itera-tively catalyzes l -lysine polymerization using free l -lysine polymer as an acceptor and Pls-bound l -lysine as a donor,thereby yielding chains of diverse length (Fig.2).Thus,-PL synthetase acts as a ligase for peptide bond formation [25].Yamanaka et al.suggested that -PL synthetase function is regulated by intracellular ATP and found that acidic pH conditions are necessary for the accumulation of intracellular ATP,rather than the inhibition of the -PL degrading enzyme [26].4.Biosynthesis and molecular geneticsThe precursor of -PL biosynthesis was identified to be l -lysine by radiolabeling studies using [14C]-l -lysine in Streptomyces72S.C.Shukla et al./Biochemical Engineering Journal 65 (2012) 70–81Fig.2.Mechanism for synthesis of -polylysine.albulus 346[18].However,a high-molecular-weight plasmid (pNO33;37kbp)was detected in -PL-producing S.albulus ,and the replicon of pNO33was used to construct a cloning vector for S.albu-lus strain [27].The order and number of NRPSs modules determine the chain length of the -PL [24,28].However,the chain length of -PL was shortened by the use of aliphatic hydroxy-compound and -cyclodextrin derivative [29,30].-PL with more than nine l -lysine residues severely inhib-ited the microbial growth while the -PL with less than nine l -lysine residues showed negligible antimicrobial activity.All the strains producing -PL from glycerol showed lower number aver-age molecular weight (M n )than those obtained from glucose [31].The -PL-degrading activity was detected in both -PL tolerant and -PL producing bacteria.The presence of -PL-degrading activity in Streptomyces strains is closely related with -PL-producing activ-ity,which indicates that tolerance against -PL is probably required for -PL producers.The presence of -PL degrading enzyme is detri-mental to industrial production of -PL.Therefore,-PL degrading enzyme of S.albulus was purified,characterized and the gene encoding an -PL degrading enzyme of S.albulus was cloned,and analyzed [32].The -PL-degrading enzyme of S.albulus is tightly bound to the cell membrane.The enzyme was solubilized by NaSCN in the presence of Zn 2+and was purified to homogeneity by phenyl-Sepharose CL-4B column chromatography,with a molecular mass of 54kDa.The enzymatic mode of degradation was exotype mode and released N-terminal l -lysine’s one by one.Streptomyces vir-giniae NBRC 12827and Streptomyces noursei NBRC 15452showed high -PL-degrading aminopeptidase activity and both strains have the ability to produce -PL,indicating a strong correlation between the existence of -PL degrading enzyme and -PL produc-ing activity [33].-PL degrading enzymes were also found in -PL tolerant microorganisms,Sphingobacterium multivorum OJ10and Chryseobacterium sp.OJ7,which were isolated through enrichmentof the culture media with various concentrations of -PL.S.mul-tivorum OJ10could grow well,even in the presence of 10mg/ml -PL,without a prolonged lag phase.The -PL-degrading enzyme activity was also detected in the cell-free extract of -PL tolerant S.multivorum OJ10.The enzyme catalyzed an exotype degradation of -PL and was Co 2+or Ca 2+ion activated aminopeptidase.This indicates the contribution of -PL-degrading enzymes to the toler-ance against -PL [34].An -PL degrading enzyme of -PL tolerant Chryseobacterium sp.OJ7,was also characterized and the purified enzyme catalyzed the endotype degradation of -PL,in contrast to those of Streptomyces albulus and Sphingobacterium multivorum OJ10.Probably,their possession of proteases enables their growth in the presence of a high -PL concentration.-PL degradation was also observed by commercially available proteases,such as Pro-tease A,Protease P and Peptidase R [34,35].5.Microbial production of -polylysinePolylysine can be synthesized by chemical polymerization start-ing from l -lysine or its derivatives.Researchers described two different routes to polymerize lysine residues without the use of protection groups.However,linear -PLL can be obtained by applying 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as an activating agent for the polycondensation of l -lysine in an aqueous medium.In contrast to this,␣-poly(l -lysine)can be obtained by using dicyclohexyl carbodiimide and 18-crown-6ether in chloro-form [36].Dendrimeric ␣,-polylysine were synthesized by using solid phase peptide synthesis method and used dendritic ␣,-polylysine as a delivery agent for oligonucleotides [37,38].Moccia et al.for the first time reported ␣,-polylysine by assembling Fmoc and Boc protected l -lysine monomers by solid phase synthesis [39].Guo et al.synthesized -PL-analogous polypeptides with not only similar ␣-amino side groups but also similar main chain throughS.C.Shukla et al./Biochemical Engineering Journal65 (2012) 70–8173microwave assisted click polymerization technique[40].Recently, Roviello et al.synthesized a cationic peptide based on l-lysine and l-diaminobutyric acid for thefirst time by solid phase synthesis [41].-PL was discovered as an extracellular material produced by filamentous actinomycetes group of micro-organism Streptomyces albulus ssp.Lysinopolymerus strain346more than35years ago [5].It is synthesized by a nonribosomal peptide synthetase and released extracellularly.In actinomycetes group of organisms l-lysine is synthesized through the diaminopimelic acid pathway. Diaminopimelate is formed via l-aspartate(Asp)produced by com-bining oxaloacetate in the tricarboxylic acid cycle with ammonium as a nitrogen source.Citrate was found to be facilitator for the production much more than other organic acids of TCA cycle[24].Studies revealed that decline in pH during the fermentation pro-cess is an essential condition for the accumulation of-PL.Shima et al.carried out two-step cultivation method for S.albulus.Strain wasfirst grown for24h in a culture medium containing glycerol as carbon source with yeast extract,then in second step medium was replaced by glucose,citric acid with(NH4)2SO4[42].It was found that the mutant of strain346decreases the culture pH from its initial value of6.8–4.2by36h,and slowly decreased thereafter to 3.2at96h.The accumulation of-PL in the broth increased signifi-cantly when the culture pH was about4.0.The fed batch cultivation was adopted to enhance the-PL production with two distinct phases.In phase I,cell was grown at pH(6.8)optimum for cul-ture growth then in phase II,the pH was kept around4.0by the addition of glucose.Depletion of glucose causes an increase in pH of the culture broth leading to the degradation of the produced -PL.Thus the pH control strategy in fed batch culture success-fully enhanced the yield of-PL to almost9fold[43].The airlift bioreactor(ABR)was also evaluated and compared with jar fer-mentor for-PL production.The results showed that the production level of-PL in a ABR with a power consumption of0.3kW/m3was similar to that in a5-l jar fermentor with power consumption of 8.0kW/m3.The leakage of intracellular nucleic acid(INA)-related substance into the culture broth in the ABR was70%less than that in the jar fermentor.Thus,ABR system with low intracel-lular nucleic acid-related substances minimize the difficulties of downstream processing for recovery and purification of the poly-mer products.Furthermore,the use of ABR is promising tool for the low-cost production of-PL of high purity[44].In some-PL producing strains,the production of-PL is unstable and depen-dent on cell density which can cause problem such as high viscosity and low oxygen transfer efficiency.Furthermore,increase of agita-tion speeds leads to the rise of shear stresses which might cause undesired effects on mycelial morphology,product formation,and product yields.Bioprocesses using immobilized cells on various inert supports can increase overall productivity and minimize pro-duction costs[45].Bankar et al.reported that aeration and agitation of the fermentation broth markedly affect-PL production,cell mass formation,and glycerol utilization.Fermentation kinetics per-formed revealed that-PL production is growth-associated,and agitation speed of300rpm and aeration rate at2.0vvm supports higher yields of-PL[46].Many efforts have been made to opti-mize the media in order to enhance the productivity of-PL.Shih and Shen applied response surface methodology for optimization of-PL production by Streptomyces albulus IFO14147[47].It was found that-PL production started on agar plated with iron two or three days earlier than that on plates without iron.Manganese and cobalt were also found to have stimulating effect on-PL produc-tion.Kitasatospora kifunense strain produces-PL of shorter chain length about8–17lysine residues[48].Metabolic precursors such as amino acids,tricarboxylic acid cycle intermediates and cofactors have been investigated for improved production of-PL.Addition of citric acid after24h and l-aspartate after36h of fermentation medium had a significant effect on-PL production[49].Zhang et al.investigated the production of-PL on immobilized cells of Kitasatospora sp.MY5-36on bagasse,macroporous silica gel,syn-thetic sponge,loofah sponge and found that loofah sponge gave highest production of-PL in shakeflask culture[50].6.Screening and detection of-PL production in microbial systemNishikawa and Ogawa developed a simple screening method to detect-PL producing microbes.Screenings were carried out on agar plates containing either basic or acidic dyes.The dyes used were,Poly R-478,Remazol Brilliant Blue-R(RBBR)and Methylene blue.The screening method was based on the rationale interac-tion that occurs between charged groups of the secreted-PL and charged group of the basic or acidic dyes.A synthetic glycerol(SG) medium containing either0.02%of acidic dye Poly R-478/RBBR or0.002%of Methylene blue was used for the primary screen-ing.The SG medium was composed of glycerol10g,ammonium sulfate0.66g,sodium dihydrogen phosphate0.68g,magnesium phosphate heptahydrate0.25g,yeast extract0.1g,and1.0ml of Kirk’s mineral solution in1l of distilled water.The pH was adjusted to7.0with1M NaOH solution,and the medium was solidified by adding1.5%agar.The plates were incubated at28◦C for about one week;microbes forming specific colonies interacting with dyes were picked up and purified after several culture transfers.The acidic dye condensed around the organism’s colonies while basic dye was excluded from the surrounding zone.A zone of at least five mm in diameter for each colony was needed to visualize the interaction between secreted substances and dyes[16].The concentrations of-PL in the culture broth can be deter-mined by using either the spectrophotometric method or HPLC method.The colorimetric method is based on the interaction between-PL and methyl orange,which is an anionic dye,and thus the interaction of cationic-PL with anionic methyl orange in the reaction mixture led to form a water insoluble complex[51].The HPLC method for-PL detection was reported by Kahar et al.in which HPLC column(Tsk gel ODS-120T,4.6mm×250mm)with a mobile phase comprising of0.1%H3PO4was used[43].7.Purification and characterization of-PL-PL a cationic polymer,can be isolated at neutral pH,and puri-fied from the culture broth by ion exchange chromatography using an Amberlite IRC-50(H+form)column[5,52].The culture super-natant can be passed through an Amberlite IRC-50column at pH 8.5with successive washing by0.2N acetic acid and water.The elution can be made with0.1N hydrochloric acid,and the eluate can be neutralized with0.1N sodium hydroxide to pH6.5.Sub-sequent purification can be done by using CM-cellulose column chromatography to get-PL in homogeneity.The purification of the product can be monitored by UV absorption at220nm and fur-ther characterized by amino acid analysis.The molecular weight of-PL can be estimated by gelfiltration on a Sephadex column [16,53].Kobayashi et al.extracted the-PL from Kitasatospora kifu-nense.The pH of the culturefiltrate wasfirst adjusted to7.0,and the aliquot was mixed with Gly-His-Lys acetate salt as an inter-nal peptide standard.The resulting mixture was then applied to Sep-Pak Light CM cartridge.The cartridge was washed with water and-PL was eluted with0.1M HCl.The eluate was lyophilized and the residue was dissolved in0.1%pentafluoropropionic acid [46].Recently,ultra-filtration technique for fractionation of-PL of different molecular weight has been applied.The-PL with molec-ular weight higher than2kDa form a-turn conformation whereas molecular weight smaller than2kDa possesses a random coil74S.C.Shukla et al./Biochemical Engineering Journal65 (2012) 70–81conformation.The fraction of-PL with molecular weight higher than2kDa was found to have significant antibacterial activity, while the fraction with molecular weight smaller than2kDa shows nominal antibacterial activity[54].8.Conformation of-PLStructure and conformation studies are prerequisite to under-stand the functional behavior of-PL.Numerous workers have investigated the conformation and the molecular structure of microbially produced-PL by NMR,IR and CD spectroscopy[55,56]. The thermal property of crystalline-PL was determined by Lee et al.[52].The glass transition temperature(T g)and the melting point(T m)was observed to be88◦C and172.8◦C respectively.The results from pH dependent IR and CD spectra,1H and13C NMR chemical shifts together with that of13C spin-lattice relaxation times T1indicated that-PL assumes a-sheet conformation in aqueous alkaline solution.-PL at acidic pH might be in an electro-statically expanded conformation due to repulsion of protonated ␣-amino group,whereas at elevated pH(above p K a of the␣-amino group)the conformation was found to be similar to the antiparallel -sheet.The molecular structure and conformation of microbial-PL was studied by FT-IR and Raman spectroscopy.-PL was found to assumed a-sheet conformation in the solid state and solid state 13C NMR also revealed that-PL existed as a mixture of two crys-talline forms.Spin-lattice relaxation times yield two kinds of T1s corresponding to the crystalline and amorphous components,with the degree of crystallinity as63%[57].Solid-state high-resolution13C and15N NMR spectra of micro-bial-PL derivatives with azo dyes have been measured.These chemically modified-PL’s Exhibit15N NMR signals characteristic of the binding mode at the␣-amino groups.The spectral analy-sis reveals that the-PL/DC sample contains a small amount of ion complexes with methyl orange(MO).It has been shown that side chain␣-amino group of-PL does not make a covalent bond with methyl orange(MO)but forms a poly-ion complex,(-PL)-NH3+SO3−-(MO).On the other hand,dabsyl chloride(DC)makes covalent bond with-PL to form sulfonamide,(-PL)-NH-SO2-(DC). However,a few tens percent of DC change to MO by hydrolysis to form a poly-ion complex,(-PL)-NH3+SO3−-(MO)[58].Rosenberg and Shoham characterized the secondary structure of polylysine with a new parameter namely,the intensity ratio of the bands of charged side chain amine NH3+and amide NH bands.The enthalpy of the secondary structure transition,which is observed in PLL at the change of pH from11to1amounts to4.7kJ mol−1[59].9.Application of polylysine in medicinePolylysine is available in a large variety of molecular weights. As a polypeptide,polylysine can be degraded by cells effortlessly. Therefore,it has been used as a delivery vehicle for small drugs[60]. The epsilon amino group of lysine is positively charged at phys-iological pH.Thus,the polycationic polylysine ionically interacts with polyanion,such as DNA.This interaction of polylysine with DNA has been compacted it in a different structure that has been characterized in detail by several workers[61–66].In addition,the epsilon amino group is a good nucleophile above pH8.0and there-fore,easily reacts with a variety of reagents to form a stable bond and covalently attached ligands to the molecule.Several coupling methods have been reported for preparation of conjugated of-PL [67–70].(a)Modification of epsilon amino groups of polylysine with bifunctional linkers containing a reactive esters,usually add a reac-tive thiol group to the polylysine molecule and consequent reaction with a thiol leads to a disulfide or thioether bond,respectively.This has been used to couple large molecules,such as proteins to polylysine.(b)Compounds containing a carboxyl group can be acti-vated by carbodiimide,leading to the formation of an amide bond with an epsilon amino group of polylysine.(c)Aldehydes,such as reducing sugars or oxidized glycoprotein,form hydrolysable schiff bases with amino groups of-PL,which can be selectively reduced with sodium cyanoborohydride to form a stable secondary amine.(d)Isothiocyanate reacts with epsilon amino groups by forming a thiourea derivative.(e)Antibody coupling can also be done specif-ically to the N-terminal amino group of polylysine[71,72].A variety of molecules such as proteins,sugar molecules and other small molecules have been coupled to polylysine by using these methods.Purification of the conjugates are usually being achieved by dialysis or gelfiltration in conjunction with ion-exchange chromatography or preparative gel electrophoresis. Fractionation of the ligand–polylysine ratio and conjugate size can be done by using acid urea gel electrophoresis in combination with cation-exchange HPLC,ninhydrin assay and ligand analysis (sugar,transferrin,etc.)[73].Galactose terminated saccharides such as galactose,lactose and N-acetylgalactosamine were found to be accumulated exclusively in the liver,probably by their hepatic receptor.These conjugates could therefore be excellent carriers for a drug delivery system to the liver.The other saccharides such as the mannosyl and fucosyl conjugates are preferentially delivered to the reticuloendothelial systems such as those in the liver,spleen and bone marrow.In particular,fucosyl conjugates accumulated more in the bone marrow than in the spleen whereas xylosyl con-jugates accumulated mostly in the liver and lung.Generally,the accumulated amount in the target tissue increased with increasing molecular weight and an increased number of saccharide units on each monomer residues of polymer[74].One of the disadvantages of polylysine from the pharmaceu-tical point of view is its heterogeneity with respect to molecular size.The size distribution of polylysine with degrees of polymer-ization(dp)can be reduced by gel permeation chromatography. Al-Jamal et al.studied sixth generation(G6)dendrimer molecules of␣-poly-l-lysine(␣-PLL)to exhibit systemic antiangiogenic activ-ity that could lead to solid tumor growth arrest.Their work showed that G6PLL dendrimer have an ability to accumulate and persist in solid tumor sites after systemic administration and exhibit antian-giogenic activity[75].Sugao et al.reported6th generation dendritic ␣-PLL as a carrier for NFB decoy oligonucleotide to treat hepatitis [76].Han et al.synthesized a new anti-HIV dendrimer which con-sisted of sulfated oligosaccharide cluster consisting with polylysine core scaffold.The anti-HIV activity of polylysine-dendritic sulfated cellobiose was found to have EC50-3.2g/ml for viral replication which is as high as that of the currently clinically used AIDs drugs. The results also indicated that biological activities were improved because of dendritic structure in comparison to oligosaccharide cluster which were reported to have low anti-HIV activity[77].9.1.Polylysine as a drug carrierPolylysine can be used as a carrier in the membrane transport of proteins and drugs.Shen and Ryser reported that␣-PLL was found to be easily taken up by cultured cells.In fact,the conju-gation of drug to polylysine markedly increased its cellular uptake and offers a new way to overcome drug resistance related to defi-cient transport[60,78,79].Resistance toward methotrexate has been encountered in the treatment of cancer patients.The poly lysine conjugates of methotrexate(MTX)were taken up by cells at a higher rate than free drugs form.This increased uptake can overcome drug resistance due to deficient MTX transport.Addi-tion of heparin at a high concentration restores growth inhibitory effect of MTX-poly lysine[11,60].Shen and Ryser worked conjuga-tion of␣-PLL to human serum albumin and horseradish-peroxidase。
郭浩，白雪媛，陈宇，等. 人参醇溶蛋白提取工艺优化、结构表征及体外抗氧化活性分析[J]. 食品工业科技，2024，45（8）：1−10. doi:10.13386/j.issn1002-0306.2023070055GUO Hao, BAI Xueyuan, CHEN Yu, et al. Optimization of the Extraction Process, Structural Characterization and Antioxidant Activity of Ginseng Alcohol Soluble Proteins[J]. Science and Technology of Food Industry, 2024, 45(8): 1−10. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023070055· 特邀主编专栏—食品中天然产物提取分离、结构表征和生物活性（客座主编：杨栩、彭鑫） ·人参醇溶蛋白提取工艺优化、结构表征及体外抗氧化活性分析郭 浩，白雪媛，陈 宇，宋文博，刘 那，王思明*（长春中医药大学东北亚中医药研究院，吉林长春 130117）摘 要：为探讨人参中醇溶蛋白提取工艺，结构表征及体外抗氧化活性，本文以人参为研究对象，采用单因素实验和响应面试验探索人参醇溶蛋白的最佳提取工艺，以紫外吸收光谱法、红外分光光度分析法、氨基酸组成、微观结构观察等方法对人参醇溶蛋白进行结构表征，并对其在不同pH 条件下的体外抗氧化活性开展一系列研究。
通过测定DPPH 自由基清除能力、羟基自由基清除能力、铁离子还原能力，评价人参醇溶蛋白的体外抗氧化活性。
结果表明，人参中醇溶蛋白最佳提取工艺为：提取时间2 h 、提取料液比1:10 g/mL 、提取pH 为7，此时人参醇溶蛋白得率为0.319%，蛋白含量为75%。
高一英语生物词汇单选题30题1.The basic unit of life is the_____.A.atomB.moleculeC.cellD.tissue答案：C。
“atom”是原子；“molecule”是分子；“cell”是细胞，生命的基本单位是细胞；“tissue”是组织。
2.Which structure is responsible for controlling what enters and leaves the cell?A.nucleusB.cell membraneC.cytoplasmD.chloroplast答案：B。
“nucleus”是细胞核；“cell membrane”是细胞膜，负责控制物质进出细胞；“cytoplasm”是细胞质；“chloroplast”是叶绿体。
3.The power house of the cell is_____.A.nucleusB.mitochondrionC.endoplasmic reticulumD.Golgi apparatus答案：B。
“nucleus”是细胞核；“mitochondrion”是线粒体，被称为细胞的动力工厂；“endoplasmic reticulum”是内质网；“Golgi apparatus”是高尔基体。
4.Which organelle is involved in protein synthesis?A.ribosomeB.lysosomeC.vacuoleD.peroxisome答案：A。
“ribosome”是核糖体，参与蛋白质合成；“lysosome”是溶酶体；“vacuole”是液泡；“peroxisome”是过氧化物酶体。
5.The storage site of genetic information is the_____.A.nucleusB.mitochondrionC.chloroplastD.cytoplasm答案：A。
Consolidated bioprocessing of cellulosic biomass:an update Lee R Lynd1,2,Willem H van Zyl2,John E McBride1and Mark Laser1Biologically mediated processes seem promising for energyconversion,in particular for the conversion of lignocellulosicbiomass into fuels.Although processes featuring a stepdedicated to the production of cellulase enzymes have beenthe focus of most research efforts to date,consolidatedbioprocessing(CBP)–featuring cellulase production,cellulosehydrolysis and fermentation in one step–is an alternativeapproach with outstanding potential.Progress in developingCBP-enabling microorganisms is being made through twostrategies:engineering naturally occurring cellulolyticmicroorganisms to improve product-related properties,suchas yield and titer,and engineering non-cellulolytic organismsthat exhibit high product yields and titers to express aheterologous cellulase system enabling cellulose utilization.Recent studies of the fundamental principles of microbialcellulose utilization support the feasibility of CBP.Addresses1Thayer School of Engineering,Dartmouth College,Hanover,NewHampshire03755,USA2Department of Microbiology,University of Stellenbosch,Private Bag XI,Stellenbosch,Matieland7602,South AfricaCorresponding author:Lynd,Lee R(lee.lynd@)Current Opinion in Biotechnology2005,16:577–583This review comes from a themed issue onBiochemical engineeringEdited by Govind RaoAvailable online8th September20050958-1669/$–see front matter#2005Elsevier Ltd.All rights reserved.DOI10.1016/j.copbio.2005.08.009IntroductionEnergy conversion,utilization and access underlie manyof the great challenges of our time,including thoseassociated with sustainability,environmental quality,security and poverty.New applications of emergingtechnologies will be required to respond to these chal-lenges[1,2 ].As one of the most powerful of thesetechnologies,biotechnology could give rise to importantnew energy conversion processes.Resources for thebiological conversion of energy to forms useful to human-ity include plant biomass and derivatives thereof(e.g.synthesis gas and animal wastes),sunlight[3,4],inorganiccompounds[5 ],and fossil resources(e.g.oil,coal andnatural gas)[6].Energy carriers that can be made fromthese resources include organic fuels,electricity[4,5 ]and hydrogen[3].Among forms of plant biomass,lignocellulosic biomass(‘biomass’)is particularly well-suited for energy applica-tions because of its large-scale availability,low cost andenvironmentally benign production[7].In particular,many energy production and utilization cycles based oncellulosic biomass have near-zero greenhouse gas emis-sions on a life-cycle basis[8–10].The primary obstacleimpeding the more widespread production of energy frombiomass feedstocks is the general absence of low-costtechnology for overcoming the recalcitrance of thesematerials[7].Biomass processing schemes involving enzymatic ormicrobial hydrolysis commonly involve four biologicallymediated transformations:the production of saccharolyticenzymes(cellulases and hemicellulases);the hydrolysis ofcarbohydrate components present in pretreated biomassto sugars;the fermentation of hexose sugars(glucose,mannose and galactose);and the fermentation of pentosesugars(xylose and arabinose).These four transformationsoccur in a single step in a process configuration calledconsolidated bioprocessing(CBP),which is distinguishedfrom other less highly integrated configurations in that itdoes not involve a dedicated process step for cellulaseproduction.Thermochemical processing options appearmore promising than biological options for the conversionof the lignin fraction of cellulosic biomass,which can have adetrimental effect on enzymatic hydrolysis but also servesas a source of process energy and potential coproducts thathave important benefits in a life-cycle context[7,11 ].Fundamental and applied topics relevant to CBP werecomprehensively reviewed in2002[12 ].Here,we pro-vide an updated perspective focusing on recent develop-ments.Ethanol production is emphasized,as this hasbeen the focus of most work pursuant to CBP to date.In principle,however,the CBP strategy is applicable toany fermentation product.The motivation for consolidatedbioprocessingAs addressed in more detail elsewhere[12 ],CBP offersthe potential for lower cost and higher efficiency thanprocesses featuring dedicated cellulase production.Thisresults from avoided costs for capital,substrate and otherraw materials,and utilities associated with cellulase pro-duction.In addition,several factors support the possibi-lity of realizing higher hydrolysis rates,and hencereduced reactor volume and capital investment,usingCBP.These include enzyme–microbe synergy(discussedbelow),as well as the use of thermophilic organismsand/or complexed cellulase systems.Moreover,cellulose-adherent cellulolytic microorganisms are likely to successfully compete for products of cellulose hydro-lysis with non-adhered microbes,including contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization.Over the past few years,much effort has been devoted to reducing the cost of producing cellulase enzymes[13]. Following greater than tenfold cost reductions,cellulase production costs have recently been reported in the range of10to20cents per gallon(¢/gal)of ethanol produced [14 ].These exciting developments could well enable a variety of formerly infeasible industrial processes,but do not diminish the potential of CBP to offer significantly lower costs than other configurations.Figure1compares the projected costs for biological processing associated with ethanol production for a CBP process and for an advanced process featuring at-site dedicated cellulase production in combination with simultaneous saccharifi-cation with co-fermentation of hexose and pentose sugars (SSCF).Adding together the9.90¢/gal ethanol for dedi-cated cellulase production and9¢/gal for SSCF gives a total cost for biological processing of18.9¢/gal,which is more than fourfold larger than the4.2¢/gal projected for CBP.For the advanced process scenarios analyzed here, identical in every respect except for the configuration assumed for biological processing and consequences thereof,the projected wholesale selling price of ethanol from a cellulosic feedstock costing$40/dry ton is77¢/gal ($1.08/gal gasoline equivalent)for the dedicated cellulase production/SSCF scenario and63¢/gal($0.88/gal gaso-line equivalent)for CBP.The difference between these projected prices,20¢/gal gasoline equivalent,is particu-larly important for fuel production with its low manufac-turing margins and price-driven competitive markets.For comparison,the average wholesale price of gasoline was $0.98/gal for the period2001to2004and$1.32/gal for the first quarter of2005[15].Organism development strategies Microorganisms with the combination of substrate-utili-zation and product formation properties required for CBP are not currently available,but could probably be devel-oped given sufficient effort.Such developments can be pursued by two strategies(Figure2).The native cellu-lolytic strategy involves engineering naturally occurring cellulolytic microorganisms to improve product-related properties,such as yield and titer.The recombinant578Biochemical engineeringFigure1The comparative cost of ethanol production by consolidated bioprocessing(CBP)and by simultaneous saccharification and co-fermentation (SSCF)featuring dedicated cellulase production.Processes are simulated assuming aggressive performance parameters intended to be representative of mature technology.Cellulase production:yield,400FPU(filter paper unit)/g carbohydrate;productivity,400FPU/L/h.Hydrolysis and fermentation yields are set at95%of the theoretical value for both CBP and SSCF.The SSCF reaction time is7days.Savings in the SSCF reactor accompanying reaction times less than7days are completely offset by the increased enzyme costs associated with higher cellulase loadings required to achieve shorter reaction times.The CBP reaction time is set at1.5days,consistent with the expectation of higher hydrolysis rates(see text).If the reaction time were increased to3days,the cost of CBP increases from4.4¢/gallon to5.5¢/gal.(Simulations were carried out using ASPEN-based models based on the modeling framework developed at the National Renewable Energy Laboratory[55,56],with modifications for mature technology as described elsewhere[2].)cellulolytic strategy involves engineering non-cellulolytic organisms that exhibit high product yields and titers so that they express a heterologous cellulase system that enables cellulose utilization.Each strategy involves con-siderable uncertainty,and different strategies could prove advantageous for different products.In light of this and other considerations[12 ],we believe that both the native and recombinant strategies for CBP organism development merit investigation.The utilization of cellulose by microorganisms involves a substantial set of fundamental phenomena beyond those associated with enzymatic hydrolysis of cellulose[12 ], many of which are incompletely understood.Advances in understanding the fundamentals of microbial cellulose utilization will both enable and draw from advances in organism development for CBP.Native cellulolytic strategyNaturally occurring cellulolytic microorganisms are start-ing points for CBP organism development via the native strategy,with anaerobes being of particular interest [12 ].The primary objective of such developments is to engineer product yields and titers to satisfy the require-ments of an industrial process.Metabolic engineering of mixed-acid fermentations in relation to these objectives has been successful in the case of mesophilic,non-cellu-lolytic,enteric bacteria[16].Far more limited work of this type has been undertaken with cellulolytic bacteria, primarily because of the absence of suitable gene-transfer techniques.Recent developments,however,appear to be removing this limitation for some organisms.Among cellulolytic anaerobes,gene-transfer systems have been described for Clostridium cellulolyticum[17,18]and for Clostridium thermocellum[19 ].Electrotransformation (ET)protocols for these organisms,first described in2000 for C.cellulolyticum and in2004for C.thermocellum,involve rather specialized conditions and,in the case of C.thermo-cellum,apparatus.ET-mediated transfer and expression of foreign genes has also been reported for Thermoanaero-bacterium thermosaccarolyticum[20]and Thermoanaerbacter-Consolidated bioprocessing Lynd et al.579Figure2Organism development strategies and related fundamentals.ium saccharolyticum[21,22],non-cellulolytic pentose-uti-lizing anaerobic thermophiles with substrate-utilizing capabilities that compliment those of C.thermocellum (which does not ferment pentoses).Efforts to engineer the end-product metabolism of cellulose-utilizing anae-robes are limited to the2002report of Guedon et al.[23], in which expression of pyruvate decarboxylase and alco-hol dehydrogenase resulted in increased growth, decreased production of lactate,and increased production of acetate and ethanol.In T.saccharolyticum,production of lactic acid has been completely eliminated by targeted gene knockout[24 ]and elimination of acetic acid production has also recently been achieved(J Shaw, unpublished).ET efficiencies for C.thermocellum($105 transformants per experiment using replicative plasmids [19 ])appear high enough to allow similar targeted gene knockouts to be obtained in this organism.Product tolerance is a key property impacting the feasi-bility of CBP through the native cellulolytic strategy. Recent studies have been carried out to select and char-acterize ethanol-tolerant strains of C.thermocellum[25 ]. Growth of a selected strain was found to occur at ethanol concentrations exceeding60g/L,a titer sufficient not to put thermophiles at a disadvantage relative to more conventional ethanol producers in the context of ligno-cellulose conversion[26].Reported ethanol titers pro-duced by C.thermocellum,as well as other thermophiles, are limited however to26g/L[12 ].We think it likely that the discrepancy between tolerance to added ethanol and the maximum titers produced can be resolved in the course of metabolic engineering to maximize ethanol anic acids and their salts are more inhibitory than ethanol for both thermophiles(T.thermosaccharoly-ticum[27])and E.coli[28],and elimination of organic acids has been shown to allow substantially higher neutral product titers in several instances[28](J Pierce,personal communication)(P Soucaille,personal communication). In addition,titers have been brought to levels consistent with tolerance to added products through a sustained effort in the case of production of both ethanol[28] and1,3-propanediol(J Pierce,personal communication) by E.coli.Recombinant cellulolytic strategyNon-cellulolytic microorganisms with desired product formation properties(e.g.high yield and titer)are starting points for CBP organism development by the recombi-nant cellulolytic strategy.The primary objective of such developments is to engineer a heterologous cellulase system that enables growth and fermentation on pre-treated lignocellulose.Given the complexity and effort required to achieve this objective,it is appropriate to examine its feasibility.On the basis of the sufficiency of expression of growth-enabling heterologous enzymes[29 ],the level of enzyme expression required to achievea specified growth rate may be calculated as a function of enzyme specific activity.For growth enabled by cellulase with specific activities in the range available,required expression levels are well within the range reported in the literature(1–10%of cellular protein;Figure3)[30,31]. Protein expression at this level has been reported in both Saccharomyces cerevisiae[30]and E.coli[32],although not to date for active cellulases.The feasibility of CBP580Biochemical engineeringFigure3Required protein expression to achieve growth on cellulose in relation to cellulase specific activity.Cellulase requirements are calculated using the following expression:percentage cell protein={(m/Y X/S)/[(U/mg cellulase)Â(0.00018g/m mol)Â(60min/h)]}Â(1g cells/0.5g cell protein), where m=specific growth rate on the substrate(0.02hÀ1)and Y X/S=cell yield on glucose(0.45g cells/g substrate for aerobic growth and0.1g cells/g substrate for anaerobic growth).Data for T.reesi were obtained from[57]and for C.thermocellum from[58].through the recombinant approach is further supported by models that combine fermentation bioenergetics and the kinetics of pretreated substrate hydrolysis with declining reactivity over the course of the reaction[33]. It is likely that evolutionary approaches will be valuable in augmenting the rational design of heterologous cellu-lase expression systems[34 ].To date,the heterologous production of cellulases has been pursued primarily with bacterial hosts producing ethanol at high yield(engineered strains of E.coli,Klebsiella oxytoca and Zymomonas mobilis)and the yeast S.cerevisiae. Cellulase expression in strains of K.oxytoca resulted in increased hydrolysis yields(but not growth without added cellulase)for microcrystalline cellulose(Avicel,FMC, Philadelphia),and anaerobic growth on amorphous cellu-lose[35,36].Although dozens of saccharolytic enzymes have been functionally expressed in S.cerevisiae[12 ], anaerobic growth on cellulose as the result of such expres-sion has not been definitively demonstrated.Recently, Kondo and coworkers expressed cellulases[37 ],xyla-nases[38],and amylases[39]on the cell surface of different S.cerevisiae strains.High cell density suspensions of the recombinant strains fermented amorphous cellulose,raw starch,and birchwood xylan to ethanol with yields of0.45, 0.44and0.3g ethanol/g substrate,respectively.Several cellobiohydrolases have been functionally expressed in S. cerevisiae[40–44].The specific activity of recombinant cellobiohydrolase was shown to be comparable to that of the native enzyme[43](R den Haan et al.,unpublished), suggesting that increased expression levels should be a target for further research.As might be expected,conferring the ability to grow on non-native substrates as a result of heterologous enzyme expression is more advanced for soluble substrates than for cellulose.Strains of E.coli[18,28],S.cerevisiae[45],and Z.mobilis[46]able to ferment xylose to ethanol were developed in the1990s.More recently,several strains of S.cerevisiae with expanded substrate utilization capability have been developed:a xylose-isomerase-expressing strain that grows well(maximum specific growth rate m max=0.09hÀ1)on xylose[47 ];a strain expressing the genes of the bacterial L-arabinose utilization pathway and overexpressing the yeast galactose permease allowing the fermentation of arabinose[48];and a b-glucosidase-expres-sing strain able to grow anaerobically on cellobiose at rates comparable to glucose[29].Strains of S.cerevisiae able to ferment starch were developed in earlier work and achieve impressive yields and titers without added amylase[49]. Selected fundamentals impacting the feasibility of CBPExperimental evidence supporting the feasibility of CBP comes from a recent study of cellulose utilization by C. thermocellum[50 ].It was shown in this study that the average degree of polymerization of cellulose hydrolysis products taken up by C.thermocellum is about four glucose units;thus,the mechanism of cellulose hydrolysis differs from that of fungal cellulase systems for which cellobiose is the primary hydrolysis product.Both physiological studies and experiments with14C-cellulose indicate that bioenergetic benefits specific to growth on cellulose are realized as a result of the efficiency of oligosaccharide uptake combined with intracellular phosphorolytic clea-vage of b-glucosidic bonds[51],and that these benefits exceed the bioenergetic cost of cellulase synthesis[52]. This work provides insight into how naturally occurring cellulolytic microorganisms are able to achieve rapid growth on cellulose(e.g.m max=0.16hÀ1on crystalline cellulose[12 ])in spite of the modest ATP available from anaerobic fermentation.A further phenomenon impacting the potential feasibility and performance of CBP is enzyme–microbe synergy—that is,the possibility that the effectiveness of the cellu-lase is enhanced when it is present as cellulose–enzyme–microbe(CEM)complexes as compared with cellulose–enzyme(CE)complexes.CEM complexes have a central role in microbial cellulose hydrolysis by anaerobic microbes[12 ],whereas cellulose hydrolysis in envi-sioned industrial processes featuring dedicated cellulase production is carried out exclusively by CE complexes that do not involve the presence of a living,cellulolytic microorganism.In observations dating back to1956, cellulase pioneers Reese and Mandels[53,54]observed that rates of hydrolysis are substantially higher when mediated by growing cellulolytic cultures as compared to enzymatic preparations,even under optimized condi-tions.Preliminary work underway in the Lynd laboratory indicates that cellulase-specific hydrolysis rates are sev-eral fold higher for C.thermocellum cultures than for cell-free cellulase preparations(Y Lu et al.,unpublished). Definitive quantitative evaluation of enzyme–microbe synergy is an important objective for future research, and could provide further evidence supporting the desir-ability of CBP.Although studies aimed at understanding the fundamen-tals of microbial cellulose utilization have necessarily focused on naturally occurring cellulolytic bacteria, insights from such studies are of importance to both the native and recombinant organism development stra-tegies.In particular,it may be highly desirable,and perhaps necessary,to incorporate features of naturally occurring cellulolytic microbes(e.g.high specific activity complexed cellulase systems,uptake and phosphorolytic cleavage of oligosaccharides,and adherence of cells to cellulose)when developing recombinant cellulolytic microbes for use in CBP.ConclusionsCBP has the potential to provide the lowest cost route for biological conversion of cellulosic biomass to fuels andConsolidated bioprocessing Lynd et al.581other products in processes featuring hydrolysis by enzymes and/or microorganisms.To realize this potential, microorganisms must be developed that utilize cellulose and other fermentable compounds available from pre-treated biomass with high rate and high conversion,and which produce a desired product at high yield and titer. Both of these capabilities are possessed by known micro-organisms,but to date have not been combined in a single microorganism or microbial system.Several lines of evi-dence support the feasibility of such combinations using biotechnology,which could proceed through two distinct strategies each with several potential host organisms. Success in this endeavor could provide a leap forward with respect to the low-cost conversion of renewable biomass into fuels as well as a variety of industrial chemicals,thereby realizing societal benefits. 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