琼胶酶研究进展

琼胶酶是一种多糖水解酶,根据其降解琼脂糖的作用方式不同,可以分为α-琼胶酶(EC 3.2.1.-)和β-琼胶酶(EC 3.2.1.81).本文结合自己的研究,从琼胶酶的生物学研究、酶的分类、晶体结构、催化机理以及酶的应用等几个方面综述了琼胶酶的研究进展.     

琼胶酶酶学研究进展

琼胶酶是一类能够降解琼胶多糖的酶的总称,根据其降解琼胶多糖的作用方式不同,分为α-琼胶酶(EC3.2.1.158)和β-琼胶酶(EC3.2.1.81)两种。本文从琼胶酶的酶学性质、晶体结构、催化机制和酶基因研究等方面,综述了近年来国内外琼胶酶酶学的最新研究进展,这将有益于琼胶酶未来的应用。     

琼胶酶研究进展*

琼胶酶是一种多糖水解酶,根据降解琼脂糖的作用方式不同,可以分为α-琼胶酶（EC3.2.1.-）和β-琼胶酶（EC3.2.1.81）。中从琼胶酶的分类及酶活测定,酶的来源,产酶微生物的酶系及酶学性质,琼胶酶的分子生物学研究,酶的应用几个方面综述了琼胶酶的研究进展。     

海洋细菌Agarivorans sp.HZ105的琼胶降解酶系

通过克隆得到菌株Agarivorans sp.HZ105中3个琼胶酶基因,长度分别为2 988 bp、1 437 bp和1 362 bp,分别编码琼胶酶HZ1、HZ3和HZ4,分别属于糖苷水解酶GH50、GH118和GH16家族。将这些琼胶酶基因与质粒p ET-32(a)构建重组表达载体,转化大肠杆菌BL21(DE3),实现了琼胶酶基因的重组原核表达,制备了重组酶,研究了琼胶酶的酶解产物。琼胶酶HZ1降解琼脂糖以及高聚合度新琼寡糖(聚合度为8、10、12和14)得到新琼二糖和新琼四糖;琼胶酶HZ3降解琼脂糖的终产物是高聚合度新琼寡糖;琼胶酶HZ4降解琼脂糖和高聚合度新琼寡糖为新琼四糖和新琼六糖。因此推测菌株HZ105主要先用琼胶酶HZ3和HZ4降解琼脂糖为较高聚合度的新琼寡糖,随后这些寡糖被琼胶酶HZ1和HZ2(课题组先前报道的另一个琼胶酶)降解为低聚合度新琼寡糖。首次研究报道了Agarivorans属中能产生4个琼胶酶的细菌菌株及其琼胶降解酶系,丰富了有关细菌降解琼胶酶体系及其中各琼胶酶作用的研究和认识,也有利于菌株HZ105琼胶酶的有效开发应用。     

罗尼氏弧菌Vibrio shilonii BY新琼胶酶基因的克隆、序列分析及表达

【目的】从海洋来源的罗尼氏弧菌菌株BY中克隆得到一个具有琼胶酶活性的新基因,并对其进行重组表达。【方法】对实验室保藏的产琼胶酶菌株BY进行16S rRNA基因序列分析,并构建系统发育树。根据已报道的琼胶酶基因序列的同源性,设计简并引物,利用降落PCR (Touch-down PCR)及染色体步移技术扩增琼胶酶基因序列全长,对基因序列进行生物信息学分析。将目的基因插入pET22a(+)载体,转化大肠杆菌BL21(DE3),对重组酶进行表达,利用DNS法测定了重组酶的酶活,对该重组琼胶酶酶学性质进行研究。【结果】克隆得到一条新的琼胶酶基因,命名为Vibrio sp. BY (GenBank登录号：AIW39921.1),Vibrio sp. BY基因序列全长2 232 bp,编码744个氨基酸,理论分子量为85 kD,Vibrio sp. BY的氨基酸序列基因库中与已知的琼胶酶氨基酸序列Vibrio sp. EJY3的相似度为86%。发酵液琼胶酶酶活力为71.73 U/mL,证明表达的蛋白为琼胶酶。酶学性质研究表明重组琼胶酶的最适温度及pH分别为50 °C和7.0,并且具有较好的稳定性。【结论】利用染色体步移技术克隆得到一条新的琼胶酶基因,并在大肠杆菌BL21(DE3)中实现了重组表达,为琼胶酶的应用奠定了基础。     

琼胶酶的研究进展

琼胶酶是一类能降解琼胶多糖的酶总称,其降解产物具有多种生理活性功能.从琼胶酶的分布来源、应用研究、酶学性质及分子生物学研究现状等方面综述了近年来国内外琼胶酶的最新研究进展.     

一株产琼胶酶细菌的分离、鉴定及其琼胶酶基本性质

【目的】分离海洋来源的琼胶酶产生菌,对其进行分类鉴定,并研究其所产琼胶酶的基本酶学性质,为琼胶酶的应用研究及开发利用奠定基础。【方法】通过以琼脂为唯一碳源的选择培养基分离产琼胶酶的菌株;利用16S rRNA基因序列分析、表型和生理生化特征对菌株进行鉴定;通过DNS-还原糖法测定琼胶酶活性;利用显色底物法测定琼胶酶的类型;对菌株所产琼胶酶粗酶的酶学性质进行初步研究。【结果】分离到一株产琼胶酶的菌株NTa,16S rRNA基因序列分析显示该菌株属于寡养单胞菌属(Stenotrophomonas sp.);该菌株主要产胞外琼胶酶,可分泌α-琼胶酶和β-琼胶酶;琼胶酶粗酶的最适反应温度和pH分别为40℃和7.0,并且琼胶酶在温度低于30℃,pH为7.0-9.0时稳定;Ca2+对琼胶酶粗酶具有促进作用,Ag+、Fe2+、Ba2+、Mn2+、Cu2+、Zn2+和Fe3+均可不同程度地抑制酶的活性;EDTA对琼胶酶粗酶活性具有抑制作用;琼胶酶粗酶对检测的抑制剂、去垢剂及变性剂有较好的抗性。【结论】海洋细菌Stenotrophomonas sp.NTa是一种新型的产琼胶酶菌株,可同时分泌α-琼胶酶和β-琼胶酶,具有潜在开发利用价值。     

封面图片介绍

正琼胶酶(agarase)是一类能够降解琼脂糖的糖苷水解酶,可催化长链琼脂糖分子内糖苷键水解,根据其作用方式可分为α-琼胶酶和β-琼胶酶两大类,目前报道的琼胶酶中β-琼胶酶占绝大多数。琼胶酶水解琼脂糖可以产生琼胶寡糖,这是一类具有抗肿瘤、抗病毒、增强免疫等作用的海洋功能性低聚糖,广泛应用于保健食品、功能饲料、医药以及化妆品领域。琼胶酶可以高效降解藻类细胞壁,是藻类遗传育种中不可或缺     

海洋细菌Agarivorans albus NBRC102603琼胶酶的分离纯化

利用盐析、分子筛和离子交换等方法对海洋细菌Agarivorans albus NBRC102603分泌的琼胶酶粗酶液进行分离纯化,得到琼胶酶A和琼胶酶B.琼胶酶A纯化倍数为17.51倍,酶比活力为881.82 U/mg;琼胶酶B纯化倍数为16.64倍,酶比活力为838.32 U/mg.纯化的琼胶酶经SDS-PAGE检测,显示为单一条带,其相对分子质量分别为酶A 8.36×104和酶B 3.68×104.     

采用随机突变方法对β-琼胶酶YM01-3活性位点的研究

【背景】琼胶酶是一种多糖水解酶,在保健食品、医药、科研及化妆品等行业极具价值。本实验室发现来源自嗜琼胶卵链菌(Catenovulumagarivorans)的β-琼胶酶YM01-3具有较高的酶活性,在最适条件下的比酶活可达到1.14×10~4U/mg。【目的】探讨不同位点的突变对β-琼胶酶YM01-3酶活力的作用,发现影响其酶活力的新位点。【方法】通过易错PCR在短芽孢杆菌(Bacillus brevis)表达系统中构建随机突变文库,从约10 000个克隆中筛选出227株有效突变体,从中选取80株进行测序。【结果】对突变体序列进行分析和定点突变验证发现,137位和237位氨基酸发生突变后酶活力丧失90%以上。【结论】位于催化腔内的137位和237位氨基酸,对于维持β-琼胶酶YM01-3酶活力具有重要作用。该研究结果为β-琼胶酶的催化机理研究及分子改造提供了借鉴。     

Effects of soil and wood depletion on biodiversity

Human depletion of soil and wood resources is dramatically altering the biodiversity of both terrestrial and aquatic ecosystems. This paper provides an overview of the numerous linkages between the depletion of soil and wood resources and the loss of biodiversity. While some of these linkages are well documented, others remain speculative or unexplored. In order to understand the full ramifications of resource depletion on biodiversity, additional research is required.     

Conformational Studies of β-glucans

Steric and energy contour diagrams have been plotted for disaccharide-like and for helical structures of linear β-D -glucans having (1 → 2), (1 → 3) and (1 → 4) linkages. The allowed conformations constitute only about. 4% of the total conformations, indicating that the freedom of rotation of glucose residues is highly restricted in all the three polysaccharides. The additional restrictions of the monomer unit, as one passes from disaccharide to polysaccaride structures, are severe in the case of (1 → 2) and (1 → 3) linked polysaccharides but not in (1 → 4) linked polysaccharide. The difference in the nature of linkages also has shown to affect the energetically preferred conformations: (1 → 2) linkages lead only to left handed helical conformations; (1 → 3) linkages lead to both right and left handed wide and extended helical conformations, (1 → 4) linkages lead to both right and left handed extended helical conformations. The possible hydrogen bonds between adjacent residues are also dependent on the nature of linkage.     

The N-terminal disulfide linkages of human insulin-like growth factor-binding protein-6 (hIGFBP-6) and hIGFBP-1 are different as determined by mass spectrometry.

The actions of insulin-like growth factors (IGFs) are modulated by a family of six high affinity binding proteins (IGFBPs 1-6). IGFBP-6 differs from other IGFBPs in having the highest affinity for IGF-II and in binding IGF-I with 20-100-fold lower affinity. IGFBPs 1-5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines. The complete disulfide linkages of IGFBP-6 were determined using electrospray ionization mass spectrometry of purified tryptic peptide complexes digested with combinations of chymotrypsin, thermolysin, and endoproteinase Glu-C. Numbering IGFBP-6 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys2, Cys3-Cys4, and Cys5-Cys6. The next two linkages are Cys7-Cys9 and Cys8-Cys10, which are analogous to those previously determined for IGFBP-3 and IGFBP-5. The C-terminal linkages are Cys11-Cys12, Cys13-Cys14, and Cys15-Cys16, analogous to those previously determined for IGFBP-2. Disulfide linkages of IGFBP-1 were partially determined and show that Cys1 is not linked to Cys2 and Cys3 is not linked to Cys4. Analogous with IGFBP-3, IGFBP-5, and IGFBP-6, Cys9-Cys11 and Cys10-Cys12 of IGFBP-1 are also disulfide-linked. The N-terminal linkages of IGFBP-6 differ significantly from those of IGFBP-1 (and, by implication, the other IGFBPs), which could contribute to the distinctive IGF binding properties of IGFBP-6.     

Coordinating theoretical and empirical efforts to understand the linkages between organisms and environments

Schwenk and colleagues challenged biologists to develop a deeper understanding of the linkages between organisms and environments. These linkages are captured by the concept of the niche, which has guided theoretical and empirical research in ecology for decades. Despite this research, we still cannot explain or predict much of the variation in niches over space and time. This shortcoming hinders efforts to forecast biological responses to environmental change. We believe that progress has been slowed by poor coordination between theoretical and empirical efforts to understand the evolution of niches. Therefore, progress should be sped by research programs that integrate modeling and experiments. Such research programs should focus on the structures of environmental variation, the constraints on phenotypes, and the relationships between phenotypes and fitness.     

Invasive plants in conservation linkages: a conceptual model that addresses an underappreciated conservation issue

Conservationists have frequently touted the merits of increased landscape connectivity, usually focusing on the efficacy of conservation linkages (corridors) for maintaining viable populations of target species. An often‐mentioned, but still greatly understudied, concern is that increased landscape connectivity via linkages may also aid the movement of undesired species. This paper provides conceptual guidance for research on one major aspect of this gap: invasive plants in conservation linkages. To guide research goals and methods, I develop a conceptual model describing eight interaction types between invasive plants and linkages, i.e. the ways that invasive plants can exist in and move into, through, and out of conservation linkages. Each interaction type within the model has three main components: linkage, matrix, and focal species. I discuss several aspects of these components, including a) differentiating among matrix types, b) understanding edge effects within the linkages, and c) incorporating relevant invasive species’ ecology (primarily dispersal ecology). Spatially‐explicit documentation of invasive plant distribution is essential to understanding these interactions. By focusing on landscape‐scale patterns in real‐world systems, this model will enhance landscape‐level knowledge of invasion ecology and aid land managers in identifying and prioritizing research and management decisions regarding invasive plants in conservation linkages.     

城市与区域生态关联研究进展

城市及其周边区域正在面临日益严峻的环境污染、生态恶化、资源短缺等问题。这些问题的出现,与长期以来城市发展过程中忽视城市与区域的生态关联密切相关。研究城市与区域的生态关联,对于解决城市与区域的生态环境问题、指导新型城镇化建设具有重要的理论意义与实践价值。主要从以下3个方面系统总结了城市与区域生态关联的研究进展:(1)城市发展对周边区域的生态环境影响,包括直接的胁迫和间接的影响;(2)区域对城市发展的生态支撑;(3)城市与周边区域社会、经济、生态关联的相互作用机制。指出了当前城市与区域生态关联研究中存在的问题和不足:1)城市对周边区域生态环境直接影响的研究较多,且多侧重城市对周边区域的负面影响,对其间接影响的研究较为缺乏;2)周边区域对城市发展生态支撑作用的研究相对缺乏、认识不够深入;3)对城市与周边区域生态关联作用机制的研究较为缺乏。未来的研究要将城市和区域作为统一整体,进一步完善城市与区域生态关联的理论框架,耦合社会经济的相互作用,定量解析城市与区域的生态关联,为城市与区域的可持续发展提供科学依据。     

alpha-L-fucosidases from almond emulsin: characterization of the two enzymes with different specificities.

Almond emulsin contains two kinds of α-l-fucosidases, which could be separated by gel filtration on Sephadex G-200. One enzyme hydrolyzed Fucα1 → 4GlcNAc and Fucα1 → 3GlcNAc linkages in milk oligosaccharides, but did not hydrolyze Fucα1→2Gal or Fucα1 → 6GlcNAc linkages. The other enzyme hydrolyzed the Fucα1 → 2Gal linkage in 2′-fucosyllactose, but did not appreciably hydrolyze other fucosyl linkages. Enzymological properties of the two α-l-fucosidases are described.     

COMPARATIVE HYDROLYSIS OF GELATIN BY PEPSIN,TRYPSIN, ACID,AND ALKALI

A comparison has been made of the relative velocity of hydrolysis of the various peptid linkings of the gelatin molecule when hydrolyzed by acid, alkali, pepsin or trypsin. It has been found that: 1. Those linkages which are most rapidly split by pepsin or trypsin are among the more resistant to acid hydrolysis. 2. Those linkages which are hydrolyzed by pepsin are also hydrolyzed by trypsin. 3. Trypsin hydrolyzes linkages which are not attacked by pepsin. 4. Of the linkages which are hydrolyzed by both enzymes, those which are most rapidly hydrolyzed by pepsin are only slowly attacked by trypsin. 5. Those linkages which are attacked by trypsin or pepsin are among the ones first (most rapidly) hydrolyzed by alkali. In general it may be said that the course of the early stages of hydrolysis of gelatin is similar with alkali, trypsin, or pepsin and quite different with acid.     

A C solid state nuclear magnetic resonance spectroscopic study of cork cell wall structure: the effect of suberin removal

Solid state ¹³C NMR measurements of cork, before and after suberin removal, showed that aliphatic suberin is spatially separated from carbohydrate and lignin and experiences higher motional freedom. Two types of chain methylenes, differing in chemical shift and in dynamic properties, were identified in aliphatic suberin. Experimental evidence indicated that the more motionally hindered methylenes are those situated nearer the linkages of aliphatic suberin to the cell wall. These linkages were shown to involve –CH₂O– groups, probably engaged in ester linkages to phenylpropane units and carbohydrate C6 carbons. Spectral intensity changes indicated that, during the first steps of alkaline desuberization, these linkages are broken and the shorter aliphatic suberin chains removed. Longer chains require hydrolysis of the ester linkages within the chains and are removed upon stronger alkaline treatment. T₁(C), T_1ρ(H) and T_1ρ(C) relaxation times have shown that the removal of suberin from cork leads to a motionally restricted and more compact environment, on the megahertz and mid-kilohertz timescales. The properties of cork suberin showed that suberin organization in cork is distinct from that in potato tissue.