海藻酸盐裂解酶研究进展

海藻酸盐裂解酶是一类降解褐藻中海藻酸盐的酶。此酶已经在多种有机体中得到分离。对海藻酸盐裂解酶的生物特性、研究方法及其生物学功能进行了介绍。在酶学特性研究的基础上 ,通过酶解构建新型海藻酸盐多聚物 ,可增强和扩展海藻酸盐裂解酶在工业、农业、医药领域中的应用 ,使其在海藻多糖的高值化应用中发挥重要的作用。概述了海藻酸盐和海藻酸盐裂解酶过去和现在的研究状况 ,展望了海藻酸盐和海藻酸盐裂解酶将来的应用前景。     

一株产褐藻胶裂解酶的需钠弧菌筛选及酶解产物分析

[背景]褐藻胶裂解酶种类丰富、降解机制多样,是高效环保降解褐藻胶、制备褐藻寡糖的工具酶,成为褐藻植物高值化开发利用的研究热点.[目的]从海泥中筛选获得褐藻胶裂解酶高效产酶菌株,确定菌株发酵产酶最优条件,鉴定和分析酶降解产物,进而解析该酶的降解特性.[方法]以褐藻胶为唯一碳源,从海带养殖场附近海泥中筛选菌株,通过形态学观...     

褐藻胶裂解酶的分子改造研究进展

褐藻胶是由β-D-甘露糖醛酸(M)以及α-L-古罗糖醛酸(G)2种单体组成的酸性多糖。褐藻胶裂解酶作为多糖裂解酶的一种,可以温和高效地将褐藻胶降解为褐藻寡糖,并用于食品、医药和农业领域。然而天然来源的褐藻胶裂解酶通常存在活性不高、催化效率低以及热稳定性差等缺点,在一定程度上限制了其工业化应用潜力。近年来分子改造策略已经开始大量应用于褐藻胶裂解酶,使得褐藻胶裂解酶的应用性能得到极大提升。本文对已报道的褐藻胶裂解酶结构与催化机制进行总结,对改善热稳定性、提高催化效率、改变底物分布等性质的褐藻胶裂解酶分子改造策略如理性设计、定向进化、结构域截短与重组等进行系统分析与综述,并展望了未来褐藻胶裂解酶分子改造的发展方向。     

褐藻胶降解菌的筛选、鉴定及产酶条件优化

【目的】筛选一株能降解褐藻胶的菌株,并优化产酶条件以提高褐藻胶裂解酶活力。【方法】从漳州海域采集到海水和海泥,以海藻酸钠为唯一碳源,通过富集培养、初筛、复筛筛选到一株能够降解褐藻胶的菌株。依据16S rRNA序列分析、生理生化特征、菌体形态及菌落特征对该菌进行鉴定。通过单因素和正交试验对该菌的产酶条件进行优化。【结果】该菌属于海科贝特氏菌,命名为Cobetiamarina HQZ08。该菌株最佳的产酶培养基组成为:海藻酸钠7.00g/L、蛋白胨3.00g/L、NaCl30.00g/L,K2HPO4·3H2O 1.25 g/L。最佳发酵条件为:接种量2%,接种龄12 h,培养基起始pH为7.0,培养温度25°C,培养时间24 h。优化后褐藻胶裂解酶活力达到68.5 U/mL,TLC法分析酶解产物为褐藻胶寡糖。【结论】HQZ08菌株可以用于降解褐藻胶,产生聚合度为2–6的褐藻胶寡糖。     

海洋弧菌褐藻胶裂解酶的分离纯化及性质

从海带糜烂物中分离到一株高产胞外褐藻胶裂解酶的海洋弧菌 (Vibriosp .QY10 1) ,利用硫酸铵沉淀、离子交换层析、凝胶过滤层析等方法从发酵液中分离纯化了褐藻胶裂解酶 (alginatelyase)。SDS PAGE电泳结果表明 ,该酶分子量为 39kD。酶反应最适pH为 7.5 ,最适反应温度为 30℃。Na 、Ca2 、Mn2 对酶活性有促进作用 ,Fe2 、Ni2 以及EDTA对酶活性有抑制作用。酶的底物专一性初步分析结果表明 ,该酶具有降解多聚古罗糖醛酸[poly(G) ]及多聚甘露糖醛酸 [poly(M) ]的活性。     

海洋细菌A78的鉴定及褐藻胶裂解酶特性的研究

对分离自日本海域海带的产褐藻胶裂解酶的细菌A78菌株进行鉴定,通过生理生化特征及16S rDNA基因序列分析,鉴定为假交替单胞菌(Pseudoalteromonas nigrifaciens).不同浓度褐藻酸钠诱导实验表明,菌株A78产酶最佳诱导物浓度为1％褐藻酸钠.不同的温度梯度(20～55℃)及pH值梯度(4～10...     

褐藻胶寡糖制备的研究进展

褐藻胶寡糖(alginate oligosaccharides,AOS)是褐藻胶降解而形成的一种功能性寡糖,具有广泛的生物活性,如促进植物生长、提高植物抗逆性、抗氧化、抗菌、抗肿瘤等。褐藻胶寡糖的制备方法主要分为:化学法、物理法和酶解法。不同的方法制备出的褐藻胶寡糖结构亦有所不同。介绍了化学法、物理法和酶解法等各种褐藻胶寡糖制备方法的研究现状、存在的问题及发展趋势。     

褐藻胶裂解酶基因的克隆表达与酶学性质

随着大型褐藻生产燃料乙醇以及褐藻寡糖重大药用价值的发现,褐藻胶裂解酶成为国内外多个领域的研究重点。文中对解藻酸弧菌上与褐藻胶降解相关的5个基因分别进行克隆表达,通过SDS-PAGE和酶活性定量测定,发现该基因簇中的4个基因有降解褐藻胶活性。对酶活最高的rAlgV3进行了诱导条件的优化、酶蛋白纯化及酶性质研究,发现优化诱导条件后重组酶rAlgV3的酶活由2.34×10~4 U/L上升为1.68×10~5 U/L,比优化前提高了7.3倍;对酶性质进行表征发现该酶在4–70℃均有活性,最适反应温度为40℃,在4–20℃酶相对稳定;该酶在pH 6.5-9.0环境下均有较高的酶活,最适pH为8.0;pH稳定性好,在pH 4.5–9.5环境下可以稳定存在;适量的NaCl浓度和Fe~(2+)、Fe~(3+)等离子具有促进酶活的作用,SDS和Cu~(2+)离子可明显抑制酶活力。对该酶的底物特性的研究发现,该酶不仅可以降解褐藻胶中的Poly-M片段,也能降解Poly-G片段,具有广泛底物特性;其降解海藻酸钠主要释放二糖和三糖,是一种内切酶。该酶对于第三代燃料乙醇的发展及褐藻寡糖的生产具有重要作用。     

褐藻胶裂解酶基因的克隆表达及重组酶酶学性质

【目的】克隆交替假单胞菌(Pseudoalteromonas sp.)BYS-2的褐藻胶裂解酶基因,实现其在大肠杆菌细胞中异源表达,对分离纯化的重组酶进行酶学性质研究。【方法】以交替假单胞菌BYS-2菌株基因组DNA为模板,克隆得到褐藻胶裂解酶基因alg738,构建重组基因工程菌BL21(DE3)/p ET22b-alg738,诱导表达,表达产物通过Ni-NTA树脂纯化后进行酶学性质研究。【结果】重组酶的最适反应p H为8.0,在p H 6.0-9.0范围内37°C保温1 h仍能保持84%以上的相对酶活力,具有较好的p H稳定性;最适反应温度为45°C,热稳定性实验显示在37°C下保温60 min其残余酶活力仍达66.6%;在5 mmol/L浓度下,Na~+、Mg~(2+)、Mn~(2+)对该酶具有明显的促进作用,Ni~(2+)、Co~(2+)、Cu~(2+)、Hg~(2+)、Zn~(2+)、EDTA、β-巯基乙醇、SDS具有明显的抑制作用。动力学参数Km、Vmax分别为1.11 g/L和0.011 g/(L·min),底物特异性分析表明该重组酶为偏好聚甘露糖醛酸钠(Poly M)裂解作用的双功能酶。【结论】重组褐藻胶裂解酶具有良好的酶学特性,为褐藻胶裂解酶的开发应用打下基础。     

褐藻胶裂解酶产生菌的分离鉴定及产酶发酵优化

对一株从腐烂海带中筛选得到的产褐藻胶裂解酶的菌株进行鉴定,并对其产酶条件进行发酵优化。经形态学、生理生化特征和分子生物学鉴定,将其鉴定为盐单胞菌属,并命名为Halomonas sp. WF6。通过在摇瓶培养水平上进行单因素和多因素正交试验,确定褐藻胶裂解酶产生菌WF6的最适产酶培养基为：褐藻酸钠6.0 g/L,蛋白胨5.0 g/L,酵母粉2.5 g/L,NaCl 30 g/L,K⁺ 5 mmol/L。进而采用最适培养基进行产酶条件的优化,优化后的发酵产酶条件为：初始pH 8.0,培养温度25℃,接种量为2%,摇瓶装液量30 ml/250 ml,培养时间39 h。优化后的褐藻胶裂解酶酶活达117.66 U/ml,是优化前的2.1倍。该酶对褐藻酸钠的酶解产物主要由聚合度为二和三的褐藻寡糖组成。     

Antibacterial activity of lyase-depolymerized products of alginate

A series of mannuronic acid (M-block) and guluronic acid (G-block) fractions (M1–M5 and G1–G5) with different molecular weights were obtained by lyase depolymerization of alginate and evaluated for in vitro antibacterial activity against 19 bacterial strains. The antibacterial data revealed that both types of fractions generally showed activity against certain tested bacteria, whereas M-block fractions showed broader spectra and more potent inhibition than G-block fractions. Among these fractions, M3 (molecular weight 4.235 kDa) exhibited the broadest spectrum of inhibition and high inhibitory activity against Escherichia coli (minimal inhibitory concentration, MIC = 0.312 μg mL⁻¹), Salmonella paratyphi B (MIC = 0.225 μg mL⁻¹), Staphylococcus aureus (MIC = 0.016 μg mL⁻¹) and Bacillus subtilis (MIC = 0.325 μg mL⁻¹).     

Kinetics and specificity of alginate lyases

A purified preparation of the extracellular alginate lyase has been used to study kinetics and specificity towards purified, homopolymeric fragments of alginate. The enzyme preparation from Bacillus circulans 1351 degraded both block types, although with different efficiency, and thus appears to be nonspecific. Addition of calcium ions markedly enhanced the reaction rate for the polymannuronate block but had little or no effect on the reaction with polyguluronate. Michaelis-Menten kinetics are not obeyed in the absence of calcium ions and only for the polymannuronate in the presence of calciumThe study of progress curves in response to variation in substrate and enzyme concentrations strongly suggests that the abalone lyase is subject to a reversible product inhibition.     

褐藻寡糖的制备方法及生物活性研究进展

褐藻胶是一类多糖聚合物,由于其独特的理化性质和有益健康的作用,已被广泛应用于制药和食品工业.然而,由于褐藻胶的水溶性低、黏度大,进而限制了褐藻胶的开发和应用.褐藻寡糖(alginate oligosaccharide,AOS)是褐藻胶的降解产物,由于其分子量低、水溶性高、安全无毒等特点,近年来受到广泛关注.AOS独特的...     

Determination of alginate composition by a simple enzymatic assay

The action of alginate lyases may be easily followed in a UV-spectrophotometer, since each cut of the alginate chain will create an unsaturated unit at the non-reducing end with a strong absorbance at 230 nm. During prolonged incubation, this absorbance will approach an apparent endpoint level that reflects the initial substrate concentration. On this basis, a standardized assay has been developed. A combination of purified mannuronate lyase from Haliotis tuberculata and purified guluronate lyase from Klebsiella pneumoniae is applied to get quantitative concentration estimates that do not depend on alginate composition. The production of alginate in Azotobacter vinelandii is included as an example of application. Most important, by applying both enzymes alone and in combination, the block composition of the alginate may be estimated. Data for a series of widely different alginates have been compared with those obtained by NMR.     

Cloning and sequencing of a Deleya marina gene encoding for alginate lyase

A bacterial strain N-1 was isolated as a decomposer of alginate and identified as Deleya marina. The alyA encoding for alginate lyase was cloned into Escherichia coli. The structural gene, located on a 1.9-kb SalI fragment, revealed 1,122 bp encoding a mature protein of 348 amino acids and a signal peptide of 26 amino acids. The deduced amino acid sequence of the D. marina alginate lyase showed high homology to AlgL of Pseudomonas aeruginosa with 63% identity and belonging to class 1 by hydrophobic cluster analysis.     

Probing the helical forms of Ca2+‐guluronan junction zones in alginate gels by molecular dynamics 1: Duplexes

A molecular dynamics investigation of the helical forms adopted by (1→4)‐α‐L ‐guluronan in explicit water environment was carried out. Single chains and duplexes were modeled at 300 K starting both from 2₁ or 3₂ helical conformations and in the presence of a neutralizing amount of Ca²⁺ ions. All systems were allowed full conformational freedom. The initial perfect helices with integral screw symmetries were lost at the very beginning of simulations and two distinct behaviors were observed: At equilibrium the 2₁ models mostly retained the 2₁ local helical conformations while exploring the 3₂ ones the rest of the time. In duplexes the two chains, which behaved similarly, were well extended and slightly twisted. By contrast, the chains in 3₂ duplex models were dissimilar and explored a much broader conformational space in which 2₁ and 3₂ local helical conformations were dominant and equally represented but the 3₁ and other conformations were also present. The wide variety of conformations revealed in this study is consistent with the general difficulty in obtaining crystals of Ca²⁺‐guluronate with suitable lateral dimensions for crystallographic studies. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 562–571, 2013.     

Hydrophobic cluster analysis and classification of sixteen bacterial alginate lyases

Sixteen alginate lyases whose primary sequences have been reported were compared, and classified into the following three groups on the basis of the identity of their primary sequences. Strong homology (>50%): A-AlgL, A-AlgL*, P-AlgL, P-AlgL*, and AlgA; weak homology (>20%): ALY, AlxM, P-Aly, K-Aly, AlyPG, AlgVGI, AlgVGII, and AlgVGIII; little homology (<20%): ALYII, Al-III, and AlgVMI. Using hydrophobic cluster analysis (HCA), a secondary structure prediction method, the sixteen alginate lyases were placed into the following classes. Class 1: AlgA, A-AlgL, A-AlgL*, P-AlgL, and P-AlgL*; Class 2: AlgVMI and Al-III; Class 3: ALY and AlxM; Class 4A: ALYII, K-Aly, P-Aly, and AlyPG; Class 4B: AlgVGI and AlgVGII; Class 5: AlgVGIII, which is put in a class of its own, because it is unlike any of the other alginate lyases.     

Bacterial alginates: from biosynthesis to applications

Alginate is a polysaccharide belonging to the family of linear (unbranched), non-repeating copolymers, consisting of variable amounts of β-d-mannuronic acid and its C5-epimer α- l-guluronic acid linked via β-1,4-glycosidic bonds. Like DNA, alginate is a negatively charged polymer, imparting material properties ranging from viscous solutions to gel-like structures in the presence of divalent cations. Bacterial alginates are synthesized by only two bacterial genera, Pseudomonas and Azotobacter, and have been extensively studied over the last 40 years. While primarily synthesized in form of polymannuronic acid, alginate undergoes chemical modifications comprising acetylation and epimerization, which occurs during periplasmic transfer and before final export through the outer membrane. Alginate with its unique material properties and characteristics has been increasingly considered as biomaterial for medical applications. The genetic modification of alginate producing microorganisms could enable biotechnological production of new alginates with unique, tailor-made properties, suitable for medical and industrial applications.     

Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae

The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.