海藻工具酶——褐藻胶裂解酶研究进展

从海洋生物中筛选提取有价值的酶类,开发海洋多糖降解产物,已成为海洋生物资源开发的一个重要方面。因此,近年来对于海藻工具酶之一的褐藻胶裂解酶及其降解产物——褐藻寡糖的研究日益受到人们的普遍关注。从褐藻胶裂解酶的来源、分类、底物专一性、作用方式及结构与机理研究、酶活力测定和酶学性质等方面,结合本课题组的研究工作综述近十年来有关褐藻胶裂解酶的研究进展。     

海藻酸裂解酶酶学与基因工程研究进展及应用

海藻中富含海藻酸盐,海藻酸裂解酶降解后产生的寡糖物质具有很强的生物活性及益生作用,酶法降解海藻酸盐的生物降解取代传统的化学降解已日益受到人们的关注,就海藻酸盐降解酶的来源、作用机制、应用效果和影响因素进行了全面综述,阐明了海藻酸盐降解酶的研究具有显著的理论意义和应用价值。     

海藻酸裂解酶异源表达研究进展

海藻酸裂解酶是通过β-消除反应切断海藻酸分子中的糖苷键,产生非还原性端具有不饱和双键的糖的酶.通常作为制备海藻酸寡糖的工具酶,广泛应用于医疗、食品和生物质能源等领域的研究.介绍了海藻酸裂解酶异源表达的研究现状、存在的问题和发展的趋势.     

噬菌体裂解酶应用研究进展

近年来,随着抗生素的滥用,导致多重耐药性菌株出现的频率加快。因细菌感染导致死亡的人数逐年增多,人类健康面临巨大挑战,因此研制新型抗菌药物刻不容缓。噬菌体裂解酶因其高效的杀菌能力及高度的宿主专一性而成为新一代抗菌制剂的候选之一。其是一种细胞壁水解酶,在双链DNA噬菌体复制后期被合成,通过水解细胞壁肽聚糖上的化学键,从而裂解细菌细胞壁,释放出子代噬菌体。本文系统地介绍了噬菌体裂解酶的研究进展,为相关裂解酶抗菌药物的研发做出有益探索。     

裂解酶治疗的研究进展与应用前景

多耐药病原细菌的不断出现和传播给公共医疗造成了严峻的威胁和挑战,开发新的抗菌分子迫在眉睫。噬菌体裂解酶来源于噬菌体,具有独特的进化和选择优势,不仅能高效快速的杀灭多耐药细菌,而且不易诱导细菌产生新的耐药性。本文对噬菌体裂解酶的结构和功能进行了简要的介绍,重点综述了裂解酶在抗细菌感染中近年的研究进展和应用前景。     

石莼多糖裂解酶的研究进展

石莼多糖(Ulvan)由3-硫酸化鼠李糖,葡糖醛酸,艾杜糖醛酸及一些随机分布的木糖组成。石莼多糖及其降解得到的寡糖在医疗、食品等领域具有广泛的应用前景。为促进对石莼多糖裂解酶这一石莼多糖利用工具的开发,对石莼多糖裂解酶的底物组成,来源分类,序列及进化关系,酶学性质及作用模式,蛋白结构以及作用机制进行了综述,以求对后续石莼多糖裂解酶研究者提供帮助。     

噬菌体裂解酶研究进展

噬菌体裂解酶是双链DNA噬菌体所特有的细胞壁水解酶。研究表明,所有噬菌体裂解酶在结构上具有相似性,即含有2个结构域：比较保守的N端催化区和差异较大的C端特异性结合区。裂解酶的高亲和性与种属特异的细胞壁糖基有关,而后常常是细菌存活的必要成分。所以,细菌难以产生对裂解酶的抗性。本简要综述噬茵体裂解酶的研究进展。     

噬菌体裂解酶——现状与未来

噬菌体裂解酶是一种由DNA噬菌体基因编码的高特异性酶, 可高效消化细菌细胞壁。革兰氏阳性菌噬菌体裂解酶的结构域相似, 裂解效率高, 与抗生素具协同抗菌作用, 且不易产生耐受性菌株, 抗体等体液因子对裂解酶的裂解活性影响小, 裂解酶作为一种潜在抗感染药物具有重要的研究价值。目前已建立了多种病原菌裂解酶应用的动物模型, 在防控耐药性病原菌感染上取得重要进展。本文就噬菌体裂解酶的抗菌作用进行综述。     

提高噬菌体裂解酶抗菌活性的研究进展

随着细菌耐药性问题的日益严重,人们开始寻求新型抗菌制剂。噬菌体裂解酶是一种由ds DNA噬菌体编码的水解酶,能高效特异性地裂解细菌细胞壁且不易使细菌产生耐药性。由于天然裂解酶具有宿主谱窄,不能裂解革兰阴性菌等缺点,研究者对裂解酶进行了大量的设计改造。本研究主要对提高噬菌体裂解酶抗菌活性的研究进展进行综述。     

聚半乳糖醛酸内切酶研究进展

论述了聚半乳糖醛酸内切酶的微生物分布、发酵机理和酶学性质、酶的固定化以及其分子生物学的研究进展。     

Preparation and structure elucidation of alginate oligosaccharides degraded by alginate lyase from Vibro sp. 510

Alginate that was purified from the fermentation solution of marine bacteria Vibro sp. 510 under specific reaction conditions was hydrolyzed by alginate lyase. Seven oligosaccharides, including di-, tri- and tetrasaccharides, were isolated through low-pressure, gel-permeation chromatography (LP-GPC) and semipreparative strong-anion exchange (SAX) fast-protein liquid chromatography (FPLC). The oligosaccharide structures were elucidated based on ESIMS and 2D NMR spectral analysis. The hydrolytic specificity of this alginate lyase to alginate is discussed.     

海藻酸转化生物乙醇研究进展

作为第三代生物燃料,大型褐藻类生物质转化燃料乙醇的研究受到广泛的关注。但是,现有的乙醇工业菌株并不能利用褐藻中的主要成分海藻酸,这个问题是海藻生物乙醇实现工业化生产的主要技术难关。近几年随着对海藻酸裂解酶和海藻酸降解菌代谢途径的深入研究,科研人员构建了不同的海藻酸发酵菌株,为高效转化大型海藻生产生物乙醇提供了可行的技术基础。这篇文章对海藻酸资源概况和海藻酸转化生物乙醇存在的科学问题及其研究进展进行了综述。     

The Surface Display of the Alginate Lyase on the Cells of Yarrowia lipolytica for Hydrolysis of Alginate

The alginate lyase structural gene (AlyVI gene) was amplified from plasmid pET24-ALYVI carrying the alginate lyase gene from the marine bacterium Vibrio sp. QY101 which is a pathogen of Laminaria sp. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying the alginate lyase could form clear zone on the plate containing sodium alginate, indicating that they had high alginate lyase activity. The cells displaying alginate lyase can be used to hydrolyze poly-β-d-mannuronate (M) and poly-α-l-guluronate (G) and sodium alginate to produce different lengths of oligosaccharides (more than pentasaccharides). This is the first report that the yeast cells displaying alginate lyase were used to produce different lengths of oligosaccharides from alginate.     

Alginate lyase from Pseudomonas aeruginosa CF1/M1 prefers the hexameric oligomannuronate as substrate

In order to investigate the catalytic properties of alginate lyase from Pseudomonas aeruginosa CF1/M1, a clinical isolate, regarding the capability to perform β-elimination on oligomannuronates of defined length (2–9), the alginate lyase was purified from periplasmic extracts. A purification method for unsaturated and saturated oligomannuronates applying anionic exchange chromatography on a FPLC apparatus was established. The alginate lyase showed the highest activity, when hexamers were provided as substrate. This indicated that the alginate lyase best accommodates a chain of six alginate residues in the active center. As a minimum chain length, the pentameric oligomannuronate was still accepted as substrate. Mannuronate oligomers shorter than the pentamer were not accepted as substrate for alginate lyase. Furthermore, oligomer pattern analysis of polymannuronate which was subjected to β-elimination by alginate lyase revealed that the trimer is the most abundant oligomer. These data indicated that β-elimination and cleavage occurred at mannuronic acid residue no. 3 of the accommodated hexameric alginate chain.     

从海洋中分离的弧菌QY102褐藻胶裂解酶的纯化和性质研究

从马尾藻(Sargassum)表面分离到一株产生高效胞外褐藻胶裂解酶的海洋弧菌（Vibrio sp.） QY102。以褐藻胶为唯一碳源发酵培养后,发酵液上清通过0.22μm滤膜过滤、DEAESepharose离子交换和Superdex75凝胶过滤得到电泳纯的褐藻胶裂解酶。酶的性质研究表明：其分子量约为28.5kD（SDSPAGE）,反应最适温度为40℃,最适pH为7.1,Ca2+、Mg2+对酶活有促进作用,而Ni2+、Al3+、Zn2+、Ba2+对酶活有抑制作用。该酶的活性明显高于已报道的褐藻胶裂解酶,pH稳定范围广（5～10）,并且对聚甘露糖醛酸的活性高于对聚古罗糖醛酸的活性。     

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。     

响应面法优化类芽胞杆菌HB172198产褐藻胶裂解酶发酵培养基

为了提高类芽胞杆菌新种HB172198产褐藻胶裂解酶活力,本研究采用响应面法对该菌株液体发酵培养基进行了优化实验。在单因素实验和Plackett-Burman试验筛选出海藻酸钠、胰蛋白胨、NaCl、MgSO4·7H2O等4个显著影响产酶因素的基础上,通过Box-Behnken设计及响应面法进行回归分析,得出产褐藻胶裂解酶最佳发酵培养基,其成分为:海藻酸钠7.50 g/L、胰蛋白胨13.57 g/L、NaCl 29.75 g/L、MgSO4·7H2O 0.08 g/L。优化条件下该菌株最大酶活性达14.60 U/mL,是优化前的1.87倍。本研究为菌株HB172198产褐藻胶裂解酶的大规模生产和工业应用提供了重要的理论依据。     

产双功能褐藻胶裂解酶菌株的筛选与初步研究

目的：双功能褐藻胶裂解酶既能降解聚β-D-甘露糖醛酸,又能降解聚α-L-古罗糖醛酸,可以用一种酶来制备不同结构的褐藻胶寡糖。本文的目的是筛选能产生双功能褐藻胶裂解酶的菌株,对其产酶曲线和降解产物作初步研究。方法：利用唯一碳源培养基筛选产生褐藻胶裂解酶的菌株,通过16SrDNA序列比对进行菌种鉴定,通过在凝胶上检测褐藻胶裂解酶活性来判断发酵上清液中褐藻胶裂解酶的数量及分子量,利用薄层层析确定降解褐藻胶的终产物组成。结果：从褐藻上筛选到一株海洋细菌QY107,鉴定为弧菌属细菌。发酵120h时褐藻胶裂解酶产量为12．32U／mL,其发酵液上清中只含有一种褐藻胶裂解酶,分子量在28kDa左右,并且对聚β—D-甘露糖醛酸和聚α-L-古罗糖醛酸都能降解,降解褐藻胶的终产物主要为三糖。结论：本文筛选到一株弧菌QY107,其发酵液上清中只有一种双功能褐藻胶裂解酶,可用于大量制备褐藻胶三糖。推测该酶具有特殊的催化腔结构,对其结构与功能相互关系的研究可能会发现新的底物结合与催化机制。酶解制备褐藻胶寡糖因其环保高效而越来越受到人们的重视,因此该菌株能促进海洋寡糖类生物制品的开发,在医药、食品、农业、生物燃料等领域具有广阔的应用前景。     

Cloning and Characterization of Alginate Lyase from a Marine Bacterium Streptomyces sp. ALG-5

A marine bacterium was isolated from seaweeds for its ability to degrade alginate. Analysis of 16S ribosomal DNA sequence and chemotaxonomic characterizations revealed that the strain belongs to Streptomyces. The alginate lyase gene of Streptomyces sp. ALG-5 was cloned by using PCR with the specific primer designed from homologous nucleotide sequences. The consensus sequences of N-terminal YXRSELREM and C-terminal YFKAGXYXQ were conserved in the ALG-5 alginate lyase gene. The recombinant alginate lyase was purified by using Ni-Sepharose affinity chromatography. The alginate lyase appears to be poly-guluronate lyase degrading poly-G block preferentially than poly-M block. The degraded products were determined to be di-, tri-, tetra- and pentasaccharides by using BioGel P-2 gel filtration chromatography and ionization mass spectroscopy method.