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。     

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。     

一株具有褐藻胶降解能力的海洋细菌的筛选鉴定及其多糖利用能力研究

旨在得到一株具有褐藻胶降解能力的菌株。利用海藻酸钠作为唯一碳源,从腐烂马尾藻中筛选纯化得到一株降解褐藻胶能力较强的海洋细菌,编号X511。根据形态观察和理化指标,结合分子生物学技术鉴定该菌株为弧菌,命名Vibrio sp.X511。X511菌株的指数生长期5-16 h,适宜生长的盐浓度为2%-6%(W/V)。能在以葡萄糖、甘露醇、淀粉等为唯一碳源的培养基中生长。4%-6%(W/V)的盐浓度、海带粉、昆布多糖能延长该菌株的生长稳定期。该菌株在筛选培养基中发酵培养24 h胞内褐藻胶裂解酶粗酶活力达到12.68±0.13 U/m L;提取X511的褐藻胶裂解酶粗酶,分别对海藻酸钠、聚甘露糖醛酸和聚古罗糖醛酸三种多糖的酶解能力依次为:海藻酸钠聚甘露糖醛酸聚古罗糖醛酸。薄层层析(TLC)结果显示,该菌株胞内酶降解海藻酸钠的产物为三糖。结果表明,X511是一株盐耐受性较强、生长周期较短且能同时以海藻酸钠、聚甘露糖醛酸和聚古罗糖醛酸为碳源生长的海洋细菌。     

褐藻胶裂解酶的结构及催化机制研究进展

褐藻胶是由β-D-甘露糖醛酸及其C_5差向异构体α-L-古罗糖醛酸所组成的酸性多糖。褐藻胶裂解酶是多糖裂解酶的一种,可以将褐藻胶降解成寡糖或者单糖。近年来随着结构生物学的发展,越来越多褐藻胶裂解酶的结构及催化机制得到解析。本文中,笔者介绍了不同家族褐藻胶裂解酶结构以及催化机制的研究进展。根据酶序列来分,褐藻胶裂解酶分属到多糖裂解酶的7个家族;从结构上来看,褐藻胶裂解酶通常采取3种结构:β果冻卷、(α/α)_n桶状结构和β螺旋结构。褐藻胶裂解酶通过β消除的方式来降解褐藻胶,根据催化过程中有无金属离子的辅助,褐藻胶裂解酶的催化机制又可进一步分为His(Tyr)/Tyr型β消除和金属离子辅助的β消除。本文可以为褐藻胶裂解酶的应用提供一定的理论依据。     

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...     

产褐藻胶裂解酶芽胞杆菌的筛选、鉴定及其降解效果评价

褐藻胶是广泛存在于褐藻中的一类多糖,降解为褐藻寡糖后能表现出更多的生物活性。从海洋样品中筛选出产褐藻胶裂解酶芽胞细菌16株,基于形态、生理生化特征和16S r DNA系统发育分析初步鉴定菌株HB12274为解淀粉芽胞杆菌植物亚种(Bacillus amyloliquefaciens subsp. plantarum)。TLC结果显示,海藻酸钠经粗酶液降解形成2～7聚合度的褐藻寡糖和单糖,菌株与马尾藻叶片共培养时能明显降解叶状体结构。为褐藻胶裂解酶的生产和工业应用提供了新的菌株来源。     

1株褐藻胶降解菌的筛选、鉴定及产酶条件优化

为获得可产生褐藻胶裂解酶并高效降解褐藻胶的菌株,以海藻酸钠为唯一碳源配制培养基,以透明圈法进行初筛,DNS法复筛,从海洋生物中筛选得到1株高酶活力褐藻胶降解菌株B12,经16S rDNA序列分析、生理生化试验、电镜观察,确定该菌为弧菌属(Vibrio sp.).通过单因素试验及响应面优化试验对影响菌株生长和产酶条件的5...     

刺参海带饲料原料褐藻胶降解菌的分离与鉴定

从海带及刺参养殖环境中筛选有效降解褐藻胶,且对刺参无致病性的微生物,对海带饲料原料进行降解处理,以降低海带饲料中刺参难以消化的褐藻胶成分,显著提高饲料利用率,增加海带原料价值。以褐藻胶为唯一碳源选择培养基初筛;DNS法测定褐藻胶裂解酶酶活;16S r DNA测序及生理生化试验对菌种进行鉴定;高浓度腹腔攻毒试验考察筛选所得菌株对刺参的潜在致病性;分子排阻色谱及高效凝胶色谱法对微生物酶解褐藻胶的终产物进行分析。系统发育树分析表明,菌株WB1与Bacillus amyloliquifaciens有最高同源性,对刺参无潜在致病性;其褐藻胶裂解酶酶解褐藻胶的终产物主要为二糖和三糖,相对含量分别为74.1%和25.9%,平均分子量为516 Da。解淀粉芽胞杆菌WB1可作为一种安全的有益微生物用于刺参海带饲料原料中褐藻胶成分的降解。     

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。     

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.     

Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.     

Cloning, sequence analysis and expression of gene alyVI encoding alginate lyase from marine bacterium Vibrio sp. QY101.

The gene (alyVI) encoding an alginate lyase of marine bacterium Vibrio sp. QY101, which was isolated from a decaying thallus of Laminaria, was cloned using a strategy of combined degenerate PCR and long range-inverse PCR (LR-IPCR), then sequenced and expressed in Escherichia coli. Gene alyVI was composed of a 1014 bp open reading frame (ORF) encoding 338 amino acid residues. The calculated molecular mass of alyVI product is 38.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 34 kDa. AlyVI was purified from culture supernatants to electrophoretic homogeneity using affinity chromatography. AlyVI was most active at pH 7.5 and 40 degrees C in the presence of 1 mM ZnCl2. A nine-amino-acid consensus region (YXRESLREM), which was only found in polyguluronate lyases, was also observed in the amino-terminal region of AlyVI. However, AlyVI could degrade both M block and G block. These results indicate that a novel alginate lyase-encoding gene has been cloned.     

Cloning and Characterization of a Novel Oligoalginate Lyase from a Newly Isolated Bacterium Sphingomonas sp. MJ-3

A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.     

Mixed carbon source control strategy for enhancing alginate lyase production by marine Vibrio sp. QY102

Medium and culture conditions for alginate lyase production by marine Vibrio sp. QY102 were first optimized using statistical methods including Plackett–Burman design and central composite design. Then, fermentation in 5-L bioreactor showed that alginate acted as easily used carbohydrate for Vibrio sp. QY102, while starch extended its growth phase and stabilized pH variations. Thus, a novel strategy using mixed carbon sources was proposed that starch supported growth while enzyme synthesis was induced by pulse feedings of solid alginate. The optimized process followed that Vibrio sp. QY102 grew on starch until the end of the logarithmic growth phase, and then solid alginate was added as 1 g/L every 3 h. Meanwhile, initial pH 5.0 and natural pH during fermentation was favorable for alginate lyase production. After optimization, the highest alginate lyase production reached 52.8 U/mL, which was 329 % higher than the control. Finally, fermentation scale-up was performed in 30-L bioreactor and the maximum alginate lyase production was obtained as 46.8 U/mL.     

Purification and Characterization of an Alginate Lyase from Marine Bacterium Vibrio sp. Mutant Strain 510-64

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.     

Extracellular Poly(α-L-guluronate)lyase from Corynebacterium sp.: Purification, Characteristics, and Conformational Properties

Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure—function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55°C, respectively. Metal compounds such as MnCl₂ and NiCl₂ increased the enzyme activity. The enzyme was identified as the endolytic poly(α-L-guluronate)lyase, which was active on poly(α-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the β-form of the enzyme molecule and resembled poly(β-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(α-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The β-sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.     

Cloning of a gene for intracellular alginate lyase in a bacterium isolated from a ditch

A DNA fragment with a gene for intracellular alginate lyase in a bacterium A1 isolated from a ditch was cloned using a vector plasmid pKK223-3 and the gene was weakly expressed in Escherichia coli DH1 cells. The alginate lyase produced by E. coli DH1 cells was thought to correspond to A1-I among three kinds of alginate lyases (A1-I, A1-I-1 and A1-I-2) produced by the strain A1. Through this study, CaCl₂ was found to be a useful agent for the screening of microbial alginate lyase-producing colonies on agar plates.