海洋细菌来源低温褐藻胶裂解酶的分泌表达和酶学性质研究

为了筛选稳定性较好的低温褐藻胶裂解酶,本研究进行了海洋细菌分离鉴定、酶的编码基因克隆与分析、酶分泌表达与纯化、不同因素对酶活力和稳定性的影响以及酶解产物分析实验。结果显示,用以褐藻胶为唯一碳源的平板,筛选出一株能分泌低温褐藻胶裂解酶的海洋细菌SJ-H-12,基于16S rDNA序列构建进化树,该菌株鉴定为Yangia sp. SJ-H-12。进而克隆酶的编码基因Alyya,ALYYA属于PL5家族褐藻胶裂解酶。将基因在食品级宿主解脂耶氏酵母(Yarrowia lipolytica)中进行分泌表达,重组ALYYA的活力达到34.2 U/mL,分子量约为39.0 kDa,具有较强的PolyM偏好性。ALYYA在25℃~35℃时表现出80%以上的活力,且在30℃时表现出最高活力;在pH为5.0~10.0的范围内稳定性较好,孵育后剩余超过60%的活力;0~2.0 mol/L的NaCl能明显激活ALYYA的活力。ALYYA降解褐藻胶的产物主要是二糖,另有少部分单糖和三糖,该酶是一种内切褐藻胶裂解酶。综上所述,本研究筛选到一株产低温褐藻胶裂解酶细菌,所产ALYYA是典型的低温褐藻胶裂解酶,具有优良的酶活力和稳定性。本研究为低温褐藻胶裂解酶的筛选和开发利用提供了参考数据。     

褐藻胶裂解酶研究进展

近年来随着海洋药物的蓬勃发展,海藻多糖的研究日益受到重视,褐藻胶便是其中之一。褐藻胶具有广泛的应用价值,褐藻胶降解物具有很强的生物活性,以酶法降解褐藻胶为代表的生物降解取代传统的化学降解已成趋势,因此褐藻胶裂解酶的研究生产有着深远的理论意义和应用价值。本文从酶的分类和来源、底物专一性和作用方式、酶的性质、酶活力测定方法、产酶菌株及酶系以及酶的应用6个方面综述了褐藻胶裂解酶的研究进展。收稿日期:19991227作者简介:刘　岩,(1971-),女,山东济南人,青岛海洋大学博士研究生.1　褐藻胶裂解酶的分类及来源1.1　褐藻胶裂解…     

基于酶技术生产低黏度及超低黏度褐藻胶的工艺

为解决当前低黏度及超低黏度褐藻胶生产工艺中存在的不足,本实验以海带为原料,利用褐藻胶裂解酶降解制备低黏度及超低黏度褐藻胶,研究了分子量、p H、温度对黏度的影响,确定了碱消化的最佳条件,探究了酶解工艺中加酶量、酶解时间及原料的初始黏度对褐藻胶产品的影响。结果显示,通过控制加酶量(100~500 U/g),酶解30 min即可得到低黏度及超低黏度褐藻胶,其中加酶量为100~330 U/g时可得到低黏度褐藻胶,加酶量增加至330~500 U/g时,可得到超低黏度褐藻胶,且酶解法得到的褐藻胶样品分子量均一度高,工艺节水率高达10%~50%;同时研究发现酶解样品黏度与原料初始黏度相关性不大,只在较短时间内表现出相关性,该工艺具有较高的原料适用性。     

锈凹螺褐藻胶裂解酶的分离纯化及性质研究

采用硫酸铵沉淀、离心超滤、DEAE-Sephadex A25、Sephadex G-100和Sephacryl S-300 HR 柱层析等方法从海洋食藻性锈凹螺(Chlorostoma rustica)消化腺中分离纯化出一种褐藻胶裂解酶(Alginate lyase),得到电泳纯酶制品,并对此酶的酶学性质进行了研究.结果表明:此褐藻胶裂解酶的分子量为28 kD,反应的最适温度为40 ℃,具有热不稳定性,最适pH为7.0;酶的反应受多种离子的影响,Mn2 、Co2 和Cd2 对此褐藻胶裂解酶活性具有明显的抑制作用,而Mg2 、Zn2 、Na 和K 则具有促进作用;采用透明圈法和底物法验证了此裂解酶对聚甘露糖醛酸和聚古洛糖醛酸的特异性,实验表明,此酶对聚古罗糖醛酸(Polyguluronate,PG)有特异性.     

皱纹盘鲍内脏酶的酶学性质及褐藻胶裂解酶的分离纯化

采用(NH4)2SO4分段盐析、透析、阴离子(DEAE-52)交换柱层析、SephadexG-200凝胶柱层析等分离纯化技术,通过SDS-PAGE电泳分析了皱纹盘鲍内脏酶的组成,结果表明鲍内脏酶主要含有两种褐藻胶裂解酶Ⅰ, Ⅱ,一种纤维素酶,一种琼脂酶。对酶的酶学性质分析结果表明两种褐藻胶裂解酶Ⅰ, Ⅱ的最适pH分别为8.6, 7.2,最适温度为35 ℃,分子量分别为35.2 ku, 67 ku;两种褐藻胶裂解酶的热稳定性比较差,且易受金属离子影响;纤维素酶的最适pH为5.0,最适温度为40 ℃。并确定了皱纹盘鲍内脏酶分离纯化的方法及参数,为进一步研究鲍内脏复合酶的性能提供了基础参数。图14表3参12 关键词：皱纹盘鲍; 褐藻胶裂解酶; 纤维素酶; 纯化 E-mail：wqk320@dlfu.edu.cn     

一株产几丁质酶菌株的筛选鉴定与产酶条件优化

几丁质酶在降解几丁质和生物防治上具有重要作用。采用平板筛选法从对虾养殖池塘底泥中筛选几丁质酶产生菌株,以培养时间、培养温度和胶体几丁质含量为自变量,响应面法优化该菌株的产酶条件,建立二次回归方程。试验结果表明,通过菌株的菌落形态、亚显微形态特征和16S rDNA序列同源性分析,确定该菌株为蜂房芽孢杆菌。3个因素对菌株产酶的影响依次为培养时间培养温度胶体几丁质含量。菌株的最佳产酶的条件为:培养基中胶体几丁质含量1.62%,培养温度40℃,培养时间72h,在此条件下,菌株产生的几丁质酶活力为13.07U/mL。该研究为筛选菌株的进一步利用提供科学依据。     

工厂化循环水养殖条件下云纹石斑鱼消化道产酶菌的分离鉴定

采用16S r DNA-PCR菌群分离鉴定的方法,对循环水养殖条件下云纹石斑鱼(Epinehelus moara)幼鱼的胃、幽门盲囊、前肠、中肠和后肠的菌群结构进行了鉴定,用产酶菌筛选培养法对产消化酶的菌株进行了分离鉴定,并测试了各菌株消化酶的活力。研究发现,云纹石斑鱼幼鱼消化道内可培养的主要菌群为假单胞菌属(Pseudomonas)、微小杆菌属(Exiguobacterium)、不动杆菌属(Acinetobacter)、寡养单胞菌属(Stenotrophomonas)和葡萄球菌属(Staphylococcus),其中产消化酶的菌株占可培养菌的55.6%。在产酶菌中,同一株菌产3种酶的有5株;产2种酶的有9株;中肠和后肠的菌株数最为丰富,胃次之,幽门盲囊和前肠菌群种类较少;产脂肪酶的菌株都集中在中肠。产消化酶的菌株主要以产蛋白酶和淀粉酶为主,且产酶量丰富,产蛋白酶活力最高达(87.732±1.134)U/m L;淀粉酶活力为(77.176±0.599)~(73.458±0.574)U/m L;产纤维素酶的菌仅一株,且酶活力较低。分析得知,消化道的菌群结构直接影响了外源性消化酶的种类与活性。本研究为工厂化循环水养殖条件下产酶有益菌的筛选提供了理论依据。     

黄鳝肠道细菌及产酶菌株的研究

从健康的黄鳝肠道中分离出细菌32株.革兰氏染色结果表明,8株为革兰氏阳性菌,24株为革兰氏阴性菌.研究了其产蛋白酶、脂肪酶、淀粉酶、纤维素酶的能力.结果表明,有53.13%的菌株能分泌蛋白酶,43.75%的菌株能分泌脂肪酶,34.38%的菌株能分泌淀粉酶,21.88%的菌株能分泌纤维素酶.其中产4种酶的有1株,产3种酶的有5株,产2种酶的有9株,产1种酶的有12株,不产酶的仅有5株.产酶菌株的比例高达84.38%.     

海洋细菌S-12-86的产溶菌酶条件

从中国东海海域底泥中筛选获得一株产溶菌酶的海洋细菌S-12-86,采用摇瓶发酵的方式,分别从培养基组分与发酵条件两个方面对海洋细菌S-12-86产酶的影响进行研究。结果表明,菌株S-12-86能够利用麦芽糖、淀粉、甘油和葡萄糖作为碳源,不能利用蔗糖与甘露醇;能够利用牛肉膏、酵母膏、蛋白胨和硫酸铵作为氮源,其中牛肉膏效果最佳,而硝酸钾、氯化铵和尿素几乎不能被利用;Zn2 、Mn2 、Cu2 对菌株S-12-86的生长和产酶均有抑制作用;Fe2 对菌体生长无明显影响,但明显抑制菌体产酶;Na 、K 对菌体的生长和产酶无明显影响;Ca2 对菌体生长有促进作用;Mg2 对菌体生长和产酶均有促进作用。菌株S-12-86最适发酵条件为:培养温度30℃、接种体积比4.0%、装液量体积比10.0%、产酶高峰在发酵开始后24 h。与白色链霉菌G(Streptomyces albusG)、灰色链霉菌P-51(S.griseusP-51)、枯草芽孢杆菌77(Bacillus subtilis77)等产溶菌酶微生物的产酶条件相比较,菌株S-12-86具有易培养、产酶量较高、生产成本较低等优点。因此,菌株S-12-86有较大的生产开发潜力。     

近江牡蛎养殖水体中细菌产酶能力的研究

为了解近江牡蛎养殖水体中细菌的产酶特性 ,筛选出有应用潜力的有益菌 ,本实验从近江牡蛎养殖水体中分离到 32株菌。革兰氏染色表明 ,10株为革兰氏阳性菌 ,2 2株革兰氏阴性菌。在此基础上研究了它们蛋白酶、脂肪酶、淀粉酶和纤维素酶的产酶能力。结果表明 ,6 1.9% ( 17株 )的菌株能分泌蛋白酶 ,71.9% ( 2 3株 )菌株能分泌脂肪酶或淀粉酶 ,34.4 % ( 11株 )的菌株产纤维素酶。这些产酶菌株均以革兰氏阴性菌为主。在这 32株菌中 ,产四种酶的有 5株菌 ,产三种酶的有 9株菌 ,产两种酶的有 9株菌 ,产一种酶的有 9株菌 ,也就是说 ,所有的菌株都能产酶。由此表明近江牡蛎养殖水体中细菌在该环境物质循环中的重要地位。     

Heat-stability and primary structure of the major alginate lyase isozyme LbAly35 from Littorina brevicula

Previously we isolated the major alginate lyase isozyme LbAly35 from a marine snail Littorina brevicula and showed that this enzyme was significantly heat stable in a broad pH range compared with other molluscan alginate lyases (Hata et al., Fish Sci 75:755?C763, 2009). LbAly35 showed practically no similarity to other molluscan alginate lyases in the N-terminal amino-acid sequence of 20 residues and no cross-reactivity with anti-abalone alginate lyase antiserum. These led us to consider that the primary structure of LbAly35 is considerably deviated from other molluscan enzymes. Thus, in the present study, we first compared the thermal stability of LbAly35 with an abalone alginate lyase, HdAly, and found that the first order inactivation rate constants for LbAly35 at 40 and 45?°C were 1/20 and 1/45 of those for HdAly, respectively. Then, we cloned cDNAs encoding LbAly35 and characterized its deduced amino-acid sequence comparing with those of other molluscan alginate lyases. The cDNAs were amplified by polymerase chain reaction (PCR) and 5??- and 3??-RACE PCRs from the L. brevicula hepatopancreas cDNA using degenerated primers synthesized on the basis of partial amino-acid sequences of LbAly35. The cDNA covering the entire translational region of LbAly35 comprised 1,093?bp and encoded an amino-acid sequence of 296 residues. The amino-acid sequence consisted of an initiation methionine, a putative signal peptide for secretion (22 residues), a propeptide-like region (10 residues), and a mature LbAly35 domain of 263 residues. Although the N-terminal region of LbAly35 was significantly deviated from those of other molluscan alginate lyases, the catalytic domain of LbAly35 showed ~45?% identity to other molluscan enzymes which had been classified under polysaccharide-lyase-family-14 (PL-14). In addition, the amino-acid residues crucially important for the catalytic actions of PL-14 enzymes were also conserved in LbAly35. Accordingly, LbAly35 was regarded as a member of PL-14 as other molluscan alginate lyases despite the significant deviation of its N-terminal region.     

Isolation of alginate lyase‐producing bacteria and screening for their potential characteristics as abalone probionts.

This study is aimed at the isolation and screening of alginate lyase‐producing bacteria from the gastrointestinal tracts of hybrid abalone, Haliotis rubra x H. laevigata, as probiotic candidates. Six bacterial isolates were detected to produce alginate lyase. Of these, the isolate with the highest alginate‐lyase activity was identified as Enterobacter ludwigii strain EN‐119, displaying 99% similarity of 16S rDNA sequence. Further assays indicated that E. ludwigii showed good viability and stability when it was incorporated into manufactured pellets and stored at 4°C for 7 days. The isolate also had high tolerance of high salinity (35 mg/L), low pH in simulated stomach juice (5) and to simulated intestinal juice containing surfactants such as bile salts and gastric enzymes (pepsin and trypsin). Additionally, a short, preliminary study indicated that supplementation of E. ludwigii via manufactured pellets improved the total weight gain and specific growth rate of hybrid abalone. These results suggest that E. ludwigii is a potential probiont for shortening the culture period of hybrid abalone.     

Chilled Storage of Pangasianodon hypophthalmus Fillets Coated with Plant Oil Incorporated Alginate Gels: Effect of Clove Leaf,Clove Bud,Rosemary and Thyme Oils

Essential oil incorporated alginate coating provides a novel way to improve the safety and shelf life of pangasius (Pangasianodon hypophthalmus) fillet. Oils from the leaves and buds of clove, flowering tops of rosemary, and dried seeds of thyme were incorporated separately in alginate coating. All the plant oils showed antibacterial activity, but the zone of inhibition was relatively larger for thyme oil. Alginate coating was performed using sodium alginate (1.5%), glycerol (10%), and calcium chloride (2%) and plant oil at 1% (v/v). The coated fillets were stored under chilled conditions and samples were analyzed for bacteriological, chemical, sensory, color, and texture parameters. Psychrotrophic counts crossed 7 log cfu/g by the 13^th day and 15^th day of chilled storage in control and plant oil treated fillets, respectively. The peroxide value of treated fillets was relatively low. Texture profile analysis indicated that plant oil incorporated alginate coating reduced the rate of loss of texture (softening) during chilled storage. Plant essential oil incorporated alginate gels were relatively better compared to control fillets in preserving pangasius fillet quality during chilled storage, and incorporation of thyme oil was relatively better compared to clove leaf oil, clove bud oil, and rosemary oils.     

Calcium-sensitive extracellular poly(α-L-guluronate)lyase from a marine bacterium Pseudomonas sp. strain F6: Purification and some properties

ABSTRACT: Poly(α- L -guluronate)lyase, as one of alginate lyases, was purified from the culture medium of a marine bacterium, Pseudomonas sp. strain F6, to an electrophoretically homogeneous state. The enzyme was shown to have a molecular mass of 36 kDa by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and was most active at around pH 7.5 and was stable between pH 6.5 and pH 8.5. In the thermal stability experiments, the enzyme's activity diminished through an intermediate state with increasing incubation temperatures and was finally lost when heated at 100°C for 15 min. The addition of hen egg-white lysozyme to the enzyme decreased thermal stability dramatically. The apparent retention of enzyme activity (approximately 50%) was observed after the addition of 6 M guanidine hydrochloride and 8 M urea. Enzyme activity was lost completely with 10 mMSDS, while the ordered structure, which is considered likely to be β-structure, was markedly created. The similar conformational feature has also been created in marine bacterial and mollusc enzymes and the β-structure is commonly observed in polyuronate lyases. The divalent cation (Ca²⁺) promoted the activity of the calcium chelator-treated enzyme significantly, suggesting that Ca²⁺ is involved in the formation of the active intermediate between the acidic uronate(s) and amino acid side-chain(s) of the enzyme.     

Structural and Rheological Characteristics of Alginate from Sargassum cristaefolium Extracted by Twin Screw Extruder

This study aimed to investigate the effects of process extrusion on the characteristics of Sargassum cristaefolium sodium alginate (SCSA) extracted using twin-screw extruder. Box-Behnken Design (BBD) from response surface methodology (RSM) was established to understand the effects of temperature, screw speed, and pH on the multiple-responses of alginate characteristics including intrinsic viscosity, yield, and molecular weight. The results revealed that temperature, screw speed, and pH significantly affected (P < .05) all responses. The optimum extraction condition was found at temperature of 58.18°C, screw speed of 77.99 rpm, and pH 10.11. At this condition, the response of residence time distribution was 7.07 ± 0.029 min, yield of 34.01 ± 0.12%, intrinsic viscosity of 460.13 ± 14.75 mL/g, and molecular weight of 217.94 ± 7.14 × 10³ g/mol. This alginate had mannuronic acid to guluronic acid (M/G) ratio of 0.28, and the L-guluronic acid block was 0.78, which was higher than the D-mannuronic acid block. Rheological characterization of SCSA in aqueous solution was shear-thinning pseudoplastic, and alginate gel in 1 M CaCl₂ was more elastic than liquid.     

Comparative study on general properties of alginate lyases from some marine gastropod mollusks

Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via β-elimination, producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino acid sequences of alginate lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate lyases. The alginate lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate lyases with molecular masses of approximately 28, 34, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate lyases with molecular masses of 35, 32, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher-order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino acid sequences, while the L. brevicula enzymes were not.     

两种海洋寡糖对仿刺参免疫活性的影响

研究了褐藻酸钠寡糖(AOS)和壳寡糖(COS)对仿刺参(Apostichopus japonicus)生长及免疫功能的影响。实验分为对照组、COS组和AOS组,对照组投喂企业常规饲料,COS组在对照组饲料基础上添加壳寡糖(1 g·kg~(-1)),AOS组在对照组饲料基础上添加褐藻酸钠寡糖(1 g·kg~(-1))。在第30、60、90天,测定各组仿刺参的质量,并在第90天随机采集50 ind仿刺参的体腔液,测定酸性磷酸酶、碱性磷酸酶、过氧化氢酶和一氧化氮合酶等免疫酶的活力。结果表明,AOS组和COS组仿刺参体质量分别比对照组增长16.4%和18.2%;AOS组仿刺参的酸性、碱性磷酸酶活性分别提高了75.1%和67.3%;而COS组仿刺参的过氧化氢酶和一氧化氮合酶的活性分别提高了190.3%和55.6%。海洋寡糖COS和AOS作为仿刺参的免疫增强剂,可有效提高幼参的生长性能和免疫力,其中COS的作用效果优于AOS。     

Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare

The virulence of eight Flavobacterium columnare strains was studied to find correlations between several virulence-related factors and virulence. Virulence was tested in vivo using rainbow trout, Oncorhynchus mykiss (Walbaum). Suggested virulence-related factors such as production of the degradative enzyme chondroitin lyase, plasmid occurrence and adhesion capability were studied in vitro. Infection with the four most virulent strains resulted in 95-100% mortality within 114 h. Chondroitin lyase activity was found to be significantly related to the virulence of the strains at 25 degrees C and it was also shown to be temperature-dependent, being higher at 25 degrees C than at 20 degrees C. Virulence was not plasmid associated. The adhesion capability of the strains in vitro varied substantially when tested on crude mucus-coated slides and no statistical relationship between adhesion and virulence was found using this method.     

响应面法优化鲅鱼抗氧化肽发酵条件

为减少加工废弃物蛋白所造成的环境污染,提高水产品的利用率,探索了回收利用鲅鱼(Spanish mackerel)加工后富含蛋白质的废弃物,以寻找制备鲅鱼抗氧化肽的最佳生产条件。借助Design-Expert数据处理软件对苏云金芽孢杆菌Hy-4发酵产抗氧化肽的条件进行优化;在单因素试验的基础上,利用PlackettBurman试验设计法对影响发酵制备鲅鱼抗氧化肽的培养条件进行筛选。确定了影响发酵液总抗氧化活性的3个主要影响因素(P0.5)为发酵温度、培养基初始p H和料液比;在此基础上,利用最陡爬坡实验逼近3个关键因素的最大响应区域,再利用Box-Behnken试验设计及响应面分析法进行回归分析,通过求解回归方程得到产抗氧化肽的最优条件为:发酵时间48 h,发酵温度30℃,培养基初始p H 6.8,接种量2%,料液比1.45 g/50 m L,菌龄24 h。经过验证,发酵液总抗氧化活性达到537.73 U,较优化前提高了57.2%,与回归方程的预测值570.11 U相比,相对误差为5.7%,说明模型能够较好地预测菌株Hy-4发酵产抗氧化肽发酵液的总抗氧化性。