卡拉胶酶产生菌的筛选及发酵条件优化

为获得卡拉胶酶高产菌株,用卡拉胶作为唯一碳源,对从海水中分离的13株菌进行筛选,进而采用分子生物学方法将3号菌株鉴定为γ变形杆菌纲的Gilvimarinus属,并应用单因素试验和正交试验对3号菌株降解活性培养条件进行优化,结果显示较优培养条件为:蛋白胨0.20%,卡拉胶0.20%,琼脂0.001%,氯化钠2%,磷酸氢二钾10%,硫酸镁0.5%,磷酸铁0.1%,氯化钙1%,25℃培养2d。     

琼胶降解菌AT-22的筛选和产酶性质

从青岛近海的红藻(Gelidium amansii)中分离筛选到1株高活力的海洋琼胶降解菌AT-22，对其生长、产酶特性和酶活力影响因素进行初步研究。结果表明，AT-22所产琼胶酶为诱导酶，0．2％葡萄糖的添加对菌株产酶有抑制作用。该酶作用的最适pH值为6．0-7．0，最适温度为40℃，最适底物质量分数为1％～1．2％，Ca^2 的添加对酶促反应有较强的促进作用，而Fe^3 、Mn^2 、Cu^2 和Hg^2 等有不同程度的抑制作用。     

九孔鲍褐藻酸酶降解褐藻胶的反应条件与酶解产物的分析

以九孔鲍(Haliotis diversicoloeaquatilis)为原料制备一定纯度的褐藻酸酶,并对影响其降解褐藻胶的条件和产物的性质进行分析。该酶的最适温度和pH分别为45℃和7.0;与磷酸盐缓冲液相比,其在Tris-HCl缓冲液中与底物的亲和力相对较高。动力学曲线表明酶解反应主要发生在1 h之内,在2 h之内达到平衡,酶的剂量和底物的浓度对该平衡起到一定的影响作用。反应2 h之后,添加同数量的酶或者底物发现,产物的反馈抑制只是反应达到平衡的一个影响因素,而酶的变性失活也起到了重要的作用。金属离子对酶的活性具有明显的影响,Co2 、Mg2 对酶的活性具有较强的促进作用,而Cu2 、Ag 和Zn2 则具有较强的抑制作用。产物特性黏度的变化主要是在1 h内,2 h后基本不再改变。1HNMR图谱分析发现随着聚甘露糖片段,特别是M-M二聚体的降解,MG二聚体、GGM和MGM三聚体的量相应增多,表明该酶属于甘露糖裂解酶(EC 4.2.2.3)。     

异枝麒麟菜酶法降解工艺的优化研究

以施氏假单胞菌分泌的卡拉胶酶为工具,采用二次回归正交旋转组合设计,对异枝麒麟菜的酶法降解工艺进行了研究,探讨pH、温度和卡拉胶酶添加量对还原糖生成量的影响。pH、温度和卡拉胶酶添加量与还原糖生成量的数学回归模型为:Y=0.3310 0.0107Z1-0.0848Z2 0.0451Z3-0.0012Z31-0.0055Z32 0.0186Z21-0.0940Z21-0.0635Z22-0.0125Z23;最佳的酶解参数为:pH 6.27,温度39.6℃,加酶量74.4 U,底物质量浓度为0.7595 g/L(总糖含量),酶解8 h。     

甲壳低聚糖酶法制备工艺的优化

为制备具有较好水溶性和生理活性的甲壳低聚糖,本实验选用纤维素酶对壳聚糖进行降解,以还原糖浓度作为衡量降解效果的指标,通过单因素实验和正交实验优化酶解制备工艺,获得最佳酶解条件为:酶浓度1600U/g、温度55℃、反应时间7h、pH值5.4,在该条件下获得的酶解产物还原糖浓度为2.52mmol/L。得到的酶解液通过超滤分离,获得分子量小于10KDa的甲壳低聚糖,其产率达到69.51%。     

南极微生物产低温蛋白酶菌株的筛选、分子鉴定及部分酶活特性

从南极获得的260株低温细菌中筛选到107株具有蛋白酶活性菌株，其中5株菌所产蛋白酶的活性高于45U／mL。对其进行16S rRNA基因序列的同源性和系统发育分析，结果表明，菌株NJ276、NJ5—9、NJ16—70、NJ345属于假交替单胞菌属(Pseudoalteromonas)，NJ341属于科尔韦尔氏属(Colwellia)。对其中NJ276、NJ341、NJ16—70、NJ345这4株产蛋白酶南极嗜冷菌的生长及分泌蛋白酶的部分酶活特性进行研究，结果表明：(1)4株菌最适生长、产酶温度均为10℃左右；培养2～5d，嗜冷菌生长、产酶量一直处于较高的状态。(2)4株南极嗜冷菌分泌的蛋白酶的酶活反应最适pH值为9。(3)菌株NJ276、NJ5—9分泌的蛋白酶最适酶活温度为50℃；菌株NJ341、NJ345分泌的蛋白酶最适酶活温度为40℃；在0℃时蛋白酶活性是最高活性的30％左右，蛋白酶热稳定性较差，因此菌株NJ341、NJ345分泌的蛋白酶属于低温蛋白酶。     

一株高产琼胶酶菌株MA-B22的分子鉴定与产酶条件优化

琼胶酶在多糖降解应用中的作用日益重要,而从海洋微生物中筛选琼胶酶高产菌株成为一个重要途径。本研究从养殖的杂色鲍体内分离到一株高产琼胶酶的菌株MA-B22。该菌为革兰氏阴性菌,经16S rRNA序列鉴定表明,与Tamlana agarivorans sp. nov.具有99%同源性,因此初步定为Tamlana sp.。在2216E培养基中该菌株在培养时间60 h,温度25 ℃,起始pH为6.0条件下产酶活性最高。基于上述培养条件,通过单因素和正交实验确定了培养基的最佳基本组成：氮源为牛肉膏（其最适浓度为6.0 ‰）,碳源为琼脂（其最适浓度为2.5 ‰）,配制培养基的人工海水的最适盐度为25。经培养条件优化后,该菌胞外琼胶酶活力可达1021.79 U/mL,较优化前提高8.82倍。实验首次对Tamlana sp.属的琼胶酶活性进行确定并优化其产酶条件,为构建琼胶酶高产工程菌株提供了基础,并可能为今后鲍养殖提供潜在的益生菌。     

琼胶寡糖的制备及其_13_C_NMR研究

天然多糖酶降解是制备寡聚糖的一个重要途径。酶降解由于具有特异性 ,可选择性地酶解切断特定链 ,从而制得特定的寡聚糖 ,并且反应条件温和 ,降解过程易于控制 ,在多糖降解中运用日益增多。在对多种微生物筛选研究基础上 ,从一种海洋微生物中分离获得了对琼胶具有较高降解活性的琼胶酶 ,并将此琼胶酶应用于琼胶糖降解修饰研究。本文研究了琼胶寡糖的制备 ,并运用波谱分析对琼胶寡糖的结构进行研究 ,为琼胶的构效关系研究提供基础实验资料。1　材料与方法1 .1　琼胶糖的酶解取 5ｇ琼胶糖 ,加入 2 5 0ｍＬ蒸馏水并加热至 10 0℃摇动使其溶解 ,…     

枯草芽孢杆菌壳聚糖酶基因在毕赤酵母中的表达及酶学性质

构建了枯草芽孢杆菌CH_2菌株(Bacillus subtilis CH_2)壳聚糖酶基因的真核表达载体p PIC9K-CH2-Cns,在毕赤酵母GS115(Pichia pastoris)中进行重组表达,获得了有生物学活性的重组壳聚糖酶,对重组壳聚糖酶的酶学特性及酶解产物进行了分析。结果显示,重组壳聚糖酶的分子量为29 k D,粗酶的比酶活达到133.60 U/mg,纯化后重组蛋白的比酶活为338.08 U/mg;该酶的最适作用温度为50℃,最适pH为4.5,酶动力学常数Vmax=24.39(μmol/mg)·min~(-1),Km=5.48 mg/m L;Fe2+、K+等对其酶活力有一定的激活作用,其中Mn~(2+)能使酶活力提升2.4倍,Ag~+、Mg~(2+)、Hg~(2+)、EDTA、EGTA和SDS等存在时则对其酶活力有强烈抑制作用。利用重组壳聚糖酶酶解壳聚糖,酶解产物主要为聚合度2~10的壳寡糖,且分布均匀。以上结果表明,枯草芽孢杆菌CH_2菌株壳聚糖酶基因在毕赤酵母GS115中成功表达,获得了一种内切型的高酶活力重组壳聚糖酶,该重组酶具有反应条件温和、酶解产物均一等特点,可被应用于海洋甲壳质加工应用中。     

黄海黄杆菌YS-9412-130固定化细胞产酶技术

3种不同的包埋材料固定化YS－9412－130菌体细胞,进行半连续发酵。结果表明,用2.5%卡拉胶固定化细胞,产酶效率最高;添加明胶对产酶不利而添加豆饼粉与玉米粉后,酶活力有显著提高;卡拉胶固定化细胞发酵液中游离菌体浓度随发酵时间变化略有上升,总体水平较低。发酵液酶活随菌体浓度的增大而呈上升趋势;固定化细胞半连续发酵效率远高于游离细胞分批发酵的效率。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.