凡纳滨对虾蛋白酶的分离纯化及生化特性

采用Tris-HCl缓冲液抽提、Sephadex G-100凝胶过滤层析、DEAE-Sepharose F.F.阴离子交换层析和SDS-PAGE等方法,分离和纯化了凡纳滨对虾蛋白酶,研究了其生化特性.试验结果表明,该蛋白酶粗提物经层析后,得到聚丙烯酰胺凝胶电泳纯的一酶组分.该蛋白酶的比活力从128.35 U/mg增至1835.65 U/mg,提高了14.3倍,产率达31.9%.SDS-PAGE显示该蛋白酶只含一条谱带,相对分子量为25 kD.以酪蛋白为底物,该酶的最适pH为7.0,最适温度为60 ℃, 在50 ℃以下,比较稳定,放置1 h后活性仍超过60%,而超过50 ℃时蛋白酶活性急剧下降,60 ℃放置1 h后,酶活性只残留4.7%.10 mmol/L EDTA、Fe~(2+)、Ba~(2+)和Zn~(2+)对该蛋白酶有较强的抑制作用,抑制率分别为84%、53%、43%和38%,10 mmol/L Mg~(2+)和Cu~(2+)对蛋白酶活性有轻微抑制作用,而10 mmol/L Ca2+能显著促进蛋白酶活性,据此推测凡纳滨对虾蛋白酶可能为一种金属蛋白酶.     

黄海黄杆菌YS-9412-130低温碱性蛋白酶性质鉴定

对黄海黄杆菌YS-9412-130菌株产低温碱性蛋白酶的理化性质研究表明，该酶由256个氨基酸组成，分子量为33000Dar，等电点pI为9.45，米氏常数Km为5×10-3mmol/L；酶的最适作用pH范围为9.5～10.5，最适作用温度30℃，具有一定的抗氧化稳定性。Ca2+、Mn2对酶有激活作用，而Hg2+、Ag+对酶有抑制作用。DFP、NBS严重抑制酶的活性,而同时该酶也能被EDTA抑制。结果表明该低温碱性蛋白酶为一新型的丝氨酸蛋白酶。     

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。     

南极磷虾粗酶液中蛋白酶的基础酶学性质研究及抑制剂的开发

本研究探讨了反应温度、pH、金属离子(Zn~(2+)、Mg~(2+)、Cu~(2+)、Fe~(3+)、Al~(3+)和Ca~(2+))对南极磷虾粗酶液中蛋白酶活性的影响,研究了苯甲基磺酰氟(PMSF)、EDTA·2Na、碘乙酰胺(IAM)和甲苯磺酰–苯丙氨酸氯甲基酮(TPCK)对蛋白酶活性的抑制效果。结果显示,南极磷虾粗酶液中蛋白酶的最适反应温度为40℃;最适反应pH值为8.0;当金属离子浓度为0.5 mmol/L时,Zn~(2+)、Mg~(2+)、Cu~(2+)、Fe~(3+)和Al~(3+)金属离子对蛋白酶酶活的抑制率分别为55.02%、55.02%、35.39%、20.67%和41.12%,而Ca~(2+)离子对蛋白酶活有明显的促进作用;PMSF和EDTA·2Na对蛋白酶酶活具有较为显著的抑制作用,PMSF的浓度为8.0 mmol/L时,抑制率为60%;EDTA·2Na浓度为0.6 mmol/L时,抑制率为86.67%;IAM在低浓度时有一定的抑制作用;TPCK低浓度时抑制效果不明显,浓度升高时有一定的抑制作用。在5~30℃的温度范围内,随着温度升高,EDTA·2Na对蛋白酶酶活的抑制效果逐渐增加。本研究阐明了影响南极磷虾粗酶液蛋白酶酶活的因素,开发了针对南极磷虾粗酶液中蛋白酶活性的抑制剂,为南极磷虾在食品领域的开发利用提供了基础理论数据。     

罗非鱼肠蛋白酶的分离纯化及其性质

以罗非鱼肠为原料,采用超声波辅助提取技术、电泳技术和层析技术等对罗非鱼肠道蛋白酶进行研究,结果表明:将鱼肠匀浆经超声波提取的粗酶液,用30%～70%的硫酸铵盐进行盐析、HitrapTM Q FF阴离子交换柱纯化及Sephadex G-100凝胶柱分离纯化,得到罗非鱼肠蛋白酶纯品,其比活为335U/mg,得率为32.8%;SDS-PAGE电泳为单一蛋白酶带,分子量为28ku。该酶最适pH为8.0～8.5,在pH7.0～9.0的条件下稳定;最适温度为37～42℃,热稳定性好;该酶的Km值和Vmax值分别为0.605g/L和9.407μg/min。金属离子Ag+、Pb2+对蛋白酶有完全抑制作用,Na+、K+对该酶无抑制作用。丝氨酸蛋白酶抑制剂能完全抑制该酶活性,胃蛋白酶抑制剂和脲素对该酶有一定抑制作用,EDTA没有明显抑制作用,DTT能激活该酶活性,该酶为丝氨酸蛋白酶。     

斑点叉尾鮰源嗜麦芽寡养单胞菌胞外蛋白酶的纯化及性质研究

采用硫酸铵盐析,DEAE Sephadex A－50凝胶层析等方法从嗜麦芽寡养单胞菌胞外产物中提纯了一种分子量为45.7 ku的单一多肽蛋白酶。该酶对小鼠和斑点叉尾鮰具有明显的致死作用,其LD50分别为4.33 μg•g-1体重和3.49 μg•g-1体重。酶的最适温度为20 ℃,热稳定性差,100 ℃作用15 min酶活完全丧失,最适pH为9.0,PMSF对酶活无影响,部分金属离子如Ca2+、Hg2+、Cu2+能使酶活性明显下降,而Co2+对酶有一定程度的激活作用,EDTA能完全抑制酶活性,表明该酶为一种金属蛋白酶。图1表4参29     

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。     

养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析

从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2 和Mg2 可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。     

驼海燕内脏中蛋白酶的提取及其酶学性质的研究

以驼海燕为原料,用丙酮沉淀法制备蛋白酶粗酶,并对其酶学性质进行研究。试验结果表明,提取蛋白粗酶的最佳条件为:原酶液与丙酮的体积比为1∶1.6;蛋白酶反应的最适温度为45℃,最适pH值为8.1;pH为7～9时可保持较高的酶活力;蛋白酶的酶活力在20～50℃基本稳定。酶促反应结果表明,Na+、K+、Li+、Mg2+对蛋白酶活力具有促进作用,而Zn2+、Cu2+、Hg2+能抑制蛋白酶的活力,EDTA对蛋白酶活力的影响甚微。酶动力学方程表明,最大反应速度vmax=5.99U/mL,米氏常数Km=0.69g/mL。     

海参内脏胶原降解酶的纯化及性质研究

针对海参自溶现象,研究海参内脏中与胶原降解相关蛋白酶的存在情况,以期了解内源性蛋白酶在海参自溶过程中发挥的作用,为解明自溶的内在机理提供理论依据。通过硫酸铵盐析、DEAE-Sepharose阴离子交换柱层析、Sephacryl S-200凝胶过滤柱层析、Phenyl Sepharose FF疏水柱层析及Mini Q离子交换柱层析等方法,从海参内脏中分离纯化出降解明胶的蛋白酶。利用荧光底物研究其酶学性质及其对海参体壁胶原蛋白的降解情况。结果表明,该酶最适温度为30 ℃,最适pH为7.5,可被金属蛋白酶抑制剂EDTA、EGTA完全抑制,亦可被PMSF、Pefabloc SC等丝氨酸蛋白酶抑制剂完全抑制。底物特异性表明,该酶只对金属蛋白酶荧光底物有分解作用,推测其为金属蛋白酶。该酶在4和37 ℃下均能有效地分解海参体壁胶原蛋白,表明其在海参自溶过程中起重要作用。     

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.     

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.     

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.