温度、pH对缢蛏(Sinonovacula constricta)消化酶活力的影响

本文研究了温度、pH对缢蛏(Sinonovacula constricta(Lamarck))三种主要消化酶(蛋白酶、淀粉酶和纤维素酶)活力的影响。结果表明：当温度在5～75℃之间时，蛋白酶、纤维素酶和淀粉酶的最适温度分别为为55℃、45℃和65℃；在不同的pH范围内，蛋白酶的最适PH值为9．2，淀粉酶的最适PH值为6．3和7．7，纤维素酶的最适PH值为5．2。缢蛏的淀粉酶和蛋白酶活力较大，纤维素酶活力极微。     

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Westem blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5-6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards （＂marker＂） so that the alterations ofprotease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10^4 cell/mL in the indirect ELISA, while 10^5 cell/mL in the dot-ELISA.     

牙鲆孵化酶的分离纯化及其部分生物化学性质研究

利用凝胶过滤柱层析和阴离子交换柱层析技术,从牙鲆（Paralichthys olivaceus）胚胎孵化液中分离纯化出了大小约为34．8ku的牙鲆孵化酶。该酶卵膜裂解的最适反应温度为35℃,最适pH为7．0;对底物酪蛋白的米氏常数Km值为1．53mmol／L。该酶对丝氨酸蛋白酶和胰蛋白酶的特异性抑制剂非常敏感,而对其他蛋白酶抑制剂不敏感,表明该酶极可能是一种丝氨酸蛋白酶类型的胰蛋白酶。此外,该酶可浓度依赖性地被EDTA所抑制,被Cu^2＋所强烈抑制,被Ca^2＋和Mg^2＋所激活,对Zn^2＋则不敏感,表明该酶很可能是一种金属蛋白酶。     

南海沉积物细菌胞外蛋白酶在家族水平上的多样性

海洋产蛋白酶细菌及其分泌的胞外蛋白酶在降解有机氮以推动海洋氮循环进行方面发挥着重要作用,但目前对这二者多样性的认识非常有限。本研究自南海沉积物中筛选得到90株产蛋白酶细菌,并通过N-端氨基酸序列,分析了其胞外蛋白酶在家族水平上的多样性。16S rRNA基因序列分析的结果表明,筛选的产蛋白酶细菌均属于Gammaproteobacteria纲,且大多数属于Alteromonadales目和Vibrionales目的不同属。对其中14株菌株的14个胞外蛋白酶的N-端氨基酸序列分析表明,所有这些蛋白酶属于金属蛋白酶的M4家族或丝氨酸蛋白酶的S8家族。本研究提供了海洋沉积物产蛋白酶细菌在类群及其胞外蛋白酶在类型上的新细节,这将有助于全面了解微生物酶促降解海洋沉积物有机氮的过程和机理。     

Salinivibrio sp.YH4胞外丝氨酸蛋白酶EYHS耐盐性及生物信息学分析

摘要：目的 探讨菌株Salinivibrio sp.YH4分泌的丝氨酸蛋白酶EYHS的耐盐性及结构特征。方法 明胶底物酶谱法分析EYHS的耐盐性。应用生物信息学手段对EYHS及6种耐盐的S8家族丝氨酸蛋白酶结构特征进行分析。结果 EYHS在4 mol/L的NaCl溶液中仍具有活性,属于耐盐蛋白酶。EYHS及6种S8家族丝氨酸蛋白酶分子表面的loop区等无规则卷曲所占比例较高,α-螺旋与β-片层则主要位于酶分子内部。EYHS分子表面酸性氨基酸含量较高,且具有弱疏水内核。多序列比对发现蛋白酶的催化三联体两侧存在高度保守的基序和保守的极性氨基酸及芳香族氨基酸,并存在多个保守的Gly与Ala。同源模建和表面电荷分布显示,α螺旋和β片层围成了蛋白酶的催化腔,EYHS活性中心包含由Asp32、His65与Ser215组成的催化三联体,且催化位点区域表面静电势为负。结论 上述结构特征可能有助于耐盐丝氨酸蛋白酶EYHS在高盐环境下维持其稳定性和适度柔性,并有助于其催化功能的发挥,为深入研究耐盐丝氨酸蛋白酶的高盐环境适应性提供了一定的理论依据。     

产碱蛋白酶海洋弧菌的筛选 及其培养条件的研究

运用酪蛋白平板法从海水中筛选分离出一株分泌高活性碱性蛋白酶的 海洋弧菌。对其产酶最适条件进行了优化试验。结果表明：在pH8,含2%NaCl,含蛋白胨、蔗 糖和酵母膏,接种量2%,23℃,培养35h条件下,产酶活性最高。发酵液中酶的活性可达719 μ/mL。     

Screening and molecular classification of low-temperature protease from Antarctic microorganism and its characterization

107 strains producing protease were screened from 260 strains of Antarctic psychrophilic bacteria, among which proteolytic activity of five strains was more than 45 U ml^-1. The 16S rRNA gcne sequences homology and phylogcnetic analysis of five Antarctic psychrophillc bacteria showed that NJ276, NJS-9, NJ16-70,NJ345 belonged tO the described genus Pseudoalteromonas and NJ341 belonged to the genus Colwellia. The growth and the protease characteristic of four Antarctic psychrophilic bacteria had been studied, and the result showed that the 6ptimal temperature for growth and protease-produeing of four strains was about 10℃. Their growth and protease-produeing were still high during incubatlng 2-5 days. The maximum proteolytic activity occurred at pH 9 for four Antarctic psychrophilic bacteria. The optimal temperature of protease action of both strains NJ276 and NJ5-9 was about 50℃, however, the optimal temperature of protease aetlon of both strains NJ341 and NJ345 was about 40 ℃, and their proteolytic activity under 0℃ exhibited nearly 30% of the maximum activity, but their thermal stabilities were weaker. These results indicated that proteases from NJ341 and NJ345 were low-temperature proteases.     

桂林会仙岩溶湿地土地利用方式对球囊霉素相关土壤蛋白分布的影响

土壤丛枝菌根真菌分泌的球囊霉素相关土壤蛋白(GRSP)是土壤碳库变化的重要指标, 为明确其在会仙岩溶湿地不同土地利用方式下的分布特征及影响因素, 以会仙岩溶沼泽, 并由其转变而来的水稻田、旱地、果园和弃耕地4种不同土地利用方式为研究对象, 采集0–10 cm、10–20 cm和20–40 cm这3个层次的土样, 对不同土地利用方式下球囊霉素相关土壤蛋白分布特征及其与土壤因子的关系进行了研究。结果表明, 不同土层总球囊霉素相关土壤蛋白(T-GRSP)含量为1.08～3.35 mg/g, 占土壤有机碳的12.33%～19.73%, 球囊霉素相关土壤蛋白是湿地土壤中的一个重要碳库。球囊霉素相关土壤蛋白在不同土地利用方式和土层之间均表现出显著差异, 随土层深度的增加表现出降低趋势。沼泽土壤中总球囊霉素相关土壤蛋白、易提取球囊霉素相关土壤蛋白(EE-GRSP)含量和有机碳(SOC)的含量均高于其它4种土地利用方式(水稻田、旱地、园土和弃耕地)。GRSP分别与蛋白酶、SOC和全氮(TN)呈极显著正相关(P＜0.01), 分别与速效氮(AN)、速效磷(AP)、粘粒和粉粒呈显著正相关(P＜0.05)。EE-GRSP与SOC和TN呈极显著正相关(P＜0.01), 分别与蛋白酶和粘粒呈显著正相关(P＜0.05)。主成分分析表明, 粉粒、SOC、AN和TN是影响球囊霉素相关蛋白分布特征和反映会仙岩溶湿地土壤营养状况的主要因子。会仙岩溶湿地土壤中的球囊霉素相关土壤蛋白对土壤碳封存有重要贡献。     

Purification and characterization of an alkaline protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca²⁺ could enhance the proteolytic activity of the protease, while Cu²⁺, EDTA and PMSF could inhibit its activity.