Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia’s hot springs

A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia’s hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70℃ and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70℃ and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.     

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。     

动物蛋白酶1058水解ti鱼可溶性蛋白质

研究了动物蛋白酶 1 0 58水解鱼是鱼蛋白时 ,酶量、温度、作用时间对鱼是鱼蛋白质水解率和苦味的影响。结果表明 :酶浓度的提高 ,水解时间的延长 ,均使鱼是鱼蛋白水解率明显提高。最适水解温度为 50℃ ,当温度高于 50℃时 ,水解率逐渐下降 ,蛋白酶开始失活。苦味值随水解率提高而增加。用正交试验的方法 ,经综合分析和试验得出 ,该蛋白酶水解鱼是鱼的最佳条件是酶量与底物之比为 1 .5∶ 1 0 0 0 ;温度为 50℃ ;水解时间为 4h。     

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。     

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15℃ and 30℃, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35 ℃ and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Ca2+ and Mn2+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.     

三疣梭子蟹(Portunus trituberculatus)肝胰腺内源酶性质的初步研究

内源酶是三疣梭子蟹(Portunus trituberculatus)体内重要的酶,往往会导致梭子蟹死后肌肉组织迅速软化,严重影响了蟹肉的贮藏品质。为了探明该酶的基本特性,本试验从三疣梭子蟹肝胰腺中提取了粗酶,优化了提取方法,并对其部分酶学性质进行初步研究。结果表明:三疣梭子蟹肝胰腺内源酶最佳浸提时间段为4～12 h,酶比活显著高于0～4 h和12～14 h;以酪蛋白为水解底物,内源酶作用最适温度为65℃、最适p H范围为7.0～8.0;丝氨酸蛋白酶特异性抑制剂,包括大豆胰蛋白酶抑制剂(SBTI)和苯甲基磺酰氟(PMSF),对内源酶活力的相对抑制率分别为100%、70.46%±6.27%,显著高于其他抑制剂的相对抑制率,推测丝氨酸蛋白酶为主要内源酶;在硫酸铵分级沉淀中,分别以酪蛋白和Boc-Phe-Ser-Arg-MCA为底物,前者最适盐析浓度为0～70%,后者为30%～70%,酶比活显著高于其他盐浓度;当硫酸铵浓度为40%～60%时,盐析蛋白质含量、粗酶酶活和丝氨酸蛋白酶活均显著高于其他盐浓度。     

米曲霉中性蛋白酶特性的研究

研究了米曲霉中性蛋白酶的特性。结果表明，米曲霉中性蛋白酶的最佳提取方法为加入pH7．2的缓冲液研匀，置恒温（30℃）培养箱中提取30min．每10min搅动1次，最后用4层纱布过滤。酶反应最适温度为55℃，最适pH值为7．2。NaCl和EDTA对其有一定的抑制作用。酶的热稳定性好．在45℃的温度下处理24h后，酶活力仍有45．5％。米曲霉中性蛋白酶与培养物一起保存时，稳定性也较好，在4℃保存一个月后，酶活力几乎无变化。     

米曲霉产中性蛋白酶的适宜条件

研究了米曲霉产蛋白酶的分布,优化了米曲霉产中性蛋白酶的适宜培养条件以及培养基的最优组成。研究发现米曲霉产中性蛋白酶的能力为最强。米曲霉产中性蛋白酶的适宜培养条件为:m(麸皮)∶m(豆粕粉)=4∶1,水的质量分数为60%,培养基中各无机盐质量分数为:KNO30.2%,MgSO40.05%,Na2HPO40.13%,pH值为6.0,接种量为每10 g培养基接种1.0×108个孢子,最佳培养温度为30℃,最佳培养时间为48 h。在此培养条件下,最高酶活力达3 999.2 U.g-1。     

Intraspecific Diversity of Aureobasidium pullulans Strains from Different Marine Environments

Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureobasidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc,A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A.pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.     

一种新的海洋微藻病毒丝氨酸蛋白酶基因的克隆表达及其活性分析

从海洋球石藻Emiliania huxleyi病毒EhV99B1的基因组中克隆了丝氨酸蛋白酶(Sp)基因(GenBank登录号:KC161207),对该基因的开放阅读框(ORF)进行系统的生物信息学分析,并在大肠杆菌中融合表达,通过亲和层析法获得了纯化的重组Sp。结果表明:EhV99B1-Sp基因的ORF为1 110bp,编码368个氨基酸,蛋白相对分子质量为39.5kDa;该基因片段与GenBank中EhV86-Sp的同源性很高,核苷酸及其对应的氨基酸序列同源性分别为95%和97%,而与其他物种Sp序列的同源性仅为28%~32%,说明其可能是丝氨酸蛋白酶家族中的一个新成员;预测的二级结构特征显示EhV99B1-Sp的蛋白结构域中具有典型的LTAGHC(组氨酸活性位点区域)和AICNGDSGGPLF(丝氨酸活性位点区域)两个丝氨酸蛋白酶催化活性位点的氨基酸基序,是一个两次跨膜蛋白;将该基因在大肠杆菌中进行低温诱导表达,得到分子量为60kDa的重组蛋白,经鲤鱼肌肉丝氨酸蛋白酶(MBSP)抗体检测证实为Sp,且重组蛋白在大肠杆菌细胞中具有明显的生物学活性。本研究结果为进一步探讨EhV99B1-Sp在病毒与宿主相互作用过程中的调节作用及其功能与应用奠定基础。