Characterization of a thermostable manganese-containing superoxide dismutase from inshore hot spring thermophile Thermus sp. JM1

A thermostable superoxide dismutase (SOD) from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The specific activity of the purified native enzyme was 1 656 U/mg. A sod gene from this strain was cloned and overexpressed in Escherichia coli (E. coli). The prepared apo-enzyme of the purified recombinant SOD (rSOD) was reconstituted with either Fe or Mn by means of incubation with appropriate metal salts. As a result, only Mn 2+ - reconstituted rSOD (Mn-rSOD) exhibited the specific activity of 1 598 U/mg. SOD from Thermus sp. JM1 was Mn-SOD, judging by the specific activities analysis of Fe or Mn reconstituted rSODs and the insensitivity of the native SOD to both cyanide and H 2 O 2 . Both the native SOD and Mn- rSOD were determined to be homotetramers with monomeric molecular mass of 26 kDa and 27.5 kDa, respectively. They had high thermostability at 50 ° C and 60 ° C, and showed striking stability across a wide pH span from 4.0 to 11.0.     

Screening of protease-producing marine yeasts for production of the bioactive peptides

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.     

A growth model for a hyalid amphipod Hyale perieri in the coastal water of Alexandria (Egypt)

Abstract-The growth rate of the hyalid amphipod Hyale perieri was studied on the bases of Ikeda'sgrowth model which is based on the inter moult period (IP) and moult increament (ΔBL). To applythis approach, laboratory experiments were carried out at three temperatures regimes (15℃, 20℃,25℃) to gain accurate data of IP and BL. The total number of specimens used in this study was 86 at15℃, 24 at 20℃ and 70 at 25℃. The number of flagellar segments of both antennae of the Hyaleperieri could not be used as an index of growth (instar criterion). The obtained results indicated that,the predicted IP of the specimens was inversely related to temperature and in good agreement with theobserved values at the experimental temperatures. IP data obtained from laboratory-reared specimes arecombined with ΔBL data to establish a growth model for Hyale perieri from its release from the mar-supium (1.64 mm BL) to the maximum size (12.67 mm BL) as a function of temperature. The maxi-mum numbers of consecutive moults     

Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1

The gene(741 bp) encoding carboxylesterase from the thermophilic bacterium Geobacillus sp.ZHl was cloned and overexpressed in Escherichia coli.The purified recombinant protein presented a molecular mass of about 40 kDa by SDS-PAGE analysis.Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity,yielding highest specific activity with p-nitrophenyl acetate.Among the p-nitrophenyl esters tested,the carboxylesterase presented preference for p-nitrophenyl caprylate,but hydrolyzed p-nitrophenyl butyrate more efficiently.When p-nitrophenyl butyrate was used as a substrate,the recombinant carboxylesterase exhibited highest activity at pH 8.0 and 60℃.Almost no decrease in esterase activity was observed at 60℃for 3 h,and over 40% of activity was still maintained after incubation at 90℃for 3 h.These results indicate that Geobacillus sp.ZH1 recombinant esterase was thermostable.The enzymatic activity was inhibited by the addition of phenylmethylsulfonyl fluoride,indicating that it contains serine residue,which plays a key role in the catalytic mechanism.Except SDS and xylene,this esterase showed stability toward other tested detergents and organic solvents.Cloning,expression,and biochemical characterization of Geobacillus sp.ZH1 carboxylesterase lay a good foundation for its structural characterization and industrial application.     

深海嗜热菌Geobacillus sp. EPT3锰超氧化物歧化酶的纯化与生物化学鉴定

Thermostable SOD is a promising enzyme in biotechnological applications. In the present study, thermophile Geobacillus sp. EPT3 was isolated from a deep-sea hydrothermal field in the East Pacific. A thermostable superoxide dismutase(SOD) from this strain was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose chromatography. SOD was purified 13.4 fold to homogeneity with a specific activity of 3 354 U/mg and 11.1% recovery. SOD from Geobacillus sp. EPT3 was of the Mn-SOD type, judged by the insensitivity of the enzyme to both KCN and H2O2. SOD was determined to be a homodimer with monomeric molecular mass of 26.0 k Da. It had high thermostability at 50°C and 60°C. At tested conditions, SOD was relatively stable in the presence of some inhibitors and denaturants, such as β-mercaptoethanol(β-ME), dithiothreitol(DTT), phenylmethylsulfonyl fluoride(PMSF), urea, and guanidine hydrochloride. Geobacillus sp. EPT3 SOD showed striking stability across a wide p H range from 5.0 to 11.0. It could withstand denaturants of extremely acidic and alkaline conditions, which makes it useful in the industrial applications.     

Degradation of malachite green dye by Tenacibaculum sp. HMG1isolated from Pacific deep-sea sediments

A deep-sea bacterium from the Pacific Ocean identified as Tenacibaculum sp. HMG1 was found to have strong malachite green(MG) degradation activity. The MG tolerance and decolorizing activities of strain HMG1 were confirmed by bacterial growth and high-performance liquid chromatography(HPLC) analyses. Strain HMG1 was capable of removing 98.8% of the MG in cultures within 12 h and was able to grow vigorously at 20 mg/L MG. A peroxidase gene detected in the genome of strain HMG1 was found to be involved in the MG biodegradation process. The corresponding recombinant peroxidase(r POD) demonstrated high degradative activity at 1 000 mg/L MG. Based on the common candidate intermediates, strain HMG1 was inferred to have one primary MG degradation pathway containing r POD. In addition, five other candidate intermediates of the r POD-MG degradative process were detected. The optimal conditions for MG degradation were determined and showed that strain HMG1 and the r POD enzyme could maintain high bioactivity at a low temperature(20°C), variable p H values(6.0–9.0), higher salinities(100 mmol/L) and other factors, such as multiple metal ions, H_2O_2 and EDTA.MG-tolerant strain Tenacibaculum sp. HMG1 and its peroxidase have prospective applications as environmental amendments for MG degradation during coastal remediation.     

Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp.AG1

An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.     

东印度洋分离的一株蓝杆藻的形态学,超显微结构以及系统发生分析

One strain of unicellular greenish algae embedded by mucilage was successfully isolated from equatorial area in the Indian Ocean. Microscopic observation, ultrastructure features and genetic identification confirmed that the strain was closely related to Cyanothece sp., which was a cyanobacteria species with great ecological significance.Cells were solitary with oval or bacilliform shapes. Diameters of this strain were relatively small, ranging from 2.5 to 6.5 μm on average. Ultrastructure of cells was simple. Thylakoids were arranged parietal and keritomized content were observed in the thylakoid region. Various electron-transparent granules with low electron-dense region as well as cyanophycin or glycogen granules-like organelle and carbonxysomes were also observed. For pigment composition, the dominant pigments were chlorophyll a, β-Carotene, Zeaxanthin and an unknown pigment, contributing 23.8%, 26.1%, 14.7% and 15.7% to total pigments respectively. The phylogenetic analysis of16 S rRNA gene and nif H gene confirmed that Strain EIO409 was closely related with Cyanothece sp..     

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.     

一个新的来自于弧菌QD-5并具有Poly-G内切功能的褐藻胶裂解酶的克隆和性质表征

Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp.QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome sequencing, and the result showed that the Vibrio sp. QD-5 containing an alginate lyase gene cluster. One of these genes, aly-IV,was cloned and characterized for the first time. After overexpression, Aly-IV, with a molecular mass of about62 kDa and a theoretical isoelectric point(pI) of 5.12, was purified to a specific activity of 1 256.78 U/mg and showed highest activity at 35°C in the Tris-HCl buffer at pH of 8.9. Moreover, the enzyme activity was enhanced by the metal ions of Na~+, K~+ and Mg~(2+) under certain concentration. Aly-IV degraded favorably polyG blocks in an endo-type, yielding monomer and dimer as the main products. Due to its high substrate specificity, Aly-IV could be used as a potential tool for production of polyG oligosaccharides with low degree of polymerization(DP) and for determining the fine structure of alginate.     

极地产适冷脂肪酶菌株的酶学性质研究

从南北极环境样品中分离到7株产脂肪酶的细菌，经16SrDNA序列分析表明，这些细菌分别属于假交替单胞菌属（Pseudoalteromonas）和嗜冷杆菌属（Psychrobacter）．采用p-NPP法对这7株细菌所产的脂肪酶进行酶学性质研究，结果显示这些酶的最适作用温度在30～40cC、最适作用pH值在7—8之间，在低温下能保持较高的剩余活力，对热敏感，属于适冷脂肪酶．其中假交替单胞菌（Psychrobacter sp．7342）所产脂肪酶具有低温下酶的剩余活力高、有效作用温度和pH范围广、热稳定性较好及对多种金属离子抗性强等特点．该菌株能利用多种单一氮源和碳源产酶，最适产酶温度为25℃．在此基础上进行正交实验分析得到了该菌株的最佳发酵条件为：蛋白胨和淀粉含量各为1．33％，酵母膏含量为0．3％，温度为25℃．     

南极深海沉积物中产低温脂肪酶菌株的筛选与基因克隆

在10℃恒温培养条件下,从第29次南极科学考察获得的南极深海沉积物样品中分离筛选到产脂肪酶细菌3株,产脂肪酶真菌1株,对其进行分子生物学鉴定,确定为假交替单胞菌属（Pseudoalteromonas arctica）、芽孢杆菌属（Bacillus pumilus）、Glaciecola sp.以及白冬孢酵母属（Leucosporidium sp.）.通过测定各酶的最适反应温度,获得产低温脂肪酶芽孢杆菌XZ18,该菌所产脂肪酶最适反应温度为30℃,在0℃仍有部分活性.从NaCl浓度和pH值2个条件对产酶条件进行研究,最终确定了该菌最适产酶NaCl浓度5.0%、最适产酶pH值为6,最高产酶量为4.65 U/cm3.通过PCR扩增,获得了脂肪酶全长序列,对该序列进行系统发育分析,发现该脂肪酶基因处于较独立的分支.     

南极菌Pseudoalteromonas sp.S-15-13引导糖基转移酶基因表达对环境因子变化的响应

为了探讨胞外多糖在南极微生物低温适应性中的作用与机制,克隆了南极菌Pseudoalteromonas sp.S-15-13的引导糖基转移酶(GTF)核心片段,并采用Real-Time PCR的方法研究了温度、冻融循环、盐浓度、pH等条件对该糖基转移酶基因(gtf)表达的影响。结果表明,低温有利于gtf的表达,短时的低温刺激(2℃)1 h后,gtf的表达量即上调为对照的1.5倍;而该菌经4和10℃培养24 h后gtf的表达量约为20℃时的8~12倍;经过冻融循环gtf的表达量上调,在第2个冻融循环后gtf 的表达量较对照提高了3.667倍;盐浓度对gtf的影响表现为低促高抑,即NaCl含量为6.0 %时gtf 的表达量是对照(3.0%)的3.59倍,当NaCl含量达9.0 %时gtf 表达量则显著下调;在一定范围内(pH5.0~8.0),pH的改变会促进gtf 的表达,当pH为6.0时gtf 表达量约为pH7.0时的2倍。该结果为探讨胞外多糖在南极微生物环境适应性中的作用与机制提供了科学依据。     

微泡菌QZHA1褐藻胶裂解酶MAAL1的酶学性质研究

为探究Microbulbifer sp. QZHA1褐藻胶裂解(Escherichia coli)酶MAAL1的酶学性质,将褐藻胶裂解酶基因maal1构建至pET-28a表达载体并利用大肠杆菌BL21(DE3)进行异源表达。研究发现：重组酶MAAL1与来源于Microbulbifer sp. ALW1菌株的褐藻胶裂解酶(WP＿23625014.1)同源性最高,为93.69%,且与PL7家族蛋白聚为一支;重组酶MAAL1最适温度为35℃,最适pH为7.5,在pH为5.5～10.5范围内保存24 h仍能保持60%以上的酶活力;MAAL1具备良好的耐有机溶剂特性,在测试的9种有机溶剂中,除异丙醇外,其他有机溶剂在添加量达到30%(体积分数)后,酶活力依然保持在59%以上;重组酶MAAL1最适条件下酶活力为4.3 U/mg,米氏常数(K_m)值为1.08 mg/mL,最大反应速率(V_max)为4.75 mg/(mL·min),催化常数(K_cat)值为4.52 s^-1;重组酶MAAL1对聚β-D-甘露糖醛酸(p...     

一株假单胞菌的亚硝酸盐降解特性及其对鱼池底泥的净化作用

从海水养殖池的底泥中筛选到一株对亚硝酸盐有强降解作用的假单胞菌（Pseudomonas sp．）SF1,在实验室条件下研究了SF1的亚硝酸盐降解特性及其对高污染鱼池底泥的净化作用。研究结果表明,SF1具有高效降解转化高浓度亚硝酸钠的能力,其适宜降解条件为：亚硝酸钠质量浓度范围为1～1000mg／L;菌种接种量为10％;...     

印尼热泉中产嗜热碱性蛋白酶菌株筛选及酶学性质研究

采用MSM寡营养培养基从印度尼西亚Pantai cermin,Kalianda和Banyuwedang三个地区的热泉水样、泥样以及沉积物样品中分离获得细菌菌株,通过检测蛋白酶产生透明圈和福林酚蛋白酶活性测定相结合的方法,从中筛选出1株产嗜热蛋白酶的菌株PBI,该菌株经初步鉴定为短杆菌属(Brevibacillus),并对其酶学性质进行了研究。结果表明,菌株PBI产蛋白酶的最适酶活温度为60℃,最适pH值为8.0～9.0,具有较好的热稳定性和pH稳定性,60℃时酶的半衰期为30min,70℃条件下20min仍保持46.1%的酶活,该酶在pH值为5.0～9.0范围的缓冲液中酶活相对稳定。其产嗜热蛋白酶的酶活力最高可达到60.53U/mL,在100℃条件下仍能保留26.37%的相对酶活。Fe2+,Fe3+,Cu2+和Zn2+对嗜热蛋白酶活性具有明显的抑制作用。该嗜热蛋白酶对EDTA敏感,苯甲基磺酰氟(PMSF)、亮抑酶肽(Leupeptin)、苄眯(Benzamidine)和胃蛋白酶抑制剂(Pepstain A)对该嗜热蛋白酶都有一定的抑制作用,说明其酶活性受到丝氨酸、半胱氨酸残基的影响。结果表明,该菌株是1株具有进一步改造利用价值的产蛋白酶菌株。     

深海适冷菌SM9913产生的低温蛋白酶

从1855m深的深海泥样中分离纯化得到200多株分泌蛋白酶的适冷菌，其中3株产低温蛋白酶，本文对其中一株Pseudomonas sp.SM9913(P.SM9913)生长的适冷性和它产生的蛋白酶的适低温特性进行了研究。该菌株能够在0℃正常生长，其最适生长温度为15℃，最高生长温度为35℃。为一株适冷菌。该菌株所产蛋白酶的比合成速率在10℃时最高，催化酪蛋白水解的最适温度为35℃，在0℃仍具有3％的酶活力。最适pH为8．0。该蛋白酶的热稳定性很低，在40℃保温10min即丧失85％的活力，40℃时的半衰期为6min，为一典型的低温酶。抑制剂试验表明，该蛋白酶为金属蛋白酶。     

细胞破碎方法对Bacillus sp. DL-2胞内蛋白酶拆分乙酸苏合香酯的影响

(R)-1-苯乙醇是手性药物合成的关键手性砌块, 在多种手性化合物的合成及医药、香料的工业生产中具有重要的作用。对筛选自西太平洋深海沉积物的Bacillus sp. DL-2进行破碎, 获取的胞内蛋白酶能有效拆分(±)-乙酸苏合香酯, 制备高光学纯的(R)-1-苯乙醇。为使拆分效果最优化, 本试验对比了表面活性剂、溶菌酶和超声法3种不同的破碎细胞方式, 并对表面活性剂和溶菌酶破碎法进行了单因素条件优化。最优方法及优化的反应条件为: 使用体积分数0.5%的表面活性剂曲拉通X-100在35℃和pH7.0条件下破碎细胞2h。经过优化后, (R)-1-苯乙醇的对映体过量值为96%, 转化率为23%, 为利用胞内蛋白酶生产手性化学品提供了参考。