栓孔菌属漆酶高产菌株的初步筛选及其产酶条件的优化

利用显色反应对栓孔菌属(Trametes)进行了漆酶高产菌株的筛选,并对目标菌株的产酶条件进行了优化,在添加愈创木酚的固体培养基中,通过显色反应初步筛选出漆酶高产菌株东方栓孔菌Trametes orientalis Cui 6300;进一步通过单因子分析、正交试验和ABTS法确定了菌株Cui 6300的最适产酶条件:麦芽糖15 g/L,蛋白胨3 g/L,pH 4.8,Cu2+2.0 mmol/L,培养温度28°C,接种饼直径1.5 cm,此时酶活最高可达19.923 U/mL;同时探索了Cu2+浓度及添加时间对其菌丝生物量和漆酶活力的影响。研究表明,Cu2+最适添加浓度为2.0 mmol/L,添加时间为接种后第3天。     

产几丁质酶菌株SWCH-6的筛选、鉴定及其产酶条件的优化研究

从汕头海湾养殖区域的海底沉积物中分离到1株几丁质酶活性较高的菌株, 命名为SWCH-6, 根据菌株的形态特征、生理生化特征和16S rDNA 序列, 确定该菌株为嗜水气单胞菌(Aeromonas hydrophlilla)。采用单因素优化方法结合正交实验, 得到菌株SWCH-6产几丁质酶的最佳发酵条件：胶体几丁质25.0 g/L, 胰蛋白胨10.0 g/L, 陈海水1.0 L, pH 8.5, 32℃, 150 r/min培养72 h; 在该条件下酶活力达0.39 U/mL。此外, 菌株所产几丁质酶的最适催化pH 5.0; 最适催化温度为40℃; Cu2+、Fe3+及表面活性剂Tween-80能增强该酶的催化活性; Zn2+、Mn2+及表面活性剂SDS、洗衣粉对该酶的催化活性有抑制作用, 与其它几丁质酶存在着一些不同。     

低温淀粉酶菌株C2的分离鉴定及其产低温淀粉酶的酶学性质

获得低温淀粉酶高产菌株,确定该菌株所产淀粉酶的酶学性质.从大黑山(大连)污泥中筛选菌株,通过菌株的形态特征、生理生化和16S rDNA序列鉴定确定其种属,对其酶学性质进行初步研究.获得1株低温淀粉酶高产菌株C2,经鉴定其为微小杆菌属,C2所产低温淀粉酶最适反应温度为25℃,酶的热稳定性比较差,最适pH为7.5,Ca2+和Fe2+对该酶有激活作用,Cu2+、Ni2+、Go2+等抑制酶活性.经薄层层析(TLC)鉴定酶解产物为葡萄糖,说明该菌株具有产生低温淀粉糖化酶的能力.菌株C2所产淀粉酶符合低温淀粉酶性质,值得进一步研究.     

产脂肪酶真菌的选育及酶学性质研究

从富含油脂土壤中筛选出一株产碱性脂肪酶酶活达6.40U/mL的真菌菌株,经显微形态及ITS序列分析鉴定为产黄青霉Penicillium chrysogenum,该菌株命名为Penicillium chrysogenum J23。该菌的最佳产酶培养条件为：蔗糖1.0%、蛋白胨2.0%、橄榄油1.0%、MgSO4·7H2O 0.05%、接种量1.0%、初始pH 9.0、摇床转速200r/min、30℃培养48h。其所产脂肪酶的最适反应温度与pH分别为33℃和7.5,在pH6.0－10.0酶具有良好的稳定性,在50℃处理2h仍可保持30%以上的酶活力,50mmol/L的Ca2+、Mg2+、K+分别对酶有较强激活作用,而50mmol/L的Fe2+、Mn2+、Cu2+、Pb2+、Li2+对酶则有不同程度的抑制作用。     

产木聚糖酶白地霉培养特性及部分纯化的酶学特性

本文对白地霉Ref1的培养特性、产酶条件和酶学特性进行了初步研究。结果表明:该菌为低温型菌株,其最佳生长条件为pH6、20℃和酵母膏作为氮源;最佳产酶条件为pH3-7、15℃及以酵母膏氮源;条件优化后产酶可达118.7U/mL,可溶蛋白含量可达到60μg/mL,酶溶液的比活可达到1250U/mg蛋白质;该木聚糖酶的最适反应温度和pH分别为50℃和5,金属离子Mg2+、Na+和8mmol/L的Fe2+、Cu2+、Zn2+等对木聚糖酶的活性有抑制作用,而Ca2+、4mmol/L的Fe2+、Cu2+、Zn2+和8mmol/L的Mn2+等对该酶反应则有促进作用;该木聚糖酶在保温2h后在15-40℃范围内能保持80%以上的酶活性,在50℃时能保持68%的酶活性;用lineweaver-Burk作图法(双倒数作图法)求得该酶的最大反应速度Vmax和Km值分别为163.38mmol/mg/min和0.75mg/mL。     

溶藻弧菌中酯酶基因的克隆表达及其酶学性质研究

旨在克隆溶藻弧菌中的酯酶基因,并在大肠杆菌中异源表达,研究酯酶酶学性质。从溶藻弧菌(Vibrio alginolyticus)7S-1的基因组DNA中克隆得到酯酶基因est Z,将该基因连接入表达载体p ET28a,并在Escherichia coli BL21(DE3)中表达,对纯化后的酯酶蛋白进行酶学性质分析。结果显示,表达菌株E.coli BL21-28a-est Z可高表达具有活性的酯酶,在18℃添加0.4 mmol/L IPTG的LB培养基中诱导22 h,酯酶的比酶活可达5.42 U/μg;重组表达的酯酶Est Z最适反应温度为25℃,最适pH为9.0。10 mmol/L Na+和Mg~(2+)对其有较好的激活作用;Cu~(2+)、Ni~(2+)、Co~(2+)对该酶活性有较大抑制。该酶对多种有机溶剂及表面活性剂具有一定的耐受性,同时具有较高的耐盐性。对溶藻弧菌中的酯酶进行研究为该菌更好的应用于生物脱墨技术奠定了理论基础。     

一株β-葡萄糖苷酶产生菌株的分离鉴定及酶学性质研究

【目的】分离获得β-葡萄糖苷酶高产菌株,确定该菌分类地位,并对其所产β-葡萄糖苷酶的酶学性质进行初步研究。【方法】采用七叶灵显色法从土壤样品中筛选β-葡萄糖苷酶产生菌,再用对硝基苯基-β-D-吡喃葡萄糖苷(PNPG)显色法进行复筛;通过形态特征、生理生化特征及16S rDNA序列相似性分析等方法确定其分类学地位;利用超滤、疏水层析、阴离子层析、分子筛层析法对β-葡萄糖苷酶进行分离纯化;以PNPG为底物,测定β-葡萄糖苷酶的最适反应pH及最适反应温度,通过双倒数作图法确定β-葡萄糖苷酶催化不同底物水解的米氏常数Km值。【结果】从土壤样品中筛选得到一株β-葡萄糖苷酶高产菌株ZF-6C,初步鉴定为Bacillus korlensis;芽胞杆菌ZF-6C所产β-葡萄糖苷酶的分子量约为90 kD,最适反应pH和温度分别为7.0和40°C,该酶具有水解β(1,4)糖苷键的活性,最适底物为邻硝基苯-β-D-吡喃葡萄糖苷,Km值为0.73 mmol/L。金属离子Ca2+、Pb2+增强酶活,而Cu2+、Fe2+抑制酶活。【结论】首次报道从Bacillus korlensis中分离得到β-葡萄糖苷酶,Bacillus korlensis ZF-6C所产β-葡萄糖苷酶在分子量、最适反应条件及底物特异性等方面均不同于已知酶,可能为一结构新颖且催化效率较高的β-葡萄糖苷酶。     

来自桔青霉的阿魏酸酯酶的分离纯化、理化性质

【目的】从桔青霉的发酵液中分离纯化了胞外阿魏酸酯酶(PcFAE)并进行了酶学性质的研究,初步探讨了PcFAE对麦糟的酶解作用。【方法】利用(NH4)2SO4沉淀、DEAE-Sepharose Fast Flow离子交换层析、Phenyl Sepharose6Fast Flow疏水层析纯化得到电泳纯的阿魏酸酯酶。【结果】从该菌株的发酵液中获得一阿魏酸酯酶,该酶亚基分子量约为31kDa,全酶分子量约为58kDa。其最适pH为6.0,最适温度为45℃-65℃,在pH5.0-6.0及25℃-55℃之间,酶保持了较好的稳定性。Mg2+、Fe2+、Mn2+、Ca2+和Na+对酶活有一定的促进作用,Zn2+对PcFAE酶活有一定的抑制作用,而Cu2+、亮抑肽素、抑肽酶有显著的抑制作用,Hg2+、苯甲基磺酰氟几乎完全抑制了酶活。EDTA对PcFAE活性无明显影响。PcFAE的kcat/Km对香豆酸甲酯、芥子酸甲酯、阿魏酸甲酯、咖啡酸甲酯的值分别为823、416、103、0,PcFAE对MpCA的催化效率最高。PcFAE作用于麦糟,当5U PcFAE/g麦糟时,其阿魏酸的释放量为7.2%。【结论】获得了一阿魏酸酯酶,其理化性质与至今报道的阿魏酸酯酶有所不同,为阿魏酸酯酶的开发提供了重要的实验依据。     

乳酸乳球菌Nisin抗性基因nisI的克隆及作为筛选标记的研究

摘要：【目的】为了优化LJ1菌株的培养条件使之产生高活性的胞外褐藻胶裂解酶。【方法】通过富集培养技术从海带筛选到一株褐藻胶裂解酶产生菌LJ1, 依据表型特征、脂肪酸组成分析及16S rRNA基因序列分析对该菌株进行鉴定。通过单因子和正交试验对LJ1 菌株产胞外褐藻胶裂解酶的培养条件进行了优化。【结果】LJ1菌株属于假交替单胞菌属（Pseudoalteromonas）。该菌株产酶的最佳培养基组成为：褐藻胶3 g/L、(NH4)2SO4 3 g/L、NaCl 20 g/L、KH2PO4 0.1 g/L、CaCl2 0.1 g/L;最佳培养条件为：250 mL三角烧瓶中装液量25 mL、接种量3%、摇瓶转速150 r/min、pH7.5、培养温度为28℃、培养时间为24 h。LJ1菌株所产褐藻胶裂解酶的最适温度为40℃,最适pH7.6,最适NaCl浓度为0.3 mol/L。1 mol/L金属离子Mg2+对酶活力有明显的促进作用,而Co2+ 和Zn2+对酶活力有较强的抑制作用。【结论】LJ1菌株是Pseudoalteromonas 新的胞外褐藻胶裂解酶产生菌,在最佳培养条件下,该菌株的酶活力提高了66%。     

一株近玫色锁掷酵母的分离鉴定及其产胞外乳糖酶性质的研究

从广州徐闻农垦丰收农场土壤样品中分离到一株产乳糖酶菌株,结合菌株形态特征和ITS基因序列同源性分析结果,表明该菌株为Saccharomycetes sp.的未定种,其系统分类学关系与近玫色锁掷酵母Sporidiobolus pararoseus strain AUMC 7791(JQ425362.1)最近,故命名为:近玫色锁掷酵母XWSP1(Sporidiobolus pararoseus XWSP1),该菌种保藏号为CCTCC NO:M2019119。该菌发酵液能水解邻硝基酚-β-D-半乳糖苷生成黄色的邻硝基酚,具有产乳糖酶性能。优化了该菌株发酵培养条件,结果表明:该菌株在蛋白胨15 g/L、酵母粉20 g/L、半乳糖20 g/L和初始pH 7.0的培养基中,以1×106 CFU/mL接种浓度、4%接种比例,28℃180 r/min恒温振荡培养54 h时,菌株分泌的胞外乳糖酶具有最高的活力。酶学性质初步研究结果表明,该胞外乳糖酶在pH 7.0的反应条件下酶活力最高,Ca^2+、Mn^2+、Mg^2+和Cu^2+对酶活有不同程度抑制作用,其中Cu^2+对酶活抑制作用最强。     

The interaction of chloroquine with transport ATPase and acetylcholine esterase in microsomal membranes of rat in vitro and in vivo

Chloroquine, an antimalarial drug has been found to inhibit Na+, K+-ATPase activity in vitro in the microsomal membranes of rat brain on time, temperature and concentration dependent manner. There have been stimulation of Na+,K+-ATPase, Ca+2-ATPase and acetylcholine esterase activities in vivo studies at lower concentration of drug or shorter period of treatment with the drug, whereas higher concentrations or longer periods of treatment lead to inhibition in the microsomal membranes of different organs.     

Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.     

金属离子对嗜热菌BF80生长及苯酚降解的影响研究

探测了17种金属离子对嗜热菌BF80菌生长和降解苯酚的影响.结果表明:与对照相比,0.01%的Cu2 、Zn2 、CO2 、Ba2 、Hg2 、Ni2 、Al 0和Al3 对嗜热菌BF80有强抑制作用;Cr2 对嗜热菌BF80的苯酚降解特性有强抑制作用,而其生长量只受到一定的抑制作用;Sn2 、Fe2 、Fe3 和Pn2 对嗜热菌BF80的生长和苯酚降解有一定抑制作用,该作用随金属粒子浓度的增加而增大;低浓度Mn2 和Mo2 可以使其生长量增大且促进苯酚降解,但超过0.1%的浓度则抑制其生长;Ca2 和Mg2 可以加速嗜热菌BF80的生长和降解苯酚的速率,但对苯酚的最大降解率却几乎没有影响;Mo2 和Mn2 的复合作用使嗜热菌BF80的生长量更大,但是苯酚降解率却比分别单独添加Mo2 和Mn2 时低.     

金属离子对嗜热菌BF80生长及苯酚降解的影响研究

探测了17种金属离子对嗜热菌BF80菌生长和降解苯酚的影响。结果表明：与对照相比, 0.01%的Cu2+、Zn2+、Co2+、Ba2+、Hg2+、Ni2+、Ag+ 和Al3+对嗜热菌BF80有强抑制作用; Cr2+对嗜热菌BF80的苯酚降解特性有强抑制作用, 而其生长量只受到一定的抑制作用; Sn2+、Fe2+、Fe3+和Pn2+ 对嗜热菌BF80的生长和苯酚降解有一定抑制作用, 该作用随金属粒子浓度的增加而增大; 低浓度Mn2+ 和Mo2+可以使其生长量增大且促进苯酚降解, 但超过0.1%的浓度则抑制其生长; Ca2+ 和Mg2+可以加速嗜热菌BF80的生长和降解苯酚的速率, 但对苯酚的最大降解率却几乎没有影响; Mo2+ 和Mn2+的复合作用使嗜热菌BF80的生长量更大, 但是苯酚降解率却比分别单独添加Mo2+ 和Mn2+时低。     

Genome sequence of Idiomarina sp. strain A28L, isolated from Pangong Lake, India

Idiomarina sp. strain A28L was isolated from the alkaline brackish water of a high-altitude lake, Pangong Lake. Here, we present the draft genome of Idiomarina sp. strain A28L, which contains 2,591,567 bp with a G+C content of 45.5 mol% and contains 2,299 protein-coding genes and 56 structural RNAs.     

Role of acetylcholine in regulation of interaction between axon and Schwann cell during rhythmic excitation of nerve fibers

Axon excitation increases the number of acetylcholine receptors (ACR) of the Schwann cell (SC) depending on the frequency of rhythmic excitation (RE) and on intercellular concentrations of K+, Ca2+, and acetylcholine. During RE, activity of axonal acetylcholine esterase is decreased, thus providing for high intercellular acetylcholine concentration. Increased intercellular concentration of acetylcholine activates phosphoinositide-specific phospholipase C (PIPLC) of the myelin nerve fiber. During RE, K+ depolarization and acetylcholine exocytosis can activate Ca2+ entry via Ca2+ channels, thus inducing SC ACR phosphorylation mediated by PIPLC stimulation.     

β-葡聚糖酶的分离纯化和特性研究

对里氏木霉所产β-葡聚糖酶粗酶液通过饱和硫酸铵沉淀、Sephadex G-100 柱层析和DEAE-Sephadex A-50 柱层析进行纯化,比活提高14.60倍,活力回收6.62%。酶特性研究表明,最适温度和pH分别为60℃和5.0,在pH低于5.0时酶较稳定,酶的热稳定性在60℃以下。 Cu～2+、 Mn～2+ 、Mg～2+ 、Fe～3+ 和K+对酶有抑制作用, Zn～2+、Ca～2+、 Co2+和 Fe～2+ 有激活作用。     

Purification and properties of pyruvate kinase from Streptococcus mutans.

Pyruvate kinase (EC 2.7.1.40) from Streptococcus mutans strain JC2 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 180,000 to 190,000, and the enzyme was considered to consist of four identical subunits. This enzyme was completely dependent on glucose 6-phosphate for activity, and the saturation curve for activation by glucose 6-phosphate was sigmoidal. In the presence of 0.5 mM glucose 6-phosphate, the saturation curves for the substrates phosphoenolpyruvate and ADP were hyperbolic, and the Km values were 0.22 and 0.39 mM, respectively. GDP, IDP, and UDP could replace ADP, and the Km for GDP (0.026 mM) was 0.067 of that for ADP. The enzyme required not only divalent cations, Mg2+ or Mn2+, but also monovalent cations, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for glucose 6-phosphate increased. However, the enzyme was immediately inactivated in a solution without Pi. The intracellular concentration of glucose 6-phosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. mutans.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.