碱性蛋白酶工程菌发酵条件及重组酶的纯化和性质的研究

在5L发酵罐中对重组碱性蛋白酶工程菌株BP071高产碱性蛋白酶的条件进行了研究,通过提高通气量和改变搅拌转速,BP071可在发酵40 h内达到产酶高峰,酶活力最高可达24480 u/mL。利用快速蛋白液相层析（FPLC）技术,建立了快速高效纯化碱性蛋白酶的方案。发酵液通过硫酸铵沉淀、DEAE-A-50脱色及聚乙二醇浓缩得粗酶,再经过CM-Sephadex-C-50、Sephadex-G-75柱层析后得到了单一组份的重组碱性蛋白酶,酶纯度提高了76.2倍。SDS-PAGE显示重组碱性蛋白酶分子量为28 kD。酶学性质研究表明,酶的最适作用pH为11,最适作用温度为60℃,具有良好的pH稳定性和热稳定性。Ca²⁺、Mg²⁺对酶的稳定性有促进作用,Hg²⁺、Ag⁺、PMFS和DFP能强烈抑制酶的活力。SDS和Urea对酶的活力无影响。     

一株产碱性蛋白酶的嗜碱芽孢杆菌的分离和鉴定

从天津塘活盐碱土壤中分离一株产碱性蛋白酶的嗜碱芽孢杆菌HAP,并对其进行了表型分类和16S rDNA序列分析.结果表明,HAP是一株革兰氏阳性芽孢杆菌,菌体大小(0.7-0.9)μm×(2-3)μm,该菌可利用木糖、葡萄糖、阿拉伯糖、甘露醇,发酵型糖代谢产酸;可以水解酪素、淀粉,但不能水解酪氨酸;能够利用柠檬酸盐;其酶活力为1.22×104U/mL.结合系统发育学分析将HAP鉴定为嗜碱芽孢杆菌(Bacillus alcalophilus).     

不同组分胶原多肽抗氧化作用的比较研究

研究发现:碱性蛋白酶+中性蛋白酶+木瓜蛋白酶三酶水解工艺(Ⅱ法)的胶原多肽中,小分子肽的比率较碱性蛋白酶+中性蛋白酶双酶水解(Ⅰ法)的胶原多肽要多;在浓度为12 mg/mL时,前者对DPPH·的清除率为78.71%,后者为57.05%;在40 mg/mL时,二者清除·OH的能力分别为27.37%和89.12%.在相同给药剂量时,Ⅱ法多肽提高小鼠GHS-Px活力和降低MDA和LF含量的效果明显优于Ⅰ法多肽.     

短小芽孢杆菌碱性蛋白酶的提纯和性质的研究

由短小芽孢杆菌(Bacillus pumilus)209产生的胞外碱性蛋白酶的粗酶制剂(5×10 4u／g),经硼砂NaOH缓冲液抽提,硫酸铵沉淀,Sephadex G一25脱盐,DEAE-纤维素柱层析,冷冻干燥获得部分纯化的酶。纯酶的活力为199x10u/g,比活力提高2,6倍。此酶的最适pH8．5—9．0,在pH6一10之间稳定,因此是属于微碱性蛋白酶“’。最适温度为50℃,在50℃以下稳定,在60℃处理10分钟活力损失95％。0.003村c丑件的存在对酶的热稳定性和pH稳定性均有显著提高。Ag+,Cu2+,Fe2+,Hg+,Zn2+ 对酶有抑制作用;Mn2+有明显的激活作用;1×10-3M的PCMB,IAA,O-PTH,PMSF对活力无影响;1×10-3M EDTA对酶有30％的抑制作用;在40℃用NBS(1×10-4M)、DFP(2．5mM)对醢进行10分钟处理,酶活力完全损失。此酶能水解多种天然蛋白,如酪蛋白、血红蛋白、蛇毒蛋白等,不水解鱼精蛋白和溶菌酶。以酪蛋白为底物测得的km值为0．66×10-2g／ml。在pH7．3进行圆盘凝胶电泳,虽然呈现九条带,但均具有大小不等的蛋白酶活力。     

AMP对蛇肌果糖-1,6-二磷酸酯酶pH9.2活力的激活作用——蛋白水解酶限制性酶解作用不是果糖-1,6-二磷酸酯酶变成碱性酶的唯一原因

低纯度的NADP⁺ 中含有一种在pH 9.2能够激活蛇肌果糖- 1 ,6 -二磷酸酯酶活力的物质 .经过分离鉴定 ,证明它就是 5′- AMP .该激活作用只有当较高浓度的Mg ²⁺ 存在时才表现出来 .在AMP的存在下 ,果糖 -1 ,6 -二磷酸酯酶的行为很像碱性酶 .Mg ²⁺ 激活动力学表明 ,AMP和Mg ²⁺ 在对蛇肌酶活力调节上存在着竞争关系 .AMP能解除高浓度Mg ²⁺对酶在碱性pH活力的抑制作用 .以往认为果糖- 1 ,6 -二磷酸酯酶由中性酶变成碱性酶均是由蛋白水解酶限制性酶解引起的 ;现报道 5′- AMP也能将蛇肌酶变成碱性酶 ,指出蛋白水解酶限制性酶解作用不是造成该酶由中性酶变为碱性酶的唯一原因 ,并且也可能有生理调节作用 .     

静态强磁场对枯草芽胞杆菌的影响研究

目的:研究静态强磁场下微生物的生物学效应.方法:以超导磁体产生的静态强磁场为基础,枯草芽孢杆菌为模式生物,通过测定生长曲线、芽胞生成率、蛋白酶表达量、蛋白酶活力等研究静态强磁场条件下微生物性状的变化.结果:强磁场可以影响枯草芽胞杆菌的芽胞形成速率,抑制营养体的死亡;测定菌体生长过程中蛋白酶的含量以及碱性蛋白酶和中性蛋白酶的酶活力,发现磁场处理前后蛋白酶的含量没有发生显著性变化,处理组碱性蛋白酶的酶活力明显高于对照组,而中性蛋白酶酶活力则低于对照组.结论:强磁场可以延长枯草芽孢杆菌的世代周期,降低菌体死亡率,对细菌酶活性的影响因酶的种类不同而异.     

土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。     

发酵性丝孢酵母蛋白酶稳定性的研究

研究了发酵性丝孢酵母所产蛋白酶的最适作用条件及稳定性。结果表明,发酵性丝孢酵母蛋白酶的最适反应温度为55℃,最适反应p H为7.0;有良好的热稳定性,50℃保温30 min,仍保留78%以上的酶活力;贮存稳定性也较好,10℃贮存60 h,仍保留80%左右的酶活力;5 mmol/L的金属离子Mn2+和Ca2+对蛋白酶有激活作用,其中Ca2+激活作用最显著,使酶活提高了0.87倍;1%(w/w)的表面活性剂吐温-100可以促进酶活力,使酶活提高了24.6%,1%(w/w)的SDS对酶活力有抑制作用,将残余酶活降低为原酶液的67.1%;2.5%(w/w)的葡萄糖、半乳糖和海藻糖等能抑制该蛋白酶活力,其中海藻糖抑制作用最显著,使残余蛋白酶活力降低到原酶液的23.6%;甘油可作为酶保护剂保护蛋白酶稳定性,且浓度为7.5%(w/w)的甘油能明显激活酶活力。     

棒曲霉Asp-195v产蛋白酶的酶学性质研究

对液体发酵的棒曲霉Asp-195v菌株所产蛋白酶的活力进行了研究,并通过分离纯化获得了电泳纯的酶蛋白。研究结果表明,该蛋白酶的最适反应温度为40℃,在30-50℃温度范围内相对活力可保持在70%以上;最适pH为7,pH稳定范围在4-8;Mn2+对该蛋白酶活力有明显的激活作用,K+、Ag+、Cu2+、Fe2+、Mg2+、Zn2+、Ca2+、Al3+和Fe3+离子则有明显的抑制作用,尤其是Hg2+和Pb2+对酶活的抑制作用更加强烈;其他试剂如葡萄糖、EDTA对酶活的抑制作用不明显,而蔗糖、SDS和Tween-20对酶活的抑制明显;以酪氨酸为底物采用双倒数作图法测得Vmax为30.40mmol/min,Km为97.53mmol/L。该酶的表观分子量为30.1kDa。     

丁鳜肠道中枯草芽孢杆菌蛋白酶的生理生化性质的研究

目的:为改良丁鳜饲料成分,研究了从丁鳜肠道中分离的枯草芽孢杆菌(Bacillus subtilis)蛋白酶的生理生化性质.方法及结果:枯草芽孢杆菌经发酵培养,破碎细胞,通过45%～85%饱和度的硫酸铵沉淀分离得到了一种碱性蛋白酶,SDS～PAGE显示该蛋白酶分子量为59kD;最适温度为40℃;温度的稳定性范围在50℃以下;最适pH9.8.低浓度的EDTA对该酶无明显抑制作用;Ca2+和Co2+完全抑制该酶的活性,Pb2+对酶活力有一定抑制作用.结论:新分离出的这种蛋白酶有助于进一步开发含有添加蛋白酶制剂的鱼饲料.     

重金属离子对凡纳滨对虾肝胰脏、鳃丝和血液SOD活力的影响

研究了3种重金属离子(Cu²⁺、Zn²⁺、Cd²⁺)在96 h内对凡纳滨对虾(Litopenaeus vannamei)对肝胰脏、鳃丝和血液超氧化物歧化酶(SOD)活力的影响.结果表明,凡纳滨对虾SOD活力在3种重金属离子作用下随取样时间变化显著(P＜0.0),Cu²⁺在实验浓度范围内(0.1～1 mg·L^-1),肝胰脏、鳃丝和血液的SOD活力随时间延长呈一峰值变化,Zn²⁺在10 mg·L^-1时对肝胰脏表现为显著抑制作用,Cd²⁺在0. mg·L^-1时对肝胰脏和鳃丝起显著抑制作用,0.2 mg·L^-1对鳃丝SOD活力无显著变化(P＞0.0),其他浓度Zn²⁺(＜10 mg·L^-1)、Cd²⁺(＜0.2 mg·L^-1)对各组织器官SOD活力的影响随时间延长均呈现先升高后下降的趋势.3种重金属离子对凡纳滨对虾肝胰脏、鳃丝、血液SOD活力的影响呈现明显的剂量-时间效应关系.其SOD活力大小顺序为肝胰脏＞鳃丝＞血液,3种重金属离子对凡纳滨对虾伤害大小顺序为Cd²⁺＞Cu²⁺＞Zn²⁺.     

Investigation of adenosinetriphosphatase activity of rat liver and thymus cell nuclei]

The activity of ATPase was studied in highly purified rat liver and thymus cell nuclei, HCO3-, CO3(2-) and SO3(2-) stimulated nuclear ATPase in 1.5--2 times. HSO3- did not affect the enzyme activity, and NO3-, J-, ClO4-,F- and SCN- inhibited it. Bicarbonate increased V and decreased Ka for ATP. SCN- inhibited HCO3--ATPase activity non-competitively with respect to HCO3-. Mg2+-ATPase activity did not depend on pH, and HCO3-component of the activity was decreased under alkaline pH. Mg2+, Mn2+ and Co2+ increased the initial ATPase activity and helped its stimulation with HCO3-. Ba2+, Ni2+ and Zn2+ inhibited the ATPase activity, and Ca2+ did not affect it, Nuclear ATPase is sensitive to 2,4-dinitrophenol and DNAase. It is suggested that cell nuclei have their own H+-ATPase differing for some characteristics from mitochondrial H+-ATPase.     

The effect of thyroid hormone on the high affinity Ca2+-ATPase in rat liver plasma membrane

The effect of thyroid hormone on the high affinity Ca2+-ATPase activity in rat liver plasma membrane was studied. The high affinity Ca2+-ATPase activity in plasma membrane was activated by 10(-7)-10(-5) M of Ca2+ and was inhibited by 70 microM trifluoperazine. Thyroidectomy of rats was associated with an increase in the activity of high affinity Ca2+-ATPase. The increased enzyme activity was normalized by T4 administration to the animals. On the other hand, Na+-K+-ATPase activity in the membrane was decreased by thyroidectomy and the decreased enzyme activity was normalized by T4 administration. The results suggest that thyroid hormone inhibits the Ca2+ extrusion system by inhibiting calmodulin-independent high affinity Ca2+-ATPase in liver plasma membrane.     

Ca2+- or Mg2+-Dependent Enzymic ATP Hydrolysis Associated with the Microsomal Fraction of Frog Sciatic Nerves

The microsomal fraction of frog sciatic nerves was found to contain Ca2+- or Mg2+-dependent hydrolytic activity toward different nucleoside di- and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p-nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18--24 degrees C in the presence of 1 mM-Ca2+ or 1 mM-Mg2+. Ca2+- and Mg2+-ATP hydrolysis displayed pH maxima around 8.0--8.5 and 7.4--8.0, respectively. Vmax values for Ca2+- and Mg2+-ATP hydrolysis similar: approx. 12 mumol Pi per h per mg protein with a Km value of approx. 0.05 mM. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10(-6)-10(-5) M) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.     

6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) from slow-growing Rhizobia.

6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) (NAD+-6PGD) was detected in several slow-growing strains of rhizobia, and no activity involving NADP+ was found in the same extracts. By contrast, fast-growing strains of rhizobia had NADP+-6PGD activity; most of them also had NAD+-6PGD activity. NAD+-6PGD was partially purified from the slow-growing strain Rhizobium japonicum 5006. The reaction was shown to be an oxidative decarboxylation.     

Calmodulin-dependent adenylate cyclase activity in rat cerebral cortex: effects of divalent cations, forskolin and isoprenaline

The role of calcium-calmodulin (Ca2+-CaM) in the modulation of beta-adrenergic adenylate cyclase activity in rat cerebral cortex has been studied. In addition, the effects of manganese (Mn2+) and forskolin on CaM-dependent enzyme activity were investigated. At 2 mM magnesium (Mg2+) low concentrations of Ca2+ stimulated the enzyme activity (Ka 0.25 +/- 0.08 microM), whereas higher Ca2+ levels (greater than 2 microM) inhibited the activity. No activating effect of Ca2+ was observed in CaM-depleted membranes, but the inhibitory effect persisted and the stimulatory action of Ca2+ could be restored by addition of exogenous CaM. The ability of Ca2+ to activate the enzyme was reduced by increasing concentrations of Mg2+. At 10 mM Mg2+ the apparent Ka of Ca2+ was 0.55 +/- 0.16 microM and half-maximal inhibition was observed at 80-120 microM Ca2+. A synergistic effect was observed between Ca2+ and isoprenaline on the adenylate cyclase activity. Calcium did not alter the apparent Ka of isoprenaline (0.9 +/- 0.27 microM) and isoprenaline did not change the apparent Ka of Ca2+. However, isoprenaline decreased the apparent Ka of CaM; 0.11 +/- 0.07 micrograms vs. 0.32 +/- 0.1 micrograms (0.5 ml assay mixture)-1, with and without isoprenaline, respectively. A synergistic effect was also observed between Ca2+ and forskolin, but no change in their apparent Ka values was found. Furthermore, Mn2+ was found to activate the enzyme through CaM. These data demonstrate that Ca2+ -CaM potentiates beta-adrenergic adenylate cyclase activity and thus is able to modulate neurotransmitter stimulation in cortex. Furthermore, both forskolin and Mn2+ affect CaM-dependent enzyme activity. Forskolin potentiates Ca2+-CaM stimulation, while Mn2+ increases the activity by activating the enzyme through CaM.     

The O2- generating oxidoreductase of human neutrophils: evidence of an obligatory requirement for calcium and magnesium for expression of catalytic activity

NADPH-dependent O2- generating oxidoreductase activity recovered from cell lysates of phorbol myristate acetate-stimulated human neutrophils exhibits dependence on Ca+2 and Mg+2 for full expression of its catalytic activity. O2- generating activity was completely abolished by exposure of the oxidoreductase to EDTA, then reconstituted by exposure of the enzyme to Ca+2 and Mg+2 in excess of the EDTA concentration used to block catalytic activity. The oxidoreductase responded maximally to either 0.25 mM Ca+2 or 0.80 mM Mg+2. The pH optimum of the oxidoreductase exposed to Ca+2 and Mg+2 is between pH 7.0 and 7.6. The molar ratio of NADPH oxidation to O2- production determined at pH 7.6 in the presence of Ca+2 and Mg+2 is 0.49, indicating 1 mole of NADPH oxidized per 2 moles of O2- formed. Particulate fractions recovered from cell lysates of resting neutrophils exhibited no oxidoreductase activity under the same conditions.     

Modulation of phospholipase A2 activity associated with human sperm membranes by divalent cations and calcium antagonists

Phospholipase A2 activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using [1-14C] oleate-labeled autoclaved E. coli and 1-[1-14C] stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates. Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids. The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4. Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively. para-bromophenacyl bromide inhibited human sperm phospholipase A2 activity, but mepacrine was without effect. In the presence of 1.0 mM added CaCl2, phospholipase A2 activity was inhibited by Zn2+ and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+ had no effect. Zn2+ stimulated activity at low concentrations (10(-6) to 10(-8) M), and inhibited activity in a dose-dependent manner at concentrations of 10(-5) M. The extent of stimulation by low concentrations of Zn2+ was dependent on Ca2+ concentration; at 10(-7) M, Zn2+ activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2. At low concentrations (10(-5) to 10(-7) M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2 activity, and trifluoperazine but not D600 produced almost complete inhibition between 10(-5) and 10(-4) M of the drug. The significance of human sperm phospholipase A2 activity and its modulation by Ca2+, Zn2+ and Mn2+ in the sperm acrosome reaction is discussed.     

Effect of taurine on the properties of myocardial sarcoplasmic reticulum guanyl cyclase

Effect of taurine on the properties of guanylate cyclase (GC) of the guinea-pig cardiac sarcoplasmic reticulum was studied. The enzymatic activity increased in the presence of Mn+2 at a concentration of 0.05 mM, reaching the maximal level at a concentration of 7 mM. Mg2+ (0.25-1 mM) did not alter the activity of GC in the absence of Mn2+, but stimulated it in the presence of Mn2+ at a concentration ranging within 0.1 to 1 mM. Taurine activated GC in the presence of Mn2+ (10 mM) and produced no effect on its activity at 0.5-3 mM of Mn2+ without Mg+2. Taurine (0.4-10 mM) potentiated the activity of GC stimulated with Mg+2. The structural analog of taurine, beta-alanine, suppressed the activity of GC 2-2.5-fold both in the absence and presence of Mg+2. Ca2+ (10(-9)--10(-4) mM) stimulated GC. Effect of Mg+2 and taurine on GC activity rose proportionally to an increase in Ca+2 concentration in the incubation medium. The data obtained evidence in favour of potential monitoring of the activity of GC through changes in the intracellular content of Ca+2, Mg+2 and taurine in the presence of Mn+2 at concentrations close to the physiological ones. The effect of taurine on GC is mediated via Mg+2 and Ca+2.     

Membrane adenosine triphosphatase activities in rat pancreas

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.