粘质沙雷氏菌产几丁质酶发酵条件的研究

目的：通过对粘质沙雷氏菌发酵条件的优化,提高其产几丁质酶的能力。方法：以实验室保存菌种粘质沙雷氏菌S418为对象,通过单因素试验和三因素三水平正交试验筛选出了菌株S418产几丁质酶的最佳培养基配方及培养条件。结果：该菌种产酶的最佳发酵条件：0.2%（w/v）胶体几丁质,1%蛋白胨,0.05%KH2PO4,在28℃、pH7.0、接种量6%,培养72h,酶活达到5.49U/mL。结论：优化后菌株S418产几丁质酶的条件。     

一株产胆固醇氧化酶的不动杆菌的筛选鉴定及特性研究

目的：筛选鉴定产胆固醇氧化酶的菌株并对其酶的性质及发酵条件进行初步的研究。方法：利用唯一碳源的胆固醇平板筛选,酶活测定比较得酶活力最高的菌株;生理生化试验结合16S rDNA序列分析鉴定其种属,单因素及正交实验优化培养基及发酵条件。结果：所得菌株H4与产不动杆菌（Acinetobacter）有最近的亲缘关系,其胆固醇氧化酶作用的最适温度和pH分别为37℃和8.0,金属离子Mg2＋、Zn2＋、Fe2＋对该酶具有一定激活作用,菌株产酶的最适培养基为（g/L）：胆固醇1.5,蔗糖5,蛋白胨7,硝酸铵3,吐温1.0,pH7.5;最适培养条件为33℃,15mL培养基/100mL三角瓶,摇床培养（200r/min）48h,优化后发酵液酶活达135.8U/L。结论：获得了1株产胆固醇氧化酶的菌株H4,并初步鉴定为不动杆菌（Acinetobacter）。     

产γ-谷氨基甲酰胺合成酶菌株的筛选及产酶条件研究

以假单胞菌(Pseudomonas sp.)为出发菌株,通过紫外诱变筛选得到一株γ-谷氨基甲酰胺合成酶高产菌株UV-19,其酶活提高32.54%。以突变株UV-19为供试菌株,对γ-谷氨基甲酰胺合成酶的发酵条件进行优化。首先利用Plackett-Burman设计筛选出影响较大的4个因素:葡萄糖、蛋白胨、起始pH值、装液量。在此基础上再利用CCD响应面分析法进行优化,得到最佳产酶培养条件为(g/L):葡萄糖15、蛋白胨12、NaCl 5.0、MgSO4.7H2O 0.2、K2HPO4.3H2O 0.5、甲胺盐酸盐1.0g/L、起始pH值6.5、装液量72mL/250mL。该优化条件下进行产酶培养,假单胞菌发酵产γ-谷氨基甲酰胺合成酶酶活力可达32.68U/mL。     

高产大豆异黄酮糖苷水解酶菌株的发酵工艺研究*

Absidia sp．R是从酒曲中分离出的一株产大豆异黄酮糖苷水解酶活性较高的菌株。该菌最佳产酶条件为：2．5％的麦麸为碳源,1％的硝酸钠为氮源,培养基起始pH为7．0,瓶装量为40mL/250mL,接种量8％,培养温度为30℃,转数为160r／min,培养时间为84h,其酶活力可达到82U／mL。除Cu^2 对该菌产酶有较强的抑制作用外,金属离子对产酶影响不大。     

表达条件对金属激活酶NAOase离子依赖的活性分析

N-乙酰鸟氨酸脱酰基酶为一种新型手性拆分酶制剂,酶活性依赖于Mg2＋、Mn2＋、Zn2＋及Co2＋ 中的某种金属离子。以高表达NAOase的重组菌DH10B/argE-pHsh为研究对象,考察不同培养条件下Mg2＋、Mn2＋、Zn2＋及Co2＋ 4种离子对重组菌的生长、酶的表达活性及表达量的影响。结果发现：同种离子在不同条件下对重组菌的生长影响不大,但对酶活性影响显著。4种离子在合适浓度时皆能提高酶活性,促进强度从高到低依次为Mn2＋、Mg2＋、Co2＋和Zn2＋。在TB培养基中,Mn2＋为15 mmol/L时：NAOase比酶活达到1 272.7 U/mL,是未添加时的4.67倍,激活作用显著高于其他离子。SDS-PAGE电泳实验表明4种离子的蛋白表达量基本相同。     

产弹性蛋白酶菌种的筛选及发酵条件优化研究

从合肥肉联厂附近的土壤和污水中分离得到19株产弹性蛋白酶菌株,初步鉴定该菌株属于假单胞菌属.经过发酵复筛有三株产酶能力超过15u/mL.实验对菌株最佳产酶发酵条件进行了优化2%干酪素、0.5%葡萄糖、0.4%酵母膏、0.2% K2HPO4、0.01% MgSO4·7H2O;起始pH值7.0;最适发酵温度为30℃;装液量为25mL/250mL;该菌株在28h左右产弹性蛋白酶的量达18u/mL.     

BA-25菌株碱性纤维素酶产酶条件优化研究

试验研究了分离自烟草调制过程中的产芽孢细菌Bacillussp.BA-25菌株碱性纤维素酶产生的优化条件.结果表明:麸皮是最佳碳源,豆饼粉是最佳的氮源,且当碳氮比为2∶1时产酶活性最高;NaCl和KH2PO4对纤维素酶的合成具有重要作用,其适宜用量分别为0.5%～1.0%和0.1%;培养液的初始pH会极大地影响产酶活性,其最适pH为10.最优化的发酵工艺参数为:种子菌龄为24h,在300mL摇瓶中,装入30mL培养基培养温度为37℃,摇床转速为160r/min,在发酵24h后补充1%的葡萄糖,培养48h,该菌产碱性纤维素酶活力可以达到68.4U/mL.     

果胶酶高产菌种的紫外诱变选育及其培养条件的响应面法优化

以果胶酶产生菌黑曲霉EIM6为出发菌株,初始果胶酶活为14 539 U/mL,经紫外诱变反复处理,摇瓶复筛和遗传稳定性试验,最终获得一株果胶酶高产菌株EIMU2。EIMU2菌株的形态特征发生了明显的改变,相较于原出发菌株EIM6,孢子色泽更黑,孢子团也较出发菌株大,菌丝与孢子上凝结有更多的液珠。复筛后EIMU2酶活为32 161 U/mL,较原出发菌株提高了1.212倍。进一步通过响应面法对EIMU2菌株的液体发酵培养条件进行优化。优化后的培养条件为甜菜渣1.83%,花生饼粉1.69%,(NH_4)_2SO_4 0.5%,K_2HPO_4 0.3%,CaCO_3 0.2%,MgSO_4 0.15%(w/v),接种量6%(v/v),装液量21.36 mL。优化的突变菌株产酶活性进一步提高至98 794.3 U/mL,提高了2.07倍。     

一株产D-海因酶菌株的鉴定及离子束诱变选育

首次报道了产D-海因酶的巨大芽孢杆菌,通过对该菌进行离子束诱变,获得酶活最高增加3倍的突变株M5,对M5的产酶条件进行优化,得到最佳的发酵条件为玉米浆1.5%,葡萄糖1%,油酸1.5%,氯化钠0.5%,并添加50 mg.L-1的Mn2+、Zn2+及500 mg.L-1的Mg2+,pH8.0,30℃发酵24 h,酶活力可达到每毫升2.119 U,比优化前突变株提高了300%,比出发菌株提高了850%。     

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%。     

一株耐热脂肪酶产生菌的筛选及酶学性质研究

从云南省富油地采取了60份土样中,利用透明圈法筛选出一株耐热脂肪酶产生菌。对其酶学性质和发酵条件进行了研究,酶学性质表明,该酶最适作用温度为50℃,最适pH6.0,在pH3.0-8.0范围内稳定,在60℃保温60 min酶活还保留70%;70℃保温60 min残余50%;具有良好的热稳定性;不同金属离子有不同的作用,Ca+,K+对酶有激活作用,Fe3+、Pb2+、Mn2、Cu2+、Al3+、Zn2+对酶活有抑制作用。EDTA对酶影响不大。产酶最佳条件为:MgSO4.7H2O 0.05 g,K2HPO40.1 g,CaCO30.25 g,可溶性淀粉2.5 g,大豆粉2.5 g,装液量50 mL。这株细菌通过培养基优化酶活达到20.3 U/mL。     

脱枝酶产生菌的分离鉴定及酶学性质研究

目的：从中国高校工业微生物资源与信息中心（CICIM-CU）细菌库中分离具有产脱枝酶酶活的细菌并鉴定,进行酶学性质的研究。方法：通过碘显色平板法筛选产酶菌株,利用16S rDNA确定其属种。对每一株产脱枝酶的细菌进行初步的酶学性质研究。结果：从4005株细菌中筛选出45株产脱枝酶的细菌,建立了产脱枝酶细菌的细菌库。酶学性质表明CICIM B272、CICIMB1-30两株菌产生的脱枝酶,酶反应的最适温度分别为70℃6、0℃,最适pH分别为6.0、5.5,来源于上述两种不同属种的脱枝酶在30-70℃反应条件下,酶在pH 4.5-8.5范围内活性稳定,Li＋、Na＋、K＋、Mg2＋、Mn2＋对两者酶活均有显著的激活作用,而Cu2＋、Fe3＋及EDTA对两者均有显著的抑制作用,Mn2＋、Ca2＋分别对两者的热稳定性具有很好的提升作用。以支链淀粉为底物的动力学常数Km分别为352.883mg/mL、4.5814mg/mL,Vmax分别为30.03mg/min.mL、0.4575mg/min.mL。结论：不同属种的脱枝酶酶学性质差别显著。     

Production of cellulase by Trichoderma reesei from dairy manure

Cellulase production by the fungi Trichoderma reesei was studied using dairy manure as a substrate. Data showed that T. reesei RUT-C30 had higher cellulase production than T. reesei QM 9414 and that a homogenized manure, treated by a blender to reduce fiber size, led to higher cellulase production. The cellulase production was further optimized by growing T. reesei RUT-C30 on homogenized manure. The effects of manure concentration, pH, and temperature on cellulase production were investigated with optimal parameter values determined to be 10 g/l manure (dry basis), 25.5 degrees C, and pH 5.7, respectively. Elimination of CaCl2, MgSO4, nitrogen sources (NH4+ and urea) and trace elements (Fe2+, Zn2+, Co2+ and Mn2+) from the original salt solution had no negative influence on the cellulase production, while phosphate elimination did reduce cellulase production. Based on above results, the final medium composition was simplified with manure additives being KH2PO4, tween-80 and CoCl2 only. Using this medium composition and a reaction time of 6-8 days, a maximum cellulase production activity of 1.74 IU/ml of filter paper activity, 12.22 IU/ml of CMCase activity, and 0.0978 IU/ml of beta-glucosidase was obtained. This filter paper activity is the highest ever reported in cellulase production from agricultural wastes.     

1株高效降解纤维素的槲寄生内生真菌研究

利用刚果红-CMC平板染色法、DNS法从槲寄生内生真菌中筛选获得1株高效纤维素酶产生菌H-3-3,通过分析其菌落形态特征和18S rDNA序列的同源性,将菌株H-3-3鉴定为拟茎点霉属Phom opsissp.;对其酶学性质研究得到：槲寄生内生真菌H-3-3中CMC酶、β-葡糖苷酶、滤纸酶这3种组分酶最适温度均为50℃,β-葡糖苷酶活和滤纸酶活最适pH值均为4.8,CMC酶最适pH值为4.4;3种酶在30～45℃,pH4.0～8.0稳定性较强。金属离子Mn^2＋、Co^2＋、Mg^2＋和Ca^2＋对3组分酶都有不同程度的促进作用,其中以Co^2＋效果最佳;而Ba^2＋、Zn^2＋、Cu^2＋对3组分酶分均表现出明显的抑制作用。     

Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis

The production of polygalacturonase (PGase) by Sporotrichum thermophile Apinis in stirred submerged fermentation (SmF) was high in comparison with that in static conditions. Yeast extract (0.25%) and citrus pectin (2%) at pH 7.0 and 45 degrees C supported a high enzyme production in flasks agitated at 200 rpm. An overall 1.75-fold enhancement in PGase production was achieved as a result of optimization. The enzyme was optimally active at pH 7.0 and 55 degrees C, and exhibited t(1/2) of 4 h at 65 degrees C. The Km and Vmax values of the enzyme (for pectin) were 0.416 mg ml(-1) and 0.52 micromol mg(-1)min(-1), respectively. The PGase activity was stimulated by Mn(2+) and Fe(2+), but inhibited strongly by Mg(2+), and slightly by Tween 80 and Triton X-100. Among the additives tested, beta-mercaptoethanol exerted a strong inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. An increase in the yield of banana, grape and apple juices was recorded due to the treatment of fruit pulps with the mixture of enzymes (pectinase, xylanases and cellulase) of S. thermophile as compared to that with only pectinase. The yield of fruit juices did not increase with enhanced titre of cellulase in the enzyme mixture.     

Dimerization of human growth hormone in the presence of metal ions

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.     

An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump,ECA1, supports plant growth and confers tolerance to Mn(2+) stress

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity.     

Effect of cellobiose, glucose, ethanol, and metal ions on the cellulase enzyme complex of Thermomonospora fusca

The saccharification of cellulosic substrates by cellulase from Thermomonospora fusca, strain YX, was influenced by the presence of various substances. Cellobiose was strongly inhibitory, reducing the activity against swollen cellulose to 25% at 5% concentration. Glucose had much less effect, reducing activity to 40% at 20% concentration. Ethanol was found to be only slightly inhibitory to the cellulase, reducing activity by about 15% at 6% concentration, but much more inhibitory to the cell-bound beta-glucosidase activity. Of the numerous metal ions examined, Ca(2+) and Co(2+) at 0.1mM-1.0mM concentration were found to be slightly activating under assay conditions, while 1.0mM Pb(2+) and Hg(2+) were the most inhibitory ions. The implications of these results for the design of commercial processes for ethanol production from cellulose are discussed.     

高产纤维素酶枯草芽胞杆菌S-16的筛选及其发酵工艺优化

利用刚果红鉴别培养基及基础液体筛选培养基进行菌种筛选,从新疆盐碱地分离得到的16株菌株中筛选获得一株产纤维素酶活力较高的菌株S-16,对该菌株进行16SrDNA鉴定,确定该菌为枯草芽胞杆菌(Bacillus subtilis)。对S-16发酵产纤维素酶的主要影响因素进行研究,分别考察了碳源、氮源、培养基初始pH和接种量等因素对发酵产纤维素酶的影响。结合单因素影响实验得到优化后的培养基配方为:羧甲基纤维素钠1.5%,酵母粉1%,NaCl 1%,MgSO_4·7H_2O 2‰,KH_2PO_4·3H_2_O 1‰。优化后的发酵条件为:初始pH为8,接种量1%,种龄8h,培养时间48h。经过发酵工艺优化,S-16产生的羧甲基纤维素酶活(CMCase)和滤纸酶活(FPase)分别达到4.64IU/mL和0.46IU/mL,与初始培养条件下的酶活相比分别提高了3.14倍和1.30倍。本研究得到的枯草芽胞杆菌S-16及其优化发酵工艺为秸秆的快速腐熟和高产纤维素酶的应用奠定了基础。     

ATP utilizing reactions of human erythrocyte membranes and the influence of modulator proteins

The ATP production of human erythrocytes in the steady state (approximately 2 mmoles . 1 cells-1 . h-1, 37 degrees C, pHi 7.2) is maintained by glycolysis and the ATP consumption is essentially limited to the cell membrane. About 25% of the ATP consumption is used for ion transport ATPases. The bulk of the ATP consuming processes in intact erythrocytes remains poorly understood. "Isotonic" erythrocyte membranes prepared under approximate intracellular conditions after freeze-thaw hemolysis have high (Ca2+, Mg2+)-ATPase activities (80% of the total membrane ATPase activity). There is a great discrepancy between the high capacity of the (Ca2+, Mg2+)-ATPase in isotonic membranes and the actual activity in the intact cell. The (Ca2+, Mg2+)-ATPase of isotonic membranes has a "high" Ca2+-affinity (Ka less than 0.5 microM) and a "low" Mg-ATP affinity (Km approximately 760 microM). This state of (Ca2+, Mg2+)-ATPase is caused by the association of calmodulin and 30000 Dalton polypeptides (ATP affinity modulator protein). Hypotonic washings of isotonic membranes result in a loss of the 30 kD polypeptides. EGTA (0.5 mM) extracts derived from isotonic membranes contain the 30 kD modulator protein and restore the properties of the (Ca2+, Mg2+)-ATPase of hypotonic membrane preparations to the isotonic characteristics. The Mg-ATP affinity modulator protein is assumed to form a complex with calmodulin and (Ca2+, Mg2+)-ATPase.