青海云杉ISSR-PCR反应体系的建立与优化(英文)

[Objective] The experiment aimed to determine the optimum ISSR-PCR reaction system of Picea crassifolia kom. [Method] Picea crassifolia kom. was used as material to select and optimize influencing factors of ISSR-PCR such as Mg2+, dNTPs, Taq DNA polymerase, template DNA, primers, annealing temperature. [Result] The optimum ISSR-PCR reaction system in 20 μl reaction system was consisted of 1 μl 10×buffer, 1.5 mmol/L Mg2+, 0.2 mmol/L dNTPs, 1.0 U Taq DNA polymerase, 40 ng template DNA, 0.6 μmol/L primers. According to gradient test of annealing temperature in optimum ISSR-PCR reaction system of Picea crassifolia kom, it was found that the optimum annealing temperature of UBC 818 was 54.2 ℃ and the annealing temperature was different for different primers.[Conclusion]The construction of ISSR-PCR reaction system provided technical basis for classification of germplasm resources, construction of genetic map, gene mapping of Picea crassifolia kom. through using ISSR technology.     

茶薪菇基因组DNA的RAPD反应体系优化(英文)

[Objective] The research aimed to screen out optimum RAPD reaction system on genomic DNA of Agrocybe chaxingu Huang.[Method] The single factor experiment was adopted to select the required Mg2+ concentration, template DNA concentration,primer concentration,dNTPs concentration,Taq enzyme concentration and anneal temperature initially.[Result] The optimum reaction system for RAPD amplification of Agrocybe chaxingu Huang was listed as follows:2.5 μl Buffer, 2 mmol/L Mg2+, 75 ng DNA, 0.5 μmol/L primer, 150 μmol/L dNTPs and 2.0 Taq enzyme.The reaction process was also listed as follows: denaturation for 5 min at 92 ℃,35 cycles(1 min at 92 ℃, 1 min for 35.5 ℃ and elongation for 2 min at 72 ℃),10 min at 72 ℃.[Conclusion] The research provided reference for conducting RAPD analysis of and studying genetic relationship and genetic diversity of Agrocybe chaxingu Huang.     

松江鲈鱼ISSR-PCR反应体系的建立及优化研究(英文)

[Objective] The aim of this study is to establish and optimize the ISSR-PCR system in roughskin sculpin(Trachidermus fasciatus Heckel).[Method] By using the primer of ISSR-63,the concentrations of Taq DNA polymerase,Mg2+,dNTPs and primer were optimized by orthogonal design for establishing the suitable ISSR-PCR system in roughskin sculpin;moreover,the suitable anneal temperature was yielded from gradient PCR on temperature.[Result]The optimized ISSR-PCR system(20 μl reaction volume)in roughskin sculpin was proved to be:2.5 mmol/L Mg2+,250 μmol/L dNTPs,0.25 μmol/L primer,1 U Taq DNA polymerase,30 ng DNA template and 1×PCR buffer;and suitable anneal temperature was determined to be 50.8 ℃.The established system was further confirmed by using 24 wild roughskin sculpin samples.[Conclusion]The results lay a foundation for the analysis of genetic diversity and germplasm resources of roughskin sculpin.     

Optimization of Solid State Fermentation Conditions Using a Mixture of Bean Curd Residue and Marc with Bacillus natto

[Objective] The aim was to optimize the appropriate solid state fermentation(SSF)conditions.[Method] The optimization of solid state fermentation using a mixture substrate of bean curd residue and the marc with Bacillus natto was developed.[Result] The best fermentation condition optimized by the test of single factor and the orthogonal design respectively was mixing ratio of bean curd residue to marc 2∶1,substrate pH value 6,fermentation temperature 39 ℃,inoculum volume 10% and fermentation time 48 h.Under this optimized fermentation condition,the content of crude fiber in the substrate decreased from 107.8 mg/g before SSF to 56.2 mg/g after SSF,and the degeneration rate of crude fiber was 47.87%.[Conclusion] The bean curd residue in its palatability was enormously improved by SSF with Bacillus natto strain,which could be expected to be widely used as raw material of health foodstuff.     

Cloning of a novel phytase from an anaerobic rumen bacterium,Mitsuokella jalaludinii,and its expression in Escherichia coli

The full length phytase gene of Mitsuokella jalaludinii was successfully cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludinii with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, p ET32 a, for expression in Escherichia coli strain Rosetta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 k Da by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, p H 4.5 and showed good p H stability from p H 3.5 to 5.5(78% relative activity). Metal ions such as Ca2+, Mg2+, and K+ were found to exert significant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+ greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced p H stability and metal ion effects should be taken into consideration during feed enzyme formulation.     

化学杀雄剂EXP对油菜膜脂过氧化及保护酶活性的影响(英文)

[Objective] The aim of this study was to investigate the characteristics and mechanism of chemical emasculation in rapeseed and to provide the theoretical basis for development and utilization of new chemical gametocides.[Method] The activity of peroxidase,catalase and the content of hydrogen peroxide,malondialdehyde in leaves and flower buds of Brassica napus cultivars Qinyou No.3 and L89 induced by the chemical gametocide EXP in the course of male sterility were studied.[Result] Protective enzyme activity and the content of hydrogen peroxide,malondialdehyde in rapeseed treated with EXP changed significantly,which indicated that active oxygen metabolism was abnormal.Furthermore,there was a significant difference in the reaction degree of different cultivars and organs treated by EXP.[Conclusion] There was a correlation between the disturbance of active oxygen metabolism and the male sterility induced by chemical gametocide EXP.     

Effect of Chemical Gametocide EXP on Membrane Lipid Peroxidation and Protective Enzyme Activity in Rapeseed (Brassica napus)

[Objective] The aim of this study was to investigate the characteristics and mechanism of chemical emasculation in rapeseed and to provide the theoretical basis for development and utilization of new chemical gametocides.[Method] The activity of peroxidase,catalase and the content of hydrogen peroxide,malondialdehyde in leaves and flower buds of Brassica napus cultivars Qinyou No.3 and L89 induced by the chemical gametocide EXP in the course of male sterility were studied.[Result] Protective enzyme activity and the content of hydrogen peroxide,malondialdehyde in rapeseed treated with EXP changed significantly,which indicated that active oxygen metabolism was abnormal.Furthermore,there was a significant difference in the reaction degree of different cultivars and organs treated by EXP.[Conclusion] There was a correlation between the disturbance of active oxygen metabolism and the male sterility induced by chemical gametocide EXP.     

Effects of Metal Ions and Organic Solvents on Alkaline Phosphatase from Rice-field Eel

[Objective]The mechanism of alkaline phosphatase(ALP) was studied to promote rice-field eel aquaculture industry.[Method] The effects of effectors such as multiple metal ions and organic solvents on ALP in viscera of rice-field eel.[Result]Na+ and K+ didn’t generate big influences on enzyme activity;Mg2+ and Ca2+ could promote ALP while Li+,Cu2+ and Zn2+could restrain ALP enzyme activity.Both HPO42-and WO43-generated by enzyme catalyzing disodium phenyl phosphate possessed strong inhibitory effects on emzyme,and 9.5 mmol/L HPO42-would make enzyme activity decline by 13% while 9.5 mmol/L WO43-would make enzyme decline by 34%.The inhibition types of them were both competitive inhibition on enzyme activity.The organic solvents such as methanol,ethanol,ethylene glycol,isopropanol all generated influences on ALP and the order according to their inhibitory effects was isopropanol>ethanol> methanol>ethylene glycol.[Conclusion]The influences of various effectors on ALP activity of rice-field eel were studied from dynamics perspective to provide theoretical basis for further clarifying ALP mechanism.     

化学杀雄剂EXP对油菜膜脂过氧化及保护酶活性的影响(英文)

[Objective] The aim of this study was to investigate the characteristics and mechanism of chemical emasculation in rapeseed and to provide the theoretical basis for development and utilization of new chemical gametocides.[Method] The activity of peroxidase,catalase and the content of hydrogen peroxide,malondialdehyde in leaves and flower buds of Brassica napus cultivars Qinyou No.3 and L89 induced by the chemical gametocide EXP in the course of male sterility were studied.[Result] Protective enzyme activity and the content of hydrogen peroxide,malondialdehyde in rapeseed treated with EXP changed significantly,which indicated that active oxygen metabolism was abnormal.Furthermore,there was a significant difference in the reaction degree of different cultivars and organs treated by EXP.[Conclusion] There was a correlation between the disturbance of active oxygen metabolism and the male sterility induced by chemical gametocide EXP.     

金属离子·有机溶剂对黄鳝碱性磷酸酶的影响(英文)

[Objective]The mechanism of alkaline phosphatase(ALP) was studied to promote rice-field eel aquaculture industry.[Method] The effects of effectors such as multiple metal ions and organic solvents on ALP in viscera of rice-field eel.[Result]Na+ and K+ didn’t generate big influences on enzyme activity;Mg2+ and Ca2+ could promote ALP while Li+,Cu2+ and Zn2+could restrain ALP enzyme activity.Both HPO42-and WO43-generated by enzyme catalyzing disodium phenyl phosphate possessed strong inhibitory effects on emzyme,and 9.5 mmol/L HPO42-would make enzyme activity decline by 13% while 9.5 mmol/L WO43-would make enzyme decline by 34%.The inhibition types of them were both competitive inhibition on enzyme activity.The organic solvents such as methanol,ethanol,ethylene glycol,isopropanol all generated influences on ALP and the order according to their inhibitory effects was isopropanol>ethanol> methanol>ethylene glycol.[Conclusion]The influences of various effectors on ALP activity of rice-field eel were studied from dynamics perspective to provide theoretical basis for further clarifying ALP mechanism.     

蒜氨酸酶动力学性质研究

[目的]研究蒜氨酸酶的动力学性质。[方法]分别测定温度、pH值、底物浓度及金属离子对蒜氨酸酶活性的影响,并计算最大反应速度及米氏常数。[结果]蒜氨酸酶为热不稳定性酶,其最适反应温度为29℃,最适pH值为6.1;以其天然抽提物为底物测得最大反应速度及米氏常数分别0.492IU/mg、0.483mmol/L;K+、Mg2+、Ca2+、Na+和Cd2+对蒜氨酸酶活性有一定的激活作用,Cu2+对酶活性有抑制作用。[结论]该研究可为开发应用蒜类药用产品提供依据。     

蒜氨酸酶动力学性质研究

大蒜中含有一类被称之为大蒜素的含硫化合物,主要由蒜氨酸酶与蒜氨酸发生酶解反应生成,但蒜氨酸酶含量低,对环境较敏感,因而活性表现不稳定,加之目前的生产条件常导致该酶活性被破坏,使得蒜类产品的药理作用不显著。因此,研究蒜氨酸酶的理化性质对于开发应用蒜类药用产品具有重要的指导作用。     

蒜氨酸酶粗提液的特性

以安徽舒城大蒜为材料,采用离心、盐析、透析等方法从大蒜匀浆中提取蒜氨酸酶粗提液,以L-半胱氨酸亚砜为底物研究蒜氨酸酶的特性及最佳反应条件。结果表明,离心、盐析、透析纯化的蒜氨酸酶的比活分别为粗提时的2.519,3.497,3.703倍。该粗酶液的最适pH值为6.4,最适温度为30℃。当底物浓度低于3μmol/ml时表现为一级反应;当底物浓度在3～5μmol/ml时表现为混合级反应;当底物浓度大于5μmol/ml时属于零级反应。Zn2+、Ca2+、Fe2+可以提高蒜氨酸酶活性170%～200%;EDTA可以提高20%;Cu2+抑制45%。     

蒲菜多酚氧化酶的酶学性质研究

[目的]研究蒲菜多酚氧化酶（PPO）的酶学性质。[方法]研究蒲菜PPO的吸收光谱和动力学性质,并对温度、pH、底物浓度、抑制剂等因素对蒲菜PPO活性的影响进行探索。[结果]蒲菜PPO催化邻苯二酚的产物在415 nm处有最大吸收峰,动力学方程为：V=0.982[S]/（4.97＋[S]）,其中,V为反应速度;[S]为底物浓度。蒲菜PPO最适温度为30℃,在95℃、2 min条件下可以有效地抑制蒲菜PPO活性;蒲菜PPO最适pH为6.8,当pH（5.4或pH（7.2时可以有效降低蒲菜PPO活性;柠檬酸、亚硫酸钠、抗坏血酸、EDTA-2Na等抑制剂对PPO均有抑制作用。[结论]蒲菜PPO的酶学性质研究结果可为蒲菜的研究开发提供依据。     

青鱼胰蛋白酶的分离纯化及部分性质研究

[目的]为青鱼的养殖和综合利用提供试验依据。[方法]对青鱼胰蛋白酶进行分离纯化,测定青鱼胰蛋白酶的分子量、热稳定性和酸碱稳定性,探讨pH值、温度、金属离子和EDTA对青鱼胰蛋白酶活力的影响。[结果]经SDS-PAGE和凝胶过滤,测得青鱼胰蛋白酶的分子量分别为44和43.5 kD。青鱼胰蛋白酶具有一定碱性耐受性,在pH值为8～10时活性较高,其最适pH值约为9,最适反应温度为50℃。以酪蛋白为底物,在pH值8.5、37℃时测得该酶Km值为4.6×103 mg/L。Mg2+对该酶有明显促进作用,而Cu2+、Mn2+、Pb2+和EDTA对青鱼胰蛋白酶的抑制作用很大。[结论]EDTA能抑制青鱼胰蛋白酶的作用,说明该酶可能为金属蛋白酶。     

脂肪酶中具有壳聚糖降解活力酶组分的性质研究

[目的]为酶法降解壳聚糖提供科学依据。[方法]从脂肪酶中分离具壳聚糖降解活力的电泳纯酶,研究pH值、温度和金属离子对壳聚糖酶活力的影响,并分析了该酶的pH值稳定性、热稳定性和底物特异性。[结果]该酶降解壳聚糖的最适pH值为4.5,最适温度为60℃。Ni2+、Co2+和Mn2+对酶活具有明显的激活作用,而Fe3+、Pb2+和Hg2+则强烈抑制酶的活力。该酶对脱乙酰度为73%~82%的壳聚糖具有较高的水解活性,而对脱乙酰度为64%和90%的壳聚糖水解活力较低。[结论]该酶在pH值为5~9范围内和60℃以下都具有较好的稳定性。     

藕带中多酚氧化酶的酶学性质研究

[目的]研究藕带中多酚氧化酶（PPO）的酶学性质。[方法]以邻苯二酚为底物,用分光光度法研究pH值、温度和抑制剂对藕带PPO活性的影响,并建立酶促褐变动力学方程。[结果]藕带PPO的最适pH值为6.0,最适温度为30℃;Km为0.0344mol/L,Vmax为172.41U/ml,70℃热保温60minPPO的活性基本丧失;抗坏血酸、柠檬酸、L-半胱氨酸和氯化亚锡均能抑制藕带PPO的活性,其中,L-半胱氨酸和氯化亚锡为最佳抑制剂,其次是柠檬酸和抗坏血酸,0.20g/LL-半胱氨酸和氯化亚锡均能有效抑制PPO的活性。[结论]该研究可为合理利用藕带资源和科学控制其深加工过程中的酶促褐变提供了参考依据。     

香樟(Cinnamomum camphora)果实酪氨酸酶的分离纯化及特性分析

[目的]检测并分离香樟果实中的酪氨酸酶。[方法]从香樟成熟果实中分离和纯化出酪氨酸酶,研究该酶的酶活、蛋白质含量、最适pH值、最适温度、动力学特性和稳定性,并分析了不同金属离子、化合物和中药对其酶活的影响。[结果]经分离和纯化,获得43 kD的酪氨酸酶。该酶的最适pH值和最适温度分别为7.5和50℃。以L-DOPA为底物,测得Km和Vmax分别为12.83 mmol/L和180.65U/mg。该酶在pH值4~7和较高温度时均较为稳定。Al3+和Cu2+对该酶活性具有抑制作用,而Zn2+、Mg2+等则有激活作用。250mmol/LEDTA对该酶有激活作用,抗坏血酸和1 mmol/Lβ-巯基乙醇有完全抑制作用。中药女贞对其有强激活作用,乌梅对其抑制作用最明显。[结论]香樟果实中酪氨酸酶同工酶的存在为该酶的纯化带来一定难度。     

黑曲霉葡萄糖苷酶的分离纯化及酶学性质研究

[目的]分离能水解獐牙菜苦苷的胞外β-葡萄糖苷酶,并研究其分子量、最适催化温度、最适pH、稳定性及催化特征。[方法]黑曲霉液体发酵3d,收集培养液,培养液经盐析、柱分离得到1个β-葡萄糖苷酶。[结果]该β-葡萄糖苷酶的分子量为86kD,最适pH为5.0～6.0,最适反应温度为50～60℃,在60℃以下时酶稳定;Ca2＋、Zn2＋、Fe3＋和Cu2＋能抑制β-葡萄糖苷酶的活性,而Mg2＋和Mn2＋能够激活β-葡萄糖苷酶的活性;纤维二糖是该β-葡萄糖苷酶的最适催化底物。[结论]该酶和大多数真菌来源黑曲霉相似,对獐牙菜苦苷的亲和性弱,对ρ-NPG的亲和力强。     

普鲁兰短梗霉N2-3菌株碱性蛋白酶的纯化及性质研究

[目的]对普鲁兰短梗霉N2-3菌株的碱性蛋白酶进行纯化,并对其性质进行研究。[方法]利用层析柱Sephadex G-75与阴离子交换柱DEAE Sepharose Fast Flow纯化菌株N2-3的胞外碱性蛋白酶,并对纯化的酶进行SDS-PAGE凝胶电泳检测,同时对纯化蛋白酶的最适反应温度、温度稳定性、最适反应pH、pH稳定性进行测定,研究金属离子及各种化合物对蛋白酶活性的影响。[结果]试验中N2-3菌株的碱性蛋白酶纯化倍数和回收率分别为2.34和25.9%;SDS-PAGE凝胶电泳显示该酶的分子量约为33 kDa;该酶的最适反应温度和pH分别为52℃和9.0;Mn2+、Mg2+、Na+离子浓度在5 mmol/L时对纯化蛋白酶的酶活有激活作用,Zn2+、Ca2+、K+、Li+对酶活的影响不大,而当存在Fe2+、Fe3+、Cu2+、Co2+、Ag+、Hg2+离子时,蛋白酶活明显降低;苯甲基硫酰氟(PMSF)强烈抑制了该蛋白酶的活性,而乙二胺四乙酸(EDTA)与碘乙酸微弱抑制了该蛋白酶的活性。[结论]为普鲁兰短梗霉N2-3菌株碱性蛋白酶的工业化生产提供了一定的理论基础。