竹笋脚料过氧化物酶的提纯及酶学性质的研究

竹笋经加工成食品后,大量脚料被丢弃,严重浪费资源,污染环境.文中采用水抽提、硫酸铵分级沉淀、丙酮分级沉淀和CM-52离子交换层析三步法从竹笋脚料中快速提取并纯化该酶;同时对该酶部分的醇学性质(温度、pH时酶促反应的影响、酶的热稳定性、酸碱稳定性)进行了考查.结果表明经过纯化后的过氧化物酶的Rz值达到2.5,纯化倍数为12倍,回收率为28%.最适反应温度为55℃,最适pH为6.8,且其温度和pH的适用范围均较宽,热稳定性和酸碱稳定性均较高的中性同工酶.这些优越的酶学性质能为竹笋脚料过氧化物酶的应用提供依据.     

竹笋脚料过氧化物酶的提纯及酶学性质的研究

竹笋经加工成食品后,大量脚料被丢弃,严重浪费资源,污染环境。文中采用水抽提、硫酸铵分级沉淀、丙酮分级沉淀和CM-52离子交换层析三步法从竹笋脚料中快速提取并纯化该酶;同时对该酶部分的酶学性质（温度、pH对酶促反应的影响、酶的热稳定性、酸碱稳定性）进行了考查。结果表明经过纯化后的过氧化物酶的Rz值达到2．5,纯化倍数为12倍,回收率为28％。最适反应温度为55℃,最适pH为6．8,且其温度和pH的适用范围均较宽,热稳定性和酸碱稳定性均较高的中性同工酶。这些优越的酶学性质能为竹笋脚料过氧化物酶的应用提供依据。     

离体稻苗叶片衰老过程中蛋白质组分的变化(简报)

离体稻苗叶片暗下衰老时,非水溶性蛋白质含量变化不大,而水溶性蛋白质含量下降迅速,其组分也同时改变。水溶性蛋白质中,Fraction Ⅰ蛋白质含量迅速下降,而Fraction Ⅱ蛋白质含量则显著增加。Fraction Ⅰ蛋白质中,RuBP羧化酶小亚基降解速率比大亚基快。叶片衰老时有两条新电泳蛋白质谱带出现。     

黑曲霉A3木聚糖酶酶学性质研究

黑曲霉A3(AspergillusnigerA3)的固体培养物浸出液,经过多步分离纯化后,获得三个组份的木聚糖酶,称为xⅠ、xⅡ和xⅢ.经7%凝胶浓度的盘状电泳分析均为单一组份.经等电聚焦电泳测xⅠ、xⅡ和xⅢ的等电点分别为6.8、5.5和6.1.SDS-PAGE测得亚基分子量(Da)分别为xⅠ,42000;xⅡ,20000;xⅢ,31000.三个酶组份的最适反应温度分别为xⅠ,40℃;xⅡ,50℃;xⅢ,50℃.最适反应pHxⅠ,3.5;xⅡ,4.5;xⅢ,5.0.保温一个小时后,酶的半失活温度分别为xⅠ,55.6℃;xⅡ,54.8℃;xⅢ,46.6℃.金属离子Ag+、Hg2+、Ni2+和脲对不同的酶组份具有一定的影响.     

蚯蚓纤溶酶组分的抗原性分析

  用热变性蚯蚓纤溶酶(earthworm fibrinolitic enzyme,EFE)组分EfP-0-2、EfP-Ⅰ-1、EfP-Ⅱ-1, 以及重组蛋白EfP-Ⅲ-2为抗原,免疫家兔获得了抗血清,利用获得的抗血清对部分组分的免疫学同一性进行了验证.EfP-0、EfP-Ⅰ、EfP-Ⅱ的免疫双扩散反应表明,EfP-Ⅰ和EfP-Ⅱ具有部分免疫学同一性. 利用DNAMAN软件对获得的EfP-0、EfP-Ⅰ、EfP-Ⅱ、EfP -Ⅲ的蛋白质序列进行了初步分析,包括整个序列相似性比对、氨基酸组成、蛋白酶消化、抗原肽、疏水性和亲水性轮廓等,并对N端氨基酸序列进行了分析.结果表明,EfP-Ⅰ和EfP- Ⅱ具有部分免疫学同一性是由它们蛋白质序列的组成和结构决定的.     

粗毛栓菌木聚糖酶的纯化及性质

以麦草粉为基质培养粗毛栓菌Trametes gallica,浸提固态培养物得浸提液,后经超滤浓缩、硫酸铵盐析、Phenyl-Sepharose CL-4B疏水层析、DEAE Sepharose fast flow阴离子交换层析和Sephadex G-150分子筛层析等分离与纯化步骤,获得部分纯化的木聚糖酶,其回收率和纯化倍数分别为1.45%和15.6。进一步经活性-PAGE回收,获得三种SDS-PAGE电泳纯级的木聚糖酶同工酶组分:XⅠ、XⅡ和XⅢ(按等电点从大到小排列)。三种组分分子量均约为19.0kDa;等电点分别为:5.6、4.7和4.0;含糖量分别为:0.25%、0.63%和3.4%;XⅠ既能降解木聚糖,又能降解纤维素;XⅡ的最适作用pH值为5.0,最适作用温度45℃;Mg2+、Fe2+对XⅡ有激活作用;Mn2+和Co2+有抑制作用;测得XⅡ的Km值为0.75mg/mL,Vmax为5,000mmoL/min·mg。     

对过氧化物酶鉴定实验的改进

过氧化物酶是生物氧化中末端氧化酶,是引导学生证明酶促反应中间产物存在的理想酶。本文对来自白菜或鲜笋壳压榨汁液直接用HAC调节pH至5.5,静置后过滤或离心、取上清液、丙酮分级沉淀,获得RZ为2．5的纯酶,颜色明亮。然后对过氧化物酶的活性进行定性和定量测定,使学生实验课的效果更为直观。     

不同注药方法对丙泊酚注射痛的影响

目的:比较不同注射部位对丙泊酚注射痛的影响.方法:选择ASA Ⅰ-Ⅱ自愿接受无痛人工流产手术患者200例,随机分为4组,(n=50).Ⅰ组手背静脉推注丙泊酚;Ⅱ组肘部静脉推注丙泊酚;Ⅲ组肘部推注20mg 2%利多卡因后即刻静脉推注丙泊酚;Ⅳ组手背推注20mg 2%利多卡因后即刻静脉推注丙泊酚.记录各组患者的疼痛分级评分.结果:Ⅱ组与Ⅳ组比较丙泊酚注射痛无差异;Ⅱ组与Ⅲ组比较丙泊酚注射痛无差异;Ⅱ组与Ⅳ组比较发生丙泊酚注射痛的程度轻.结论:选择肘部静脉静推丙泊酚且无需预推利多卡因注射痛最轻.     

里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。     

不同溶剂分级提取的茶多糖的组成及降血糖活性

比较绿茶中不同溶剂分级提取的茶多糖的得率、含量、单糖组成及降血糖活性的差异,以从茶叶中分离出高活性的茶多糖.粗老绿茶采用去离子水低温提取茶叶中的水溶性多糖TPSⅠ,水不溶性多糖先用草酸铵提取其果胶类多糖TPSⅡ,再用碱提取,所得到碱提取液回调至pH中性后可溶解部分为碱溶性多糖TPSⅢ,并分析其得率、含量、采用气相色谱和离子色谱分析了其单糖组成,通过四氧嘧啶腹腔注射造成高血糖模型,连续灌胃茶多糖12 d,对比研究两种茶多糖的降血糖活性.并比较复合纤维素酶提取与去离子水提取在得率、含量、单糖组成及其降血糖活性的差异.TPSⅠ单糖组成以鼠李糖、半乳糖和半乳糖醛酸为主;TPSⅡ以半乳糖和半乳糖醛酸为主;TPSⅢ以阿拉伯糖、葡萄糖和半乳糖为主.降血糖活性顺序为:TPSⅠ＞TPSⅢ＞TPSⅡ,但TPSⅠ得率最低.复合纤维素酶提取的多糖得率、糖醛酸提取率比热水提取的多糖高1.53倍和1.42倍.酶提茶多糖较水提茶多糖降血糖效果更明显些.表明茶叶中水溶性多糖降血糖活性最好,复合纤维素酶提取可以提高其得率,增强其活性.     

The Partial Characterization of the Major Seed Storage Proteins in Arabidopsis thaliana L.

This study was aimed at the characterization of the major storage proteins in Arabidopsis thaliana. Two major protein fractions, i.e., the fraction Ⅰ and Ⅱ proteins, were isolated from the extract of mature seeds of this plant by molecular seive gel filtration chromatography. Various polyacrylarnide gel electrophoretic techniques were used to study the properties and polypeptide compositions of these two protein fractions. In was shown that during the SDS gel electrophoresis, fraction Ⅰ protein was separated into 6 major bands with the mol. was. of 34, 31, 29, 28 and 19-20 kD, respectively, whereas Fraction Ⅱ protein migrated as 3 low mol. wt. bands (10-12 kD) on the same gel. Non-denaturing native gel electrophoresis revealed that fraction Ⅰ was a neutral protein and Fraction Ⅱ was a positively charged basic protein with an isoelectric point (pI) higher than 8.8. Fraction I protein was further separated into at least 16 polypeptides in isoelectric focusing/SDS two-dimensional gel electrophoresis, i.e. each SDS band contained 3-4 polypeptides with the same mol. wt. but different pis. This suggested a more complex polypeptide composition of this protein. The properties of fraction Ⅰ and Ⅱ proteins were in good accordance with that of the 12s and 1.7s storage globulins in seeds of many other dicotyledonous plants, and therefore had been characterized as the two major seed storage proteins in this species. These two storage globulins were shown to be accumulated within a defined period during the late stage of seed development (12-14 DAF) and became predominant protein components in mature seeds. In the mean time, a few points in relation to the polypeptide composition and subunit molecular configuration of the 12s globulin were noted.     

三疣梭子蟹两种养殖模式浮游植物与产量的比较

为获知三疣梭子蟹（Portunus trituberculatus）两种养殖模式浮游植物与产量是否存在差异,于2012年6月—12月在宁波市象山县东盛水产养殖有限公司进行。结果显示：浮游植物共7门32属57种。模式Ⅰ浮游植物7门30属54种;模式Ⅱ5门19属32种。藻类平均细胞密度、平均生物量均为模式Ⅰ〉模式Ⅱ。Margalef指数、Shannon-Wiener指数均值模式Ⅰ〉模式Ⅱ,Pielou指数均值模式Ⅰ〈模式Ⅱ。养殖前期（8月）、中期（10月）、起捕（12月）时,对三疣梭子蟹壳长、壳宽、体重测量取其平均值,均为模式Ⅰ〉模式Ⅱ。三疣梭子蟹产量模式Ⅰ〉模式Ⅱ。研究表明,模式Ⅰ优于模式Ⅱ。     

苦荞种子胰蛋白酶抑制剂的分离纯化及部分性质研究

采用凝胶层析及离子交换层析等方法,从苦荞种子中分离出一组胰蛋白酶抑制剂（ＴＢＴＩ－Ⅰ、Ⅱ）．对其性质研究表明：两个组分均对胰蛋白酶有较强的抑制作用,对胰凝乳蛋白酶抑制作用较弱,其中ＴＢＴＩ－Ⅱ的抑制作用大于ＴＢＴＩ－Ⅰ,两者对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用．用ＳＤＳ－聚丙烯酰胺凝胶电泳和ＳｅｐｈａｄｅｘＧ－１００凝胶层析分别对纯化产物进行分析得出ＴＢＴＩ－Ⅰ和ＴＢＴＩ－Ⅱ的近似分子量分别为１５．０ｋＤ和１８．０ｋＤ．ＴＢＴＩ－Ⅰ、Ⅱ都具有较高的热稳定性,在１００℃处理１０ｍｉｎ后可保留８６％左右的抑制活性．ＴＢＴＩ在酸性环境下较为稳定,在ｐＨ２．０条件下保温１ｈ,仍保留７５％的抑制活性．用Ｌｉｎｅｖｅａｅｒ－Ｂｕｒｋ作图法得知,该抑制剂属竞争性抑制类型,ＴＢＴＩ－Ⅱ的Ｋｉ值为３．５９×１０－７ｍｏｌ／Ｌ（以ＢＡＰＮＡ为底物）,对胰蛋白酶的摩尔抑制比为１∶１．４．     

嗜热毛壳菌CT2纤维二糖水解酶Ⅰ在毕赤酵母中的高效表达

嗜热毛壳菌ChaetomiumthermophilumCT2可产生具有重要工业生产价值的纤维素酶类。RT-PCR扩增cbh1成熟蛋白的编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.42mg/mL。表达蛋白分泌到培养基中,分子量约80kD;以脱脂棉为底物测得酶活为21U/mL。表达蛋白在60℃稳定,70℃保温60分钟仍保持90%的酶活力,具有较高的热稳定性。     

兔肝金属硫蛋白结合铅离子的圆二色性光谱研究

从锌诱导的家兔肝脏中分离纯化得到金属硫蛋白两种亚型：ＺｎＭＴ－Ⅰ和ＺｎＭＴ－Ⅱ．在酸性条件下脱金属,经Ｓｅｐｈａｄｅｘ Ｇ－２５柱层析得到脱金属硫蛋白（ａｐｏＭＴｓ）．用圆二色性（ＣＤ）光谱法研究,发现两种亚型ａｐｏＭＴｓ 与Ｐｂ２＋ 的结合依赖于Ｐｂ２＋ 的加入比例及ｐＨ 值．ａｐｏＭＴ－Ⅰ在ｐＨ３～５之间,ａｐｏＭＴ－Ⅱ在ｐＨ４～６之间与Ｐｂ２＋ 结合形成特征簇合物Ｐｂ７ＭＴｓ,其ＣＤ谱图特征峰位于３１６ｎｍ （－ ）,２７０ ｎｍ （＋ ）,２４５ ｎｍ （＋ ）及２２５ ｎｍ （－ ）,提示解铅中毒的最佳条件应控制在弱酸性环境．不同亚型ａｐｏＭＴｓ 与Ｐｂ２＋ 的结合方式各不相同：Ｐｂ２＋ 与ａｐｏＭＴ－Ⅰ的结合采取平均分配的方式,而与ａｐｏＭＴ－Ⅱ则为选择性结合方式,表明这两种亚型在解铅毒功能上存在差异．     

Mobilization and Acquisition of Sparingly Soluble P-Sources by Brassica Cultivars under P-Starved Environment Ⅱ. Rhizospheric pH changes, Redesigned Root Architecture and Pi-Uptake Kinetics

Non-mycorrhizal Brassica does not produce specialized root structures such as cluster or dauciform roots but is an effective user of P compared with other crops. In addition to P-uptake, utilization and remobilization activity, acquisition of orthophosphate (Pi) from extracellular sparingly P-sources or unavailable bound P-forms can be enhanced by biochemical rescue mechanisms such copious H~+-efflux and/or carboxylates exudation into rhizosphere by roots via plasmalemma H~+ ATPase and anion channels triggered by P-starvation. To visualize the dissolution of sparingly soluble Ca-phosphate (Ca-P), newly formed Ca-P was suspended in agar containing other essential nutrients. With NH_4~+ applied as the N source, the precipitate dissolved in the root vicinity can be ascribed to rhizosphere acidification, whereas no dissolution occurred with nitrate nutrition. To observe in situ rhizospheric pH changes, images were recorded after embedding the roots in agar containing bromocresol purple as a pH indicator. P-tolerant cultivar showed a greater decrease in pH than the sensitive cultivar in the culture media (the appearance of typical patterns of various colors of pH indicator in the root vicinity), and at stress P-level this acidification was more prominent. In experiment 2, low P-tolerant class-Ⅰ cultivars (Oscar and Con-Ⅱ) showed a greater decrease in solution media pH than low P-sensitive class-Ⅱ (Gold Rush and RL-18) cultivars, and P-contents of the cultivars was inversely related to decrease in culture media pH. To elucidate P-stressinduced remodeling and redesigning in a root architectural system, cultivars were grown in rhizoboxes in experiment 3.The elongation rates of primary roots increased as P-supply increased, but the elongation rates of the branched zones of primary roots decreased. The length of the lateral roots and topological index values increased when cultivars were exposed to a P-stress environment. To elucidate Pi-uptake kinetics, parameters related to P influx: maximal transport rate (V_(max)), the Michaelis-Menten constant (K_m), and the external concentration when net uptake is zero (C_(min)) were tested in experiment 4. Lower K_m and C_(min) values were better indicative of the P-uptake ability of the class-Ⅰ cultivars, evidencing their adaptability to P-starved environmental cues. In experiment 5, class-Ⅰ cultivars exuded two- to threefold more carboxylates than class-Ⅱ cultivars under the P-stress environment. The amount and types of carboxylates exuded from the roots of P-starved plants differed from those of plants grown under P-sufficient conditions. Nevertheless, the exudation rate of both class-Ⅰ and class-Ⅱ cultivars decreased with time, and the highest exudation rate was found after the first 4 h of carboxylates collection. Higher P uptake by class-Ⅰ cultivars was significantly related to the drop in root medium pH, which can be ascribed to H~+-efflux from the roots supplied with sparingly soluble rock-P and Ca_3(PO_4)_2. These classical rescue strategies provided the basis of P-solubilization and acquisition from sparingly soluble P-sources by Brassica cultivars to thrive in a typically stressful environment.     

Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 μM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells.     

菠菜中的乙醇酸氧化酶是一个同工酶

乙醇酸氧化酶（ＥＣ．１．１．３．１５．ＧＯ）是光呼吸途径的关键酶,降低其活性可提高Ｃ３植物如水稻的产量,在目前中国乃至世界人口不断增加和可耕种土地日益减少的情况下,对ＧＯ的研究具有重要的理论意义和实际应用价值．在光呼吸途径被提出后的几十年间,人们对如...     

Some chemical, physical and histochemical properties of three diamine fractions obtained by gel chromatography from the high-iron diamine staining solution used for localizing sulphated mucosaccharides

Synopsis The coloured components in the high-iron diamine dye bath were separated into three fractions using column chromatography on Sephadex G-10. These fractions were called Fraction I, II and III in order of their emergence from the column. From atomic absorption measurements, part of Fraction I was found to be free of iron. Most of Fraction II and the whole of Fraction III contained only trace amounts of iron. Therefore, the three Fractions were investigated further. All experiments were carried out at pH 1.4 (corresponding to the pH of the original high-iron diamine bath).Fraction I was violet, Fraction II red-violet and Fraction III aniline-red; the extinction maxima in the visible region were 560, 526 and 540 nm respectively. On electrophoresis, the Fractions were not quite homogeneous, although most of Fractions I and II migrated in the same front and much faster than Fraction III. The high-iron diamine solution separated into two main fractions, one of which corresponded in colour and velocity to Fractions I and II and the other to Fraction III.In histochemical experiments, Fractions I, II and III bound to tissue sites containing sulphated mucosaccharides or nucleic acids; from histochemical enzyme digestion tests or by using purified materials in spot tests on cellulose acetate membrane, it was confirmed that the diamines were bound to RNA and DNA. However, when ferric chloride was added to any of the Fractions in an amount corresponding to that in the original high-iron diamine dye bath, the binding to tissue sites containing nucleic acids was inhibited but the reaction with sulphated mucosubstances was not affected. Also, in the presence of added ferric chloride, the anomalous binding of Fraction I to carboxyl groups of mouse sublingual gland sialomucin was prevented.It is concluded that ferric chloride in the high-iron diamine dye bath prevents diamine complexes from binding with nucleic acids, and apparently with carboxymucins too. Further, this conclusion substantiates our previous observations of the central role ferric chloride plays in making the histochemical high-iron diamine technique specific for sulphated mucosubstances.     

Inhibition of progesterone synthesis and cAMP accumulation in small bovine luteal cells by a low molecular weight component of bovine follicular fluid

Follicular fluid from large follicles of cows was extracted with charcoal and filtered through an Amicon XM-50 membrane. The XM-50 filtrate was further fractionated on a column of Fractogel TSK HW-40 (s) using Krebs-Ringer-phosphate buffer (1/100th dilution), pH 7.2, as an eluant. Two fractions (1 and 2) were obtained. Inhibition of progesterone secretion by small luteal cells was associated with the XM-50 filtrate and Fraction 2. Whole follicular fluid, the XM-50 retentate and Fraction 1 had no significant inhibitory activity. Fraction 2, which contained about 1/100,000th of the original follicular fluid proteins, inhibited the LH- or (Bu)2cAMP-induced progesterone production during a 2-h incubation. This inhibition was dose-dependent. Fraction 2 also inhibited LH-induced cAMP accumulation, but did not affect the conversion of pregnenolone to progesterone or the basal progesterone production. The molecular weight of the inhibitory factor was estimated to be less than 10,000 and its ability to inhibit steroidogenesis was lost after digestion with protease but retained after heating for 60 min at 75 degrees C. These results demonstrate that bovine follicular fluid contains a heat-stable factor likely to be a polypeptide and which suppresses the steroidogenic response of small luteal cells to LH. The action of this inhibitory factor could involve both an inhibition of the LH-induced synthesis of cAMP and an inhibition of the action of cAMP.