草菇低温诱导蛋白的分离与纯化

低温4℃诱导草菇（Volvariella volvacea (Bull.ex Fr)Sing.)菌丝体2h后，对其可溶性蛋白进行分析，发现草菇在低温胁迫下有新的可溶性蛋白质产生。应用硫酸铵分级沉淀和电泳分离制备技术，分离和纯化得到了草菇菌丝体中的一个低温诱导蛋白，用聚丙烯酰胺凝胶电泳和等电聚焦电泳等方法分析该蛋白，结果均呈现为单一条带。SDS－聚丙烯酰胺凝胶电泳结果表明蛋白是由分子量为51kD的一个亚基组成，等电聚焦电泳结果发现该蛋白的等电点为3．73。     

家蚕消化液35K蛋白酶基因的克隆及序列同源性分析

根据纯化的家蚕35K蛋白酶N末端氨基酸序列设计简并引物，从家蚕中肠组织cDNA库中分离并克隆该蛋白酶基因,对由DNA推测的氨基酸序列进行同源性检索。结果表明：存在于家蚕消化液中的35K蛋白酶基因长939bp，可编码313残基的多肽链，其中信息肽为22aa，活性肽为22aa，成熟的蛋白酶由269aa组成。与其他昆虫消化液的类胰凝乳蛋白酶具有同源性，是存在于家蚕消化液中的一种新发现的蛋白酶。     

用含有凝胶的基质进行等电点聚焦电泳分离和检测蛋白酶

介绍用等电点聚焦电泳分离并检测蛋白酶的一种精确方法。向含有明胶铁聚丙烯酰胺凝胶中加入０．３％辛基－Ｄ－吡喃葡糖苷或８Ｍ尿素，同时向样品溶液中加入细胞色素Ｃ和ＰＩ标记蛋白，这样可避免电泳过程中蛋白酶与基质的互作。在木瓜蛋白酶，胰凝乳蛋白酶和蛋白酶Ｋ等常用蛋白酶的估计等电点进行检测，检测极限可达微微克至毫微克。     

苏云金芽孢杆菌4.0718菌株晶体毒素性质的研究

以苏云金芽孢杆菌（Bacillus thuringiensis，Bt）4．0718菌株伴孢晶体65kD原毒素为材料，比较了几种染色-脱色方法对电洗脱回收蛋白的影响。蛋白纯化的步骤包括SDS-PAGE分离、采用自制蛋白回收装置电洗脱回收杀虫晶体蛋白及抗胰蛋白酶核心多肽、超滤法脱盐、冷丙酮沉淀法除去考马斯亮蓝及SDS。经SDS—PAGE检测，呈现出均一的蛋白带，回收蛋白达到了电泳纯。以双向凝胶电泳（2-DE）技术分析了苏云金芽孢杆菌库斯塔克亚种HD—1和4．0718菌株伴孢晶体内的蛋白质组分。结果表明，两者相互匹配的蛋白点有54个，HD—1菌株的130kD亚基有2个点，其等电点在5．25～5．75之间，65kD亚基在等电点3．4～8．3之间具有广泛的分布，130与65kD之间的亚基的等电点集中在3．5～4．2之间，65kD以下亚基的等电点集中在3．5～6．2之间；4．0718菌株的130kD亚基仅有1个点，65kD亚基的蛋白点比HD-1菌株更多，130与65kD之间的亚基、65kD以下的亚基蛋白点均比HD-1菌株多且等电点分布范围更广。     

铁蟹过敏原的分离、鉴定和快速纯化

应用免疫印迹(Western blot) 的方法鉴定铁蟹过敏原组分,然后利用电泳洗脱的方法快速纯化主要过敏原。取磷酸盐缓冲液制备的铁蟹肉浸出液,经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,测定各组分的相对分子量,然后利用18 例蟹过敏患者的血清进行免疫印迹,鉴定出主要和次要过敏原,利用普通垂直电泳槽快速洗脱主要过敏原蛋白,并鉴定活性。结果显示,SDS-PAGE显示铁蟹肉可辨条带有16条,相对分子质量在16.5～168 kD 之间。Western blotting结果表明,铁蟹肉浸出液共有10条过敏条带,其中相对分子质量为76kD的蛋白条带,阳性反应率为100%,纯化后获取了76 kD的主要过敏原,经过免疫印迹鉴定其具有免疫活性。表明相对分子质量76 kD的组分为铁蟹主要过敏组分,快速电洗脱可以纯化相对分子量为76kD的过敏原组分。     

海洋枯草芽孢杆菌UMBR1027产抑金黄色葡萄球菌活性蛋白分离鉴定及发酵条件优化

金黄色葡萄球菌(Staphylococcus aureus)是一种能引起严重传染性疾病的人兽共患病原菌,由于其严重的致病性及其易产生耐药性的特点,使得探索新的具有活性的药物的需求越发迫切.为了研究对S.autrgus有抑制作用的活性蛋白,本实验采用硫酸铵沉淀法对海洋枯草芽孢杆菌(Bacillus subtilis)UMBR1027产抑S.aureus活性蛋白进行提取,并利用葡聚糖凝胶SephadexLH-20柱层析、十二烷基硫酸钠聚丙烯酰胺(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)凝胶电泳进行分离、ABI 4800 plus MALDI-TOF进行鉴定;为了提高活性蛋白在B.subtilis UMBR1027发酵过程中的产量实验采用滤纸片扩散法,以抑菌圈直径为指标,分别从培养基pH、培养基的盐度以及培养温度这3个关键因素对B.subtilis UMBR1027产抑S.aureus活性蛋白的影响进行考察,并设计Box-Behnken实验以优化其发酵条件.结果显示活性蛋白主要含有碱性丝氨酸蛋白酶、α-淀粉酶、枯草杆菌蛋白酶和内切-β-1,3-1,4葡聚糖酶,优化实验得出B.subtilis UMBR1027产活性蛋白的最佳发酵条件组合为培养基pH为6.95、培养基盐度为10.38％、发酵培养温度为34.95℃;拟合实验中选取发酵培养基pH为6.95,发酵培养基盐度为10.38％,发酵温度为35℃进行验证,抑菌圈直径为20.4 mm,与理论值基本吻合.本研究对B.subtilis UMBR1027所产抗菌粗蛋白进行了分离,并成功对抗菌粗蛋白分离纯化后的各个部分进行了鉴定.响应曲面法有效提高了B.subtilis UMBR1027产抑制S.aureus的抗菌蛋白类物质,此研究为探索来自海洋的活性抗菌蛋白类天然药物分子提供了理论依据.     

香蕉束顶病毒海口分离物核穿梭蛋白（NSP）的纯化及抗血清制备

本研究以本实验室保存的含重组载体pDEST17-NSP的原核表达菌株E．coliBL21（DE3）为材料，于25℃、0．1mmol／LIPTG条件下诱导4h，集菌后超声波破碎，获得以包涵体形式表达的约20kD的融合蛋白。实验结果表明，将沉淀的融合蛋白溶于含6mol／L尿素的Binding Buffer中，再经Ni^2＋-NTA亲和层析纯化后，可获得高纯度的融合蛋白。将纯化融合蛋白经12％SDS-PAGE电泳，切胶回收目的带，液氮研磨并按1：1（W／V）混合佐剂，4次免疫家兔，获得BBTV病毒核穿梭蛋白的特异性抗血清。以融合蛋白作抗原，间接ELISA法测定其抗血清效价为1：5000。田间检测样品的最佳抗血清工作浓度为1：500。Western Blot鉴定结果表明抗血清能与目的蛋白特异性结合。本研究的结果将为下一步NSP基因转录调控和蛋白功能研究奠定一定基础。     

红星苹果花柱S-核酸酶的分离与纯化

采用多种蛋白分离纯化技术，结合生物鉴定方法，成功地从红星苹果（Malus domestica Borkh．）花柱中纯化出了一类与自交不亲和性识别反应相关的花柱蛋白。通过聚丙烯酰胺凝胶和等电聚焦电泳分析，确定该蛋白分子量为26和28kD，等电点为10．2和10．4，为碱性蛋白；核糖核酸酶活性染色和N-末端序列分析结果显示具有核酸酶活性，且与其它蔷薇科植物花柱S-核酸酶N-末端氨基酸序列同源性较高。     

棉铃虫中肠.Bt 毒素受体蛋白(APN )的分离与纯化

用Mg／EGTA方法，以不同的速度进行离心，成功地分离了棉铃虫(Bacillus thuringiensis)中肠刷状缘膜囊泡（BBMVO，BBMV中保留了大部分氨肽酶N(aminopeptidase N，APN)活性，BBMV中APN的特异活性较粗匀浆液相比提高了3．29倍；CHAPS3-[(3-chlor-amidopropyl)dimethylammonio]-1-propane sulphonate可以增加BBMV的溶解，加入1％(CHAPS)之后APN特异活性增加至5．21倍；利用磷酸酰肌醇特异性的磷脂酶(PI-PLC)能将APN从膜上解离下来，BBMV经PI-PLC处理后，SDS-PAGE电泳结果仅有120kD的1条带；Mono-Q和FPLC结合的方法可以纯化部分APN，SDS-PAGE电泳结果显示，经Mono-Q柱处理后的BBMV中分子量约100kD的蛋白条带加粗；棉铃虫3个抗性品系与敏感品系相比，APN特异活性差异不显著，说明棉铃虫对Bt产生抗性与APN活性的变化无关，抗性的产生可能与Bt毒素和APN结合能力的改变有关。     

多肽抗生素apidaecin和Shiva—Ⅰ在烟草胞外液中的稳定性

本研究利用电泳分析法和活性分析法测定了多肽抗生素apidaecin和Shiva-Ⅰ在烟草胞外液中的稳定性。apidaecin的活性半衰期为1．6h;Shiva-Ⅰ的活性半衰期为8．0h.apidaecin的C－末端酰胺化后稳定性增加1倍，而酰胺化对Shiva-Ⅰ的稳定性影响不大。利用MALDI－TOF质谱分析了两种多肽抗生素在烟草胞外液中的降解位点，apidaecin有两个蛋白酶降解位点，而Shiva-Ⅰ有5个。利用蛋白酶抑制剂法测定了烟草胞外液中降解两种多肽抗生素的蛋白酶种类。apidaecin主要被丝氨酸蛋白酶和金属蛋白酶降解；Shiva-Ⅰ可被丝氨酸蛋白酶、金属蛋白酶、巯基蛋白酶和精氨酸蛋白酶降解。     

Analysis of the Thermal Energy Requirements for the Extraction of Leaf Protein Concentrate from some Green Plants

Extraction of protein from the leaves of green plants is very important because of the high cost of conventional forms of protein such as meat, milk and fish. In order to design machinery for this extraction, and also to embark on leaf protein concentrate extraction, it is necessary to measure and analyse the energy requirements to carry out each process involved in the extraction, using different plant species.Experiments were carried out to determine the amount of crude protein, and the thermal energy required to extract leaf protein concentrate, from juices obtained from the leaves of some plant species. Leaves from the following plants were selected: cassava (Manihot esculanta), Siam weed (Chromolaena odorata), bitter leaf (Vernonia amygdalina), gliricidia (Gliricidia maculata) and thorny tree (Hura crepetans). The leaves from the plant species were macerated in a laboratory pulper. Juice was obtained from the samples using perforated cylinders and a hydraulic press. The specific heat capacity of the juices was determined using the cooling curve method. The values of the heat capacities were used to calculate the amount of thermal energy required to raise the temperature of each juice from its normal temperature of about 25°C to a total protein coagulation temperature of about 80°C. The crude protein content of the extract was determined using the Kjeldal method.Results indicate that the green coagulum extracted from all the juices all have a protein content of at least 37%. The thermal energy required to coagulate protein from the juices ranges from 1·59 kJ kg⁻¹ for Hura crepetans to 2·7 kJ kg⁻¹ for Vernonia amygdalina. The energy requirement to obtain crude protein (CP) ranges from 8 kJ kg⁻¹ [CP] with Hura crepetans to 182 kJ kg⁻¹ [CP] with Vernonia amygdalina. Both results are statistically significant at the 0·01 confidence interval. It is concluded that the choice of plant species can significantly lower the thermal energy requirement for the extraction of leaf protein concentrate.     

Processing and storage effects on orange juice aroma: a review

Freshly squeezed orange juice aroma is due to a complex mixture of volatile compounds as it lacks a specific character impact compound. Fresh hand-extracted juice is unstable, and thermal processing is required to reduce enzyme and microbial activity. Heating protocols range from the lightly heated not from concentrate, NFC, to the twice heated, reconstituted from concentrate, RFC, juices. Thermal processing profoundly effects aroma composition. Aroma volatiles are further altered by subsequent time-temperature storage conditions. Heating reduces levels of reactive aroma impact compounds such as neral and geranial, and creates off-flavors or their precursors from Maillard, Strecker, and acid catalyzed hydration reactions. Off-flavors such as 4-vinylguaiacol, p-cymene, and carvone are the products of chemical reactions. Other off-flavors such as butane-2,3-dione, guaiacol, and 2,6-dichlorophenol are indicators of microbial contaminations. Since most orange juice consumed worldwide is processed, the goal of this review is to summarize the widely scattered reports on orange juice aroma differences in the three major juice products and subsequent aroma changes due to packaging, storage, and microbial contamination with special emphasis on results from GC-O studies.     

杨梅澄清汁及浓缩汁中花色苷热降解动力学的研究

杨梅花色苷易受温度、pH值等因素的影响而发生降解,是导致产品外观品质变劣的主要原因。本文研究了杨梅澄清汁及浓缩汁内花色苷在不同pH值和不同加热温度下的热稳定性。花色苷降解动力学数据的分析结果表明：杨梅花色苷热降解属动力学一级反应,随着pH值和温度的升高,杨梅花色苷降解的半衰期(t_1/2)和热降解活化能(Ea)显著下降,即花色苷的降解速度增大;同一处理条件下,浓缩果汁的t_1/2明显低于澄清汁。而反应速率常数k和Ea值大于澄清果汁,说明澄清汁花色苷的热稳定性优于浓缩汁。     

利用苹果浓缩汁生产中的蒸发冷凝水制备营养水饮品

为再利用苹果冷凝水,该文以苹果浓缩汁实际生产中的蒸发冷凝水为原料,采用化学及仪器分析方法确定其成分组成与含量;在此基础上利用超滤、反渗透等膜分离技术手段对其进行净化和营养物质的浓缩,继而将此冷凝水开发成一种天然的植物水饮料。试验结果表明：苹果浓缩汁生产中的冷凝水其还原糖、总酸、香气成分、总酚质量浓度依次为150、98.5、166、1.046μg/mL;未检出铅、砷、汞,而嗜酸耐热菌超标,为43 CFU/100mL,但采用超滤膜分离技术（40℃,0.06 MPa）可去除苹果冷凝水中的嗜酸耐热菌,使其含量小于1 CFU/100mL;采用反渗透膜分离技术（0.5 MPa、5℃）可使苹果汁加工的冷凝水中营养成分浓缩,对还原糖、总酸、香气、总酚成分的截留率分别为95.07%、97.61%、87.71%、94.52%;以此浓缩液为原料,再适量添加维生素B和C即可制备富含苹果香气的营养水饮品。研究结果可为苹果浓缩汁企业再利用其浓缩工序的蒸发冷凝水和节约水资源提供思路以及试验基础数据。     

红颊草莓离体培养及其增殖过程中可溶性蛋白研究

本文以红颊草莓为试材,对红颊草莓茎段进行组织培养,筛选出离体快繁最适的培养基条件,并诱导再生植株,也初步探索了培养物增殖过程中可溶性蛋白的动态变化。结果表明：红颊草莓适宜的诱导分化培养基为MS＋6-BA0.5mg/mL＋IAA0.25mg/mL,不定芽分化率可达100%;继代增殖最适宜培养基为MS＋6-BA0.25mg/L＋KT0.25mg/L＋IAA0.05mg/L,增殖倍数达到6.44;最佳生根培养基为1/2MS＋IAA0.05～0.1mg/L,生根数为5～6条,平均根长2cm左右,且生长健壮;最佳移栽基质为河沙,移栽成活率达88.9%。对红颊草莓增殖过程中的可溶性蛋白研究结果表明：可溶性蛋白含量在第10天、第30天呈现明显的两个高峰,SDS-PAGE电泳结果显示16.7kD、15.6kD和14.4kD的蛋白是草莓生长所必须的,而分子量为26.2kD和23.7kD的蛋白则只在继代增殖旺盛时期出现,为增殖时的特异蛋白组分。本研究结果为红颊草莓快繁育苗提供了一条高效途径,也为进一步研究草莓离体培养过程中体内生理变化的分子机理提供了有意义的参考。     

Effect of juice extractor settings on juice cloud stability.

Juice was extracted from Valencia oranges using three different extractor settings. Differential juice cloud stability was observed. Soft-extracted juice was the most stable, and hard-extracted juice was the least stable. The medium-extracted juice had intermediate cloud stability. Yearly (1997 versus 1998) differences were observed, but the relationship among the juices did not change. Addition of protein extracts, obtained from each juice, to pasteurized juice also resulted in differential cloud stability. Using pectinmethylesterase (PME) activity estimated at pH 4.5, the effects of the protein extract mirrored results from raw juice. Estimating PME activity at pH 7.5 produced contradictory results, indicating that predicting consequences of PME activity estimated at pH 7.5 is unreliable.     

苏云金芽孢杆菌cry1Ia基因的克隆、表达与活性研究

根据cry1Ia类基因的全长序列设计引物，以苏云金芽孢杆菌（Bacillus thuringiensis）菌Btc008的总DNA为模板扩增出片段长为2.1kb的cry1Ia的全长基因，插入大肠杆菌（Escherichia coli）表达载体pET-21b，转化大肠杆菌BL21（DE3）菌株，诱导表达出81kD的蛋白。该蛋白由719个氨基酸组成，推导的分子量为81.2kDa。该蛋白的氨基酸序列不同于已知的12种Cry1Ia蛋白，是一种新的Cry1Ia蛋白，该基因已被国际基因命名委员会正式命名为cry1Ia8。杀虫活性测定结果表明：Cry1Ia8对亚洲玉米螟（Ostrinia furnacalis）、小菜蛾（Plutella xylostella）有很强的杀虫活性，LC50分别为0.268 µg/g、2.227 µg/ml，其杀虫效果与Cry1Ab、Cry1Ac相当。对大豆食心虫（Leguminivora glycinivorella）也有较好的活性，但对鞘翅目叶甲科害虫榆兰叶甲（Pyrrhalta aenescens）没有活性。该基因的获得将为我国抗虫转基因作物和工程菌的研制提供新的基因来源，为筛选延缓昆虫抗性产生的基因组合提供了极为重要的依据。     

高透光率青梅浓缩汁贮藏过程颜色的动力学研究

该文研究了高透光率青梅浓缩汁在贮藏过程中吸光度与贮藏温度、贮藏时间的关系,建立了颜色变化动力学模型,为高透光率青梅浓缩汁贮藏条件的优化控制及保质期预测提供了科学依据。结果表明：高透光率青梅浓缩汁的吸光度变化(A₀-A)符合Arrhenius模型,且为零级反应,其反应常数K₀为1.13×10⁷,活化能Ea为59.89 kJ/mol。经验证,该模型预测值与试验实测值的相关系数达0.999,表明该模型是合适有效的。     

Effects of processing and of storage on the stability of pantothenic acid in sea buckthorn products (Hippophaë rhamnoides L. ssp. rhamnoides) assessed by stable isotope dilution assay

A stable isotope dilution assay for quantification of pantothenic acid in sea buckthorn berries, juice, and concentrate using a four-fold labeled isotopologue of vitamin B5 as the internal standard was adopted using reversed phase liquid chromatography-mass spectrometry with electrospray ionization. Because of a rapid sample clean up procedure without the necessity of external calibration, this methodology permits the accurate analysis of a high number of samples within a short time. Sea buckthorn juice was stored at 25 and 40 degrees C for up to 7 days to determine the effects of storage temperature on the stability of pantothenic acid. Analysis of kinetic data suggested that the degradation follows a first-order model. The results of the experiments showed that storage of sea buckthorn juice for 7 days at ambient temperature (25 degrees C) already resulted in a significant degradation of pantothenic acid of about 18%. The processing effects of juice production and subsequent concentration revealed a decrease of about 6-7% in the juice and of 23% in the juice concentrate.     

Rheology of sea buckthorn (Hippophae rhamnoides L.) juice.

Juice extracted from sea buckthorn fruits was subjected to dynamic rheological measurements in a controlled stress rheometer. Sea buckthorn juice exhibited wide variations in flow behavior from pseudoplastic to dilitant with increasing temperature. The power law model suitably (r >/= 0.975, P相似文献