骆驼蓬种子凝集素粗品活性的初步研究

采用硫酸铵分级沉淀法提取骆驼蓬种子凝集素粗品,并进行凝血活性和抗菌活性检测.凝血实验结果表明:(1)50%～60%硫酸铵饱和度下提取骆驼蓬种子的凝集素粗品对鸡红细胞的凝集活力最强,最低凝集浓度为0.6 mg/L,且其凝集素粗品的活力受温度、pH值、金属离子及糖溶液的影响.(2)不同硫酸铵饱和度沉淀获得的凝集素粗品对6种人体致病细菌及5种能引起水果腐烂和粮食霉变的真菌均有不同程度的抑制作用,其中50%～60%硫酸铵饱和度下提取的凝集素粗品抑菌活性最强,其浓度为1.071 mg/mL时对鲍曼不动杆菌和意大利青霉菌的抑菌环半径分别可达8.1 mm和4.9 mm.     

姬松茸凝集素的分离纯化及性质

凝集素是一类能够识别特异性糖并与之非共价结合的蛋白或糖蛋白,因其具有特定的识别受体,故在免疫系统和发育过程中发挥了重要的作用。使用DEAE-Sepharose FF弱阴离子交换层析、SP-Sepharose强阳离子交换柱层析、Sephadex G-75分子筛凝胶层析对姬松茸凝集素进行分离纯化,研究理化因素对其凝血活性的影响及其抗菌活性。结果表明:纯化的样品经SDS-PAGE电泳分析得到单一条带,姬松茸凝集素是含有2个相同亚基的同源二聚体,其分子量都为1.20×10~5。纯化后的姬松茸凝集素相对粗提液的回收率为28.99%,纯化倍数为19.06倍。姬松茸凝集素对热有较好的耐受性,但温度为90℃时失去凝血活性;在强酸或强碱下丧失活性,最适pH为7～9;凝集活性不受金属离子的影响;葡萄糖是其专一性结合糖。姬松茸凝集素对褐腐菌的生长起到了明显的抑制作用,而对大肠杆菌、枯草芽孢杆菌、绿色木霉、西瓜枯萎病菌和黑曲霉的生长没有明显的影响。     

大黑花芸豆凝集素的分离纯化及温度、pH、色氨酸修饰对其活性的影响

大黑花芸豆(Phaseolus multiflorus.Wiud)种子经匀浆、浸取、硫酸铵分级沉淀、阴离子交换层析(DEAE-Sepharose)、阳离子交换层析(CM-Sepharose)和Sepllacryl S-200分子筛层析得到凝集素样品(PML).经SDS-PAGE检测为一分子量约为28k的单一条带,Sephacryl S-100凝胶过滤测得其表观分子量约为56 kD表明PML是由两个相同亚基组成的蛋白.温度低于60℃时,PML较为稳定,当温度达80℃时,其凝血活性完全丧失;pH为5.6～9对活性影响不大,pH为12时,活性大部分丧失;高温和强碱对荧光光谱有较大影响.NBS修饰Trp结果表明,在天然状态下有3个色氨酸分子被修饰,其中第二和第三个色氨酸分子对其活性至关重要.     

枯草芽胞杆菌FB123细菌素的理化性质及抑菌谱的研究

研究了细菌素产生菌枯草芽胞杆菌(Bacillus subtilis)FB123 所产细菌素的理化性质及其抑菌谱.枯草芽胞杆菌FB123经过 28 ℃、32 h的发酵得到发酵上清液,用饱和度为50%的硫酸铵溶液沉淀发酵上清液中的细菌素.以革兰阴性菌大肠杆菌和革兰阳性菌金黄色葡萄球菌为指示菌,采用牛津杯法检测细菌素抑菌活性,对细菌素粗品进行理化性质的研究.结果表明该细菌素最适作用pH为 6.0,最适作用温度 40 ℃,具有较宽的pH作用范围和较好的热稳定性.各种蛋白酶、金属离子对其活性有不同程度的影响.抑菌谱试验结果表明,该细菌素对多种革兰阳性菌和革兰阴性菌有明显的抑制作用,虽然对部分真菌有抑制作用,但抑制作用较弱.     

冬虫夏草菌丝体γ-谷氨酰转肽酶的探测和部分性质研究

本研究首次发现冬虫夏草发酵菌丝体含有较高活力的γ-谷氨酰转肽酶（简称CSGT）,并且通过硫酸铵分级沉淀、疏水层析、凝胶过滤层析、阴离子交换层析和制备电泳的提取纯化程序,将CSGT纯化了2300倍,然后对CSGT的基本酶学性质进行了研究。CSGT的稳定pH范围和温度范围分别为pH8-11和0-20℃, 当pH 9-10 、30℃并且以L-谷氨酸-对-硝基苯胺（简称GpNA）和双甘肽为底物时CSGT的活力达到最大值。几种还原剂均能激活CSGT,说明其活性中心含有巯基。Zn2+, Cu2+, Hg2+ , Mn2+ 等金属离子均强烈抑制CSGT活性,而K+, Ca2+, Mg2+ 和Na+等对CSGT活性没有影响。     

虎血清免疫球蛋白（IgG）的纯化及纯度、活性鉴定

目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。     

正红菇菌丝体凝集素的分离纯化及生化特性

正红菇菌丝体经磷酸缓冲液浸提、硫酸铵分级沉淀、DEAE-Sepharose FF离子交换层析和Sephadex G-100分子筛层析纯化得到红菇凝集素（Russula vinosa Lectin,RVL）。经SDS-PAGE检测为单一蛋白带,其亚基相对分子质量为55kDa,Sephadex G-100凝胶过滤测得相对分子质量为55.25kDa,提示RVL分子只有一个亚基。RVL中性糖含量为3.87%,经酸水解测定含15种氨基酸。温度在20–60℃、pH在5–9的范围内,凝集活性保持相对的稳定。RVL的凝血活性受Mn2+、Zn2+、Ca2+的影响。糖抑制实验表明,在供试的11种糖中,D-甘露糖强烈抑制RVL的凝血活性。抑菌实验显示,RVL对供试的细菌没有抑制作用,对稻瘟病菌、绿色木霉、红色链包霉、黑曲霉菌丝生长有显著的抑制作用。     

狗脊蕨凝集素的分离纯化与部分性质

狗脊蕨叶组织经磷酸缓冲液抽提,硫酸铵分级沉淀,DEAE-Sepharose(fast flow)离子交换层析和Sephadex G-100分子筛层析,获得具有凝集活性的狗脊蕨凝集素.用PAGE和SDS-PAGE检测均显示1条蛋白带,提示狗脊蕨凝集素分子只有一个亚基.Sephadex G-100层析和SDS-PAGE测得其相对分子质量在20 kD左右.该凝集素对红细胞有种属专一性,其凝集活性可被木糖、卵粘蛋白所抑制.狗脊蕨凝集素对热相当稳定,以100℃温度加热10 min仍保存部分凝集活性.凝集活性不依赖于金属离子Mg2 、Ca2 、Mn2 .它的中性糖含量为3.1%,Phe含量较高,His含量较低,不含Tyr和Pro.     

一株产甘露聚糖酶菌株的分离鉴定及酶的纯化与性质

【目的】筛选性能良好的产碱性甘露聚糖酶的菌株,对菌株进行多项分类鉴定,分离纯化所产甘露聚糖酶并进行性质研究。【方法】利用碱性魔芋粉培养基分离纯化产甘露聚糖酶的嗜碱菌,通过形态特征观察、生理生化测定、16S rRNA序列分析等实验确定菌株的分类地位。利用硫酸铵沉淀、阴离子交换层析和分子筛层析得到电泳纯的酶,分析了酶的最适温度、最适pH、温度和pH稳定性、NaCl以及金属离子等的耐受性。【结果】从我国内蒙古碱湖样品中分离得到一株产碱性甘露聚糖酶的菌株HMTS15,经过多项分类鉴定显示其是与Bacillus agaradhaerens DSM 8721不同的新菌株。菌株HMTS15所产的甘露聚糖酶反应的最适pH为10.0,最适温度75℃。【结论】多项分类结果鉴定菌株为Bacillus agaradhaerens HMTS15。该菌株产生的碱性甘露聚糖酶与同类其他来源的酶相比具有更好的热稳定性和pH适应性,有进一步的研究价值。     

海洋微生物溶菌酶的纯化与性质研究

海洋微生物溶菌酶发酵上清液经超滤、CM-SepharoseFF阳离子交换层析和SephadexG-10 0凝胶过滤层析纯化得到电泳纯的溶菌酶,纯化倍数为34.7,活力回收为24.1%。对纯化溶菌酶性质研究表明,该酶分子量约为39kD ,对溶壁微球菌的最适作用温度为35℃,最适作用pH为8 0 ,在5 0℃以下和pH 5 0～10 0之间都有较好的稳定性,与常见金属离子和化学试剂有良好的配伍性,广谱杀菌,对多种致病菌也有较强的溶菌作用。     

cDNA Cloning and Functional Expression of the α-<Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin Frutalin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

cDNA clones encoding frutalin, the α-d-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa), were isolated and sequenced. The deduced amino acid sequences indicated that frutalin may be encoded by a family of genes. The NCBI database searches revealed that the frutalin sequence is highly homologous with jacalin and mornigaG sequences. Frutalin cDNA was re-amplified and cloned into the commercial expression vector pET-25b(+) for frutalin production in Escherichia coli. An experimental factorial design was employed to maximise the soluble expression of the recombinant lectin. The results indicated that temperature, time of induction, concentration of IPTG and the interaction between the concentration of IPTG and the time of induction had the most significant effects on the soluble expression level of recombinant frutalin. The optimal culture conditions were as follows: induction with 1 mM IPTG at 22°C for 20 h, yielding 16 mg/l of soluble recombinant frutalin. SDS-PAGE and Western blot analysis revealed that recombinant frutalin was successfully expressed by bacteria with the expected molecular weight (17 kDa). These analyses also showed that recombinant frutalin was mainly produced as insoluble protein. Recombinant frutalin produced by bacteria revealed agglutination properties and carbohydrate-binding specificity similar to the native breadfruit lectin.     

Ethanol-Induced Gastric Injury: Microscopic Analysis of the Protective Effect of Frutalin

Obtained from breadfruit seeds (Artocarpus incisa), frutalin (FTL) has a range of important pharmacological properties. FTL activates and modulates lymphocytes and neutrophils and possesses gastroprotective effects. The purpose of this study was to evaluate the ability of FTL to protect the gastric mucosa of mice submitted to ethanol-induced gastric injury. The gastroprotective effect of frutalina was evaluated using a murine model of ethanol-induced gastric injury. Damage of the gastric mucosa was assessed morphologically by light, scanning electron and atomic force microscopy. Lymphocyte infiltration, necrosis, disruption of the mucosal structures and epithelial desquamation were observed in the control group. In the group pretreated with frutalina at 0.5 mg/kg, gastroprotection, reduced tissue damage and preserved submucosal and mucosal structures were observed. In conclusion, FTL displayed gastroprotective activity against ethanol-induced gastric injury, preventing the formation of gastric ulcer and reducing epithelial desquamation, glandular damage, mucosal necrosis and infiltration. The observed effects appear to be due to mechanisms previously described for frutalin.     

Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).     

响应面法优化水酶法提取辣木籽蛋白质的工艺及其功能性质研究

为了提取和利用辣木籽蛋白质,本文通过水酶法优化其提取条件,并探讨其功能性质。以辣木籽为原材料,以蛋白质提取率为指标,首先确定了最佳使用酶为Alcalase碱性蛋白酶,再通过单因素试验考察料液比、时间、温度、酶添加量和pH等因素对蛋白质提取率的影响,在此基础上,利用响应面试验设计优化水酶法提取辣木籽蛋白质的工艺。结果表明,最佳工艺条件为:使用Alcalase碱性蛋白酶,在料液比为1∶10,酶添加量为4.5%,pH为9.0,温度为60℃,时间为4.5 h,此时辣木籽蛋白质的提取率最高为68.23%。在pH为10、温度为55℃时辣木籽蛋白质的氮溶解指数最高;辣木籽蛋白质持水性随着pH的增加而增加,在温度为40℃时持水性最好;在温度为55℃时,辣木籽分离蛋白的吸油效果最明显。     

Expression of frutalin, an alpha-D-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

Frutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.     

Purification and characterization of ACC oxidase from Artocarpus altilis

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase was purified to homogeneity from breadfruit (Artocarpus altilis) by a six-step chromatography procedure to yield an 87-fold increase in purification with a recovery of 9.5%. SDS-PAGE revealed that the purified enzyme had a molecular mass of 42.3 kDa and a Km of 28.2 μM. The enzyme showed marked inhibition by cobalt sulphate, sodium metabisulphite, sodium dithionite, n-propyl gallate, zinc sulphate and hydrogen peroxide. Dithiothreitol (DTT) enhanced enzyme activity when it was included in the assay medium. Optimum activity of ACC oxidase was obtained at a pH of 7.2 at 28 °C.     

膜荚黄芪种子中2种几丁质酶的分离纯化及活性测定

运用传统的层析技术对膜荚黄芪种子中两种几丁质酶进行分离纯化,并对分离纯化中各个步骤得到的蛋白进行了活性研究.结果表明:(1)粗提液的硫酸铵沉淀经再生几丁质亲和柱和凝胶过滤层析Sephadex G-75得到几丁质酶A.(2)粗提液的硫酸铵沉淀经离子交换色谱DE-52、CM、凝胶过滤层析Sephadex G-75得到几丁质酶B.(3)几丁质酶A、B的比活性分别为35.6 U/mg和4.1 U/mg.(4)从膜荚黄芪种子中分离纯化的几丁质酶A和B均为糖蛋白,含糖量分别为6.1%和5.8%.(5) SDS-PAGE显示,几丁质酶A、B的分子量分别为35.5 ku、39.6 ku;凝胶过滤层析测定几丁质酶A、B的分子量分别为36.9 ku、40.8 ku;表明几丁质酶A、B均为单亚基蛋白.     

How many seeds in a seeded breadfruit,Artocarpus altilis (Moraceae)?

Despite their demonstrated nutritional value, yield of seeded breadfruit trees (Artocarpus altilis) has not been investigated in any detail. In the present study, the average number of seeds per seeded breadfruit harvested from a single 6-yr-old tree over a period of 7 months was 59. Individual fruits contained as many as 151 or as few as 12. The average seed weight/fruit was 348 g. Fruit production from March to September totalled 33 and ranged from one to eight per month.     

Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato

Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato (Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20 kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.     

Purification of glandular kallikrein in maxillary mucosa from humans suffering from chronic inflammation

Glandular kallikrein was purified from human maxillary mucosa with chronic inflammation and its biochemical properties were characterized. The purification procedure consisted of extraction with 3 mol/l KCl, saturation of ammonium sulfate (66%), DEAE-Sepharose, arginine-Sepharose and Sephadex G-150 chromatographies. Maxillary mucosa with chronic inflammation contains considerable activity of glandular kallikrein, which is a serine protease with limited proteolytic activity and its biochemical properties resemble those of pancreatic kallikrein.