白地霉Cryytococcus neoformans脂肪酶的双水相萃取

本文研究了不同无机盐的双水相体系对白地霉脂肪酶的萃取分离效果,对PEG/（NH4）2SO4成相系统进行了系统的研究,通过考察体系PEG分子量、不同的无机盐、PEG浓度、（NH4）2SO4浓度、离子强度、pH值及（NH4）2SO4浓度对反萃取的影响,并通过正交实验进一步优化了实验条件,初步确定在PEG浓度15%,（NH4）2SO4浓度22.5%,pH8.0,不加NaCl的条件下进行双水相萃取,脂肪酶分离系数和纯化倍数分别为6.8和7.5,比活力达到40.3 U/mg蛋白。     

双水相体系萃取人参根中人参皂苷的研究

建立稳定的聚乙二醇(PEG)与(NH4)2SO4双水相体系以分离人参根中人参皂苷。通过上下相体积比(R)、分配系数(K)和回收率(Y)分析双水相体系对人参皂苷的萃取效果,研究了PEG分子量、PEG/(NH4)2SO4质量分数、pH值和温度等因素对双水相成相及人参皂苷萃取的影响。结果表明:PEG分子量为3350、PEG3350的质量分数为12%、(NH4)2SO4质量分数为16%、溶液pH为7.0、温度为60℃时,双水相体系对人参皂苷有较高的萃取率,回收率可到达88.94%。     

双水相萃取法分离竹叶黄酮的工艺研究

以竹叶黄酮水提溶液为原料,采用PEG(聚乙二醇)/(NH4)2SO4双水相体系对竹叶黄酮进行萃取,考察了PEG平均相对分子质量、PEG质量分数、(NH4)2SO4质量分数、pH值、NaCl质量分数、原液质量分数、萃取温度等对双水相及竹叶黄酮萃取效果的影响。双水相萃取法提取竹叶黄酮的最优条件为:PEG 400 31%,(NH4)2SO411%,pH 3.9,NaCl 0.7%,原液51.5%,萃取温度20℃,在此条件下得到的竹叶黄酮萃取率为97.8%。结果说明,双水相萃取法操作简单方便,成本低,不会引起生物质失活或变性,适合于黄酮类化合物的萃取分离。     

聚乙二醇共沉淀物复溶萃取辣根过氧化物酶的条件优化

为了改进辣根过氧化物酶的提纯方法,在双水相萃取的基础上使用聚乙二醇(PEG6000)在高饱和度(NH4)2SO4中沉淀辣根提取液中的过氧化物酶,利用磷酸盐溶液复溶解共沉淀物形成的双水相萃取体系能高效回收高纯度酶蛋白。研究了pH、PEG浓度和(NH4)2SO4饱和度对酶活性的影响,并考察不同液固比、pH和NaCl浓度对目标酶在双水相体系中的分配行为,并通过响应面法优化出最优萃取条件。结果表明:在液固比0.3 m L/g、pH7.02和NaCl 42 g/L的优化萃取条件下,辣根过氧化物酶回收率达88.1%,酶纯度较优化前提高了21.7倍。该方法的建立对于微量蛋白质的高效率提纯具有重量的参考价值。     

双水相萃取丽江山慈菇中的秋水仙碱

建立了由聚乙二醇(PEG6000)与(NH4)2SO4形成的双水相体系萃取丽江山慈菇中秋水仙碱的新方法。考察了PEG分子量、PEG的浓度、(NH4)2SO4的浓度和pH值对双水相成相及秋水仙碱萃取率的影响,并结合HPLC对萃取相进行检测。结果表明:PEG6000质量分数为8%,(NH4)2SO4质量分数为20%,pH为7.0时,双水相体系对丽江山慈菇粗提液中秋水仙碱萃取率达82.09%,富集倍数为6.84倍。此方法可用于丽江山慈菇中秋水仙碱的初步分离富集,且操作简单,绿色无污染。     

鸡冠花种子蛋白质的提纯及分析

鸡冠花种子干燥后用石油醚脱酯,用凯氏定氮法测定蛋白质含量。按Osbern系统分别提取白蛋白、球蛋白、醇溶蛋白、谷蛋白;用Folin一酚试剂法测定各自相对含量,并用单向和双向SDS—PAGE方法分析种子总蛋白和4类不同溶性蛋白质的组成成分及这些组份的热稳定性。实验发现:鸡冠花种子蛋白质含量达26.04％,白蛋白、球蛋白、醇溶蛋白、谷蛋白的相对含量分别为11.5％、72％、4.6％、12％。SDS—PAGE表明;其种子蛋白中20KD球蛋白成份含量最高,其它一些次要组分为63KD、61KD、15KD、13KD白蛋白成份,58KD、37KD、23KD球蛋白成份,39KD、34KD、25KD谷蛋白成份及26KD醇溶蛋白成份,其中58KD由37KD和20KD两亚基经二硫键连接而成,39KD组份由24.5KD和18KD两亚基通过二硫键连接而成,23KD球蛋白组份遇热沉淀。     

海洋真菌Caldariomyces fumago产氯过氧化物酶的双水相提纯条件

采用聚乙二醇(PEG6000)在(NH4)2SO4高饱和度下沉淀夹带蛋白质富集氯过氧化物酶,再利用磷酸盐溶液复溶解共沉淀物形成的双水相萃取体系高浓度回收酶蛋白,最后再经Sephadex G100柱层析纯化获得高纯度酶试样。结果显示:氯过氧化物酶与PEG共沉淀总活力回收率达85.5%,酶在优化的PEG/磷酸盐双水相系统中上下相分配系数k在0.341以下,酶活力回收率达到69.1%,纯度提高了21.57倍,柱层析可使酶纯度进一步提高到24.79倍,总回收率为37.75%。     

Separation of Flavonoids from Bamboo Leaves by Aqueous Two-Phase Extraction

以竹叶黄酮水提溶液为原料,采用PEG(聚乙二醇)/(NH4 )2SO4双水相体系对竹叶黄酮进行萃取,考察了PEG平均相对分子质量、PEG质量分数、(NH4)2SO4质量分数、pH值、NaCl质量分数、原液质量分数、萃取温度等对双水相及竹叶黄酮萃取效果的影响.双水相萃取法提取竹叶黄酮的最优条件为:PEG 400 31％,(NH4)2 SO4 11％,pH3.9,NaCl 0.7％,原液51.5％,萃取温度20℃,在此条件下得到的竹叶黄酮萃取率为97.8％.结果说明,双水相萃取法操作简单方便,成本低,不会引起生物质失活或变性,适合于黄酮类化合物的萃取分离.     

一种新的蘑菇凝集素的生化特征和氨基酸分析

用 (NH4) 2 SO4分级沉淀、离子交换和分子筛等方法 ,从食用蘑菇中分离纯化出一种凝集素 ,经SDS -PAGE测定其亚基的相对分子量为 15 .8kDa,质谱分析法测定其分子量为 32kDa ,说明该凝集素由两个亚基组成。氨基酸分析发现 ,该凝集素不含半胱氨酸。热稳定试验和耐酸碱试验显示 ,该凝集素活性具很高稳定性     

毕赤氏酵母植酸酶工程菌高密度培养条件的研究

研究了毕赤氏酵母植酸酶工程菌高密度生长的培养条件 ,包括不同碳源、酵母粉、(NH4 ) 2 SO4 、KH2 PO4 等不同用量对菌体生长的影响。结果是甘油 4 %、蛋白陈 2 %、酵母粉 0 5%、(NH4 ) 2 SO4 0 .8%、K2 HPO4 0 1%、KH2 PO4 0 6 %。在此基础上 ,对温度、起始pH、接种量等影响该工程菌菌体生长的因素也作了初步研究。     

In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin

Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% alpha-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 microg/ml. Refolded His-rMcnapin exhibited approximately 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.     

苦瓜种子蛋白及其抑菌作用

SDS-PAGE分析表明,苦瓜种子含有多种蛋白组分,其中57kD和49kD蛋白可能由多个或多种亚基以二硫键结合形成。苦瓜种子蛋白对真菌和细菌有显著抑制效应。     

Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies

A ribosome inactivating protein from Saponaria officinalis, SO-6, was purified and the N-terminus sequenced. The sequence shows extensive homology with Pokeweed antiviral protein, Pokeweed antiviral protein II, Pokeweed antiviral seed protein and dodecandrin. SDS gel electrophoresis in the Laemmli system revealed two bands of similar intensities with a smear between them, probably an artifact due to the high pI of the protein. Use of a harsher denaturing gel system resulted in one band in electrophoresis. Immune antisera was raised in rabbits against this protein and it cross reacted with other proteins (SO-5, SO-8 and SO-9) from seeds of Saponaria officinalis, but not with gelonin, Momordica charantia inhibitor and dianthin 32.     

苦瓜叶提取物对亚洲玉米螟的生物活性及对斜纹夜蛾卵巢细胞的毒力

【目的】为研究苦瓜(Momordica charantia L.)叶正丁醇萃取物对亚洲玉米螟Ostrinina furnacalis(Güenée)生长发育及生殖力的影响,并探讨其作用机理。【方法】采用饲料混药法测定了苦瓜叶正丁醇萃取物对亚洲玉米螟幼虫存活率、发育历期和雌成虫产卵量的影响。【结果】用0.025%、0.1%和0.2%浓度的苦瓜叶正丁醇萃取物混合饲料饲喂亚洲玉米螟2龄幼虫3 d后,幼虫存活率明显降低,校正死亡率分别为27.43%、39.23%和54.93%,且发育历期明显延长,雌成虫产卵量与对照相比分别下降了12.63%、22.74%和37.27%。进一步的研究结果表明,苦瓜叶正丁醇萃取物对斜纹夜蛾卵巢细胞(SL)的形态、增殖和生长密度有明显的抑制作用。处理后的SL细胞皱缩变圆,细胞核偏移甚至消失,胞质外泄。处理24、48和72 h后,对SL细胞增殖的IC50值分别为180.96、158.54和122.87μg/mL;处理6~72 h后,SL细胞相对生长密度随药剂浓度和处理时间的增加而明显下降;用125μg/mL浓度处理SL细胞2、3、4和5 d后,SL细胞对葡萄糖吸收的抑制率分别为7.66%、13.97%、19.45%和27.30%。【结论】苦瓜叶正丁醇萃取物对SL细胞表现出的较强毒杀作用可能是其导致亚洲玉米螟生殖能力降低的主要作用机制之一。     

9种植物水浸提液对空心莲子草的化感作用

【背景】空心莲子草是难以防除的恶性入侵杂草,因此,探索高效无毒的化感防治方法具有重要意义。【方法】利用水浸提法研究了博落回、苦瓜、樟树、柳杉、凤尾蕨、柑橘、夹竹桃、洋葱及大蒜9种植物不同器官对空心莲子草的化感作用。【结果】与对照相比,博落回叶、苦瓜果肉与种子、凤尾蕨、樟树叶和柳杉叶的水浸提液对空心莲子草具有较强的化感作用,主要表现为空心莲子草叶片数和茎节数减少,株高与生物量的增长受到抑制。【结论与意义】不同植物水浸提液对空心莲子草的化感作用有所差异。本研究为利用植物的化感作用控制空心莲子草提供了依据。     

Anti-hyperglycemic and anti-oxidative effect of aqueous extract of Momordica charantia pulp and Trigonella foenum graecum seed in alloxan-induced diabetic rats

Diabetes is an oxidative stress disorder and oxidative damage to tissues such as heart, kidney, liver and other organs may be a contributory factor to several diabetic complications. Momordica charantia (family: Cucurbitaceae) and Trigonella foenum graecum (family: Fabaceae) are used traditionally in Indian folk medicine to manage diabetes mellitus. In the present study, the anti-hyperglycemic and anti-oxidative potential of aqueous extracts of M. charantia pulp and seed powder of T. foenum graecum were assessed in alloxan (150 mg/kg body weight) induced diabetic rats. Alloxan treatment to the rats could induce diabetes as the fasting blood glucose (FBG) levels were > 280 mg/dl. Treatment of diabetic rats for 30 days with M. charantia and T. foenum graecum could significantly (p < 0.001) improve the FBG levels to near normal glucose levels. Antioxidant activities (superoxide dismutase, catalase, reduced glutathione content and glutathione-s-transferase) and lipid peroxidation levels were measured in heart, kidney and liver tissues of normal, diabetic and experimental animals (diabetics + treatment). TBARS levels were significantly (p < 0.001) higher and anti-oxidative activities were found low in diabetic group, as compared to the control group. Significant (p < 0.001) improvement in both the TBARS levels and antioxidant activities were observed when M. charantia and T. foenum graecum were given to diabetic rats. Our results clearly demonstrate that M. charantia and T. foenum graecum are not only useful in controlling the blood glucose levels, but also have antioxidant potential to protect vital organs such as heart and kidney against damage caused due to diabetes induced oxidative stress.     

Plant extracts as an alternative to control Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae)

We evaluated the effects of crude extracts from the plantain Plantago lanceolata and the bitter gourd Momordica charantia on the oviposition preference and development of the coffee leaf miner Leucoptera coffeella Guérin-Mèneville & Perrottet under laboratory and/or greenhouse conditions. The ovicidal effects of these extracts were also studied in a greenhouse. Plantago lanceolata and M. charantia extracts also underwent fractionation directed by oviposition tests with the coffee leaf miner. The extracts of both plants reduced L. coffeella oviposition and egg hatching, apparently as a result of action of plant metabolites on the embryo. Adults originating from eggs treated with the extracts exhibited similar survival rates, but a higher female/male ratio. Fecundity was reduced for females obtained from eggs treated with the M. charantia extract. Partial chemical analysis indicated that both extracts produced polar fractions that reduced the oviposition of L. coffeella on coffee leaves under laboratory conditions. The extracts of P. lanceolata and M. charantia have potential for use in the development of new products to control the coffee leaf miner.     

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.     

小麦种子在不同保存条件下的生活力丧失特性研究

近年来种子在低温种质库和室温保存条件下的生活力丧失特性研究日益受到关注,但是与其重要性相比,目前很少见有文献报道此方面进展。本文中我们监测了在中期库、室温和人工加速老化条件下小麦种子的生活力变化,以了解种子在这些保存条件下的生活力变化特性。结果表明,三种条件下所有种子的存活曲线均呈反S形,这种特点不受保存条件的影响。随着保存温度的降低,种子生活力丧失速度降低,反S形曲线的平台期和种子寿命延长。我们采用T检验来判断平台期和种子生活力快速下降期之间的转折点（Pt）,发现小麦种子在不同条件下保存时,其Pt均十分接近,在82.2%到83.6%之间。本文讨论了将Pt以及其他生理指标作为种子保存过程中生活力快速下降的预警指标的可能性。     

Proteomic Analysis of Sauvignon Blanc Grape Skin,Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest.