植物半胱氨酸合成酶复合体(SAT/OAS-TL)研究进展

综述了植物半胱氨酸的合成调节和半胱氨酸合成酶复合体的结构、调节机理及其植物转基因的研究进展。     

Source of soil protease based on the splitting sites of a polypeptide

Abstract

Soil protease is an important enzyme in the nitrogen cycle which plays an essential role in the growth of various crops. We have attempted to identify a microbial source of soil protease. Selective soil incubation using antibiotics was suitable for a rough estimation of the groups of microorganisms responsible for the production of soil protease (Hayano and Watanabe 1990; Hayano 1993). Enumeration of proteolytic microorganisms in the field and analysis of their protease-producing ability enabled to evaluate the potential of various soil microorganisms for soil protease production (Watanabe and Hayano 1993a; Watanabe et al. 1994). For the accurate estimation of the soil protease source, the characteristics of the soil protease and microbial protease must be compared directly based on enzymatic properties as indices.     

Distribution of Enzymes in Wheat Flour Mill Streams

The distribution of alpha -amylase, protease, lipoxygenase, polyphenol oxidase and peroxidase in wheat roller flour mill streams was studied. Break flours had relatively less alpha -amylase and protease activity than reduction flours both on flour weight and a protein basis. Among the different flour streams, the 5^thand 6^threduction passage had the highest alpha -amylase activity, while the 4^threduction passage had the highest protease activity. The lipoxygenase activity was concentrated mostly in the last break and the reduction streams, whereas polyphenol oxidase activity was highest in break flour streams. Peroxidase activity was distributed unevenly among the different mill streams. The lipoxygenase, polyphenol oxidase and peroxidase were highly concentrated in different bran fractions. Except for protease, the other enzymes were concentrated in the «atta», a milling by-product comprising refined flour, bran and shorts; and are least active in semolina (farina).     

中华鲟半胱氨酸蛋白酶抑制剂C基因的原核表达

为了研究鱼类半胱氨酸蛋白酶抑制剂(cystatin)的功能并探索其在水产品加工和病害防治中的应用潜力，将改造后的中华鲟(Acipenser sinensis)cystatin C cDNA亚克隆到原核表达载体pBV220，构建表达cystatinc的大肠杆菌基因工程菌。该工程菌经温度诱导、SDS—PAGE检测，在约12．4kD处有一特异蛋白带，该特异蛋白的含量约为菌体总蛋白的25％。重组半胱氨酸蛋白酶抑制剂经洗涤、溶解、透析、复性后纯度为85％，实现了中华鲟cystatinC在大肠杆菌中的高效表达。木瓜蛋白酶活性抑制实验结果表明该重组cystatinc具有明显的酶活抑制作用。     

菌根伴生真菌对外生菌根真菌生长及其中性蛋白酶活性的影响

【目的】筛选产中性蛋白酶活性较高的外生菌根真菌及最佳诱导底物。【方法】以酵母粉(对照)和4种菌根伴生真菌(Trichoderma harzianum HDTP-1、T.harzianum HDTP-3、Mucor hiemalis SA10-6 HDTP-4和M.hiemalis XSD-98 HDTP-5)灭活菌丝体为诱导底物,研究其对粘盖牛肝菌(Suillus bovines)、褐环粘盖牛肝菌(Suil-lus luteus)、褐黄牛肝菌(Boletus luridus)和铆钉菇(Gomphidius viscidus)4种外生菌根真菌生物量及其所产中性蛋白酶活性的影响。【结果】4种菌根伴生真菌均明显促进了褐环粘盖牛肝菌、粘盖牛肝菌和褐黄牛肝菌的生长,其中Mucorhiemalis SA10-6HDTP-4对褐环粘盖牛肝菌的促进效果极显著,而4种菌根伴生真菌对铆钉菇的生长均有不同程度的抑制,其中Trichoderma harzianum HDTP-3的抑制作用最强。4种菌根伴生真菌诱导的4种外生菌根真菌所产的中性蛋白酶活性,均以褐黄牛肝菌最高,铆钉菇最低;M.hiemalis XSD-98 HDTP-5诱导褐环粘盖牛肝菌所产中性蛋白酶活性最高,T.harzianum HDTP-3最低;M.hiemalis XSD-98 HDTP-5诱导粘盖牛肝菌所产中性蛋白酶活性极显著高于酵母粉和其他3种菌根伴生真菌,但其诱导褐黄牛肝菌和铆钉菇所产中性蛋白酶活性仅极显著高于其他3种菌根伴生真菌。【结论】4种菌根伴生真菌中,M.hiemalis XSD-98 HDTP-5是外生菌根真菌产中性蛋白酶的最佳诱导底物;而4种外生菌根真菌中,褐黄牛肝菌所产中性蛋白酶活性最高。     

Cytotoxic,Cytostatic and HIV-1 PR Inhibitory Activities of the Soft Coral Litophyton arboreum

Bioassay-guided fractionation using different chromatographic and spectroscopic techniques in the analysis of the Red Sea soft coral Litophyton arboreum led to the isolation of nine compounds; sarcophytol M (1), alismol (2), 24-methylcholesta-5,24(28)-diene-3β-ol (3), 10-O-methyl alismoxide (4), alismoxide (5), (S)-chimyl alcohol (6), 7β-acetoxy-24-methylcholesta-5-24(28)-diene-3,19-diol (7), erythro-N-dodecanoyl-docosasphinga-(4E,8E)-dienine (8), and 24-methylcholesta-5,24(28)-diene-3β,7β,19-triol (9). Some of the isolated compounds demonstrated potent cytotoxic- and/or cytostatic activity against HeLa and U937 cancer cell lines and inhibitory activity against HIV-1 protease (PR). Compound 7 was strongly cytotoxic against HeLa cells (CC₅₀ 4.3 ± 0.75 µM), with selectivity index of SI 8.1, which was confirmed by real time cell electronic sensing (RT-CES). Compounds 2, 7, and 8 showed strong inhibitory activity against HIV-1 PR at IC₅₀s of 7.20 ± 0.7, 4.85 ± 0.18, and 4.80 ± 0.92 µM respectively. In silico docking of most compounds presented comparable scores to that of acetyl pepstatin, a known HIV-1 PR inhibitor. Interestingly, compound 8 showed potent HIV-1 PR inhibitory activity in the absence of cytotoxicity against the cell lines used. In addition, compounds 2 and 5 demonstrated cytostatic action in HeLa cells, revealing potential use in virostatic cocktails. Taken together, data presented here suggest Litophyton arboreum to contain promising compounds for further investigation against the diseases mentioned.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

草鱼鱼肉蛋白酶解物抗氧化性及功能特性研究

为全面了解水解度(DH)、蛋白酶种类对草鱼鱼肉蛋白酶解产物抗氧化性和功能特性的影响,采用木瓜蛋白酶及Alcalase 2.4L在各自最适条件下进行酶解,制备水解度为10%和20%的酶解产物,对其功能特性进行分析。结果显示:随着水解度升高酶解产物的亚铁离子螯合能力增强,但还原力和清除DPPH自由基的能力下降(P<0.05)。相同水解度下与Alcalase 2.4L酶解产物相比,木瓜蛋白酶酶解产物具有较强的清除DPPH自由基能力和还原力(P<0.05)。2种蛋白酶酶解产物的溶解性、乳化性、起泡性均在pH4时达到最低,而后随pH升高而增大。相同pH下随着水解度的升高酶解产物的溶解性增强,乳化性下降。相同pH及水解度下木瓜蛋白酶酶解产物的溶解性和起泡性小于Alcalase 2.4L酶解产物,但乳化性优于Alcalase 2.4L酶解产物。酶解产物的抗氧化性及功能特性受水解度及蛋白酶种类的影响。     

鲮鱼鱼皮胶原蛋白酶解物分子量分布与抗氧化性的研究(英文)

[目的]为了研究鲮鱼鱼皮胶原蛋白水解物的分子量的分布及抗氧化活性。[方法]分子量分布采用分子排阻色谱法和基质辅助激光解吸附飞行时间质谱(MALDI-TOF-MS),鱼皮用碱性蛋白酶2709处理。[结果]最佳水解条件为:pH 10.0、反应温度55℃、底物浓度为80 g/L、酶和底物比4%,水解时间3 h。两种方法分析水解产物的相对分子质量分布范围为400~1 800 Da,大多数胶原蛋白短肽的分子量在1 400 Da以下。[结论]结果表明,鱼皮水解物是一种潜在的抗氧化物。