龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

海绵硅蛋白研究进展

海绵是生物进化过程中最古老的多细胞动物,其中大部分能够利用二氧化硅在常温水环境下合成形状、大小和结构极为丰富的硅质骨骼。随着近年来人们发现其骨骼的基本组成单位骨针具有优异的光导性能和机械性能,海绵生物硅化过程及仿生纳米和微米硅质生物材料合成的研究成为生物技术和材料科学的热点。在海绵生物硅化过程中,一类被称为硅蛋白(Silicatein)的蛋白质表现出了特殊的催化活性,也因此得到了生物学家、化学家和材料学家的关注。以下将对硅蛋白的国际研究现状进行了评述,以期促进国内相关领域的研究。     

Enhanced production of harringtonine and homoharringtonine in Cephalotaxus fortunei callus culture by periodic temperature oscillation

Total production of harringtonine, homoharringtonine and isoharringtonine in solid cultures of Cephalotaxus fortunei was 1.22 mg/l by periodic oscillation in temperature between 10 and 25°C every 12 h for 45 days. Production was enhanced 1.8 and 1.3-fold compared to the controls at constant temperatures of 10 or 25°C. For suspension cultures subjected to such an oscillation every 24 h for 30 days, total alkaloid production of 0.18 mg/l was achieved, a 2.0 and 1.1-fold improvement compared to the suspension culture controls, correspondingly.     

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca²⁺/Mg²⁺ artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

几丁质酶和壳聚糖酶对部分乙酰化壳聚糖作用方式的比较

通过对几丁质酶和壳聚糖酶降解部分乙酰化壳聚糖的作用方式的比较,得到几丁质酶切断壳聚糖的ＧｌｃＮＡｃ－ ＧｌｃＮＡｃ 和ＧｌｃＮＡｃ－ ＧｌｃＮ 或ＧｌｃＮ－ ＧｌｃＮＡｃ 糖苷键,而壳聚糖酶切断壳聚糖的ＧｌｃＮ－ ＧｌｃＮ 和ＧｌｃＮ－ ＧｌｃＮＡｃ 或ＧｌｃＮＡｃ－ ＧｌｃＮ 糖苷键,为得到较高聚合度的壳寡糖提供理论基础。     

Identification, bioinformatic analysis and expression profiling of candidate mRNA-like non-coding RNAs in Sus scrofa

Messenger RNA-like non-coding RNAs （mlncRNAs） are a newly identified group of non-coding RNAs （ncRNAs） that may be involved in a number of critical cellular events. In this study, 93 candidate porcine mlncRNAs were obtained by computational prediction and screening, among which 72 were mapped to the porcine genome. Further analysis of 8 representative candidates revealed that these mlncRNA candidates are not highly conserved among species. Remarkably, one of the candidates, sTF35495, was found to be precursor of a putative porcine microRNA. By RACE PCR, we determined that the full length of sTF35495 was 3 kb. The protein-coding potential of this RNA was tested in silico with no significant finding. Semi-quantitative RT-PCR analysis of the subgroup of 8 candidates revealed two distinct expression profiles and two molecules were further validated by real-time PCR. The predicted pre-microRNA sequence in this study provides a potentially interesting insight into the in vivo function of porcine mlncRNAs and our findings suggest that they play key biological roles in Sus scrofa.     

葡萄悬浮细胞长期培养过程中不同继代条件下花青素生产的不稳定性

为了深入研究植物细胞培养生产次生代谢产物不稳定性的机制,以葡萄细胞作为模式体系,研究悬浮培养过程中花青素合成的不稳定性。除了用常规的花青素总含量来表征花青素的生物合成之外,还采用HPLC测定花青素不同组分的含量。结果表明,在长期的继代培养过程中,不仅花青素的含量而且花青素的组成也表现出明显的不稳定性。首次采用了不稳定系数 (δ) 和因素得分 (Factor scores) 来表征植物细胞培养过程中次生代谢生产的不稳定性。培养条件对花青素生物合成的影响实验结果表明,继代周期和接种量均能诱发次生代谢的不稳定性表达,其中接种量的影响相对更大。在考察的 (6.5 d,2.00 g),(7 d,2.00 g),(7.5 d,2.00 g),(7 d,1.60 g) 和 (7 d,2.40 g) 五种不同的继代周期和接种量组合条件中,7 d继代周期和1.60 g接种量最有利于保持花青素的稳定生产。     

青霉素酰化酶固定化前后动力学行为的比较

在优化的固定化条件下,通过戊二醛交联直接将青霉素酰化酶固定化。在优化的环境条件下测定游离酶和两种固定化酶的动力学常数。结果表明,尽管固定化酶的米氏常数增大,但产物抑制作用减弱,裂解青霉素的实验结果表明,固定化酶更适合在工业上应用。     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.