厌氧菌Sunxiuqinia sp. SH-52外切型海藻酸裂解酶的异源表达及酶学特征

【背景】目前已报道的海藻酸分解菌多数为好氧菌,未见有关厌氧菌的报道。从分离的海藻酸分解菌中表征的海藻酸裂解酶大多为内切型海藻酸裂解酶,外切型较少。【目的】研究来自厌氧海藻酸分解菌的海藻酸裂解酶基因,表征新型海藻酸裂解酶并阐明其酶学性质,为海藻酸裂解酶的多样性和微生物降解海藻酸机制提供理论依据。【方法】对来自厌氧海藻酸分解菌Sunxiuqinia sp. SH-52的海藻酸裂解酶SHA-4编码基因进行克隆,分析基因序列,构建重组质粒PGEX-4T-1-SHA-4并在大肠杆菌中实现异源表达,经纯化后对其酶学性质及降解特征进行研究。【结果】该酶在28°C、用0.1 mmol/L IPTG (异丙基-β-D-硫代半乳糖苷)条件下诱导6 h达到最大表达量,纯化后酶的比活力达到21 U/mg。酶学性质分析表明SHA-4的最适温度为37°C;最适pH为7.5;对PolyMG (杂聚古罗糖醛酸-甘露糖醛酸嵌段)具有底物偏好性;Na+对该酶的活性具有抑制作用,而金属离子Cu~(2+)具有明显促进作用,使活性提高了约168%;SHA-4催化海藻酸的Km值为2.5 mg/mL,Vmax为8.7 mg/(mL·min);SHA-4为外切型海藻酸裂解酶,降解海藻酸终产物为单糖。【结论】异源表达了来自一株厌氧海藻酸分解菌Sunxiuqiniasp.SH-52的海藻酸裂解酶SHA-4,该酶是PL6家族中第一个对PolyMG有底物偏好性的外切型海藻酸裂解酶,而且活性较高,作为工具酶有很好的应用前景,为海藻酸降解机制的探索提供了新的线索。     

SYSTEMATIC REVISION OF THE GENERA LIAGORA AND IZZIELLA (LIAGORACEAE,RHODOPHYTA) FROM TAIWAN BASED ON MOLECULAR ANALYSES AND CARPOSPOROPHYTE DEVELOPMENT,WITH THE DESCRIPTION OF TWO NEW SPECIES1

Some Liagora and Izziella distributed in Taiwan display a wide range of morphological variation and can be difficult to distinguish. To clarify species concepts, we applied DNA sequence analyses and examined carposporophyte development in detail. These studies revealed two new species, which are described herein as Izziella hommersandii sp. nov. and Izziella kuroshioensis sp. nov. I. kuroshioensis superficially resembles Izziella formosana and Izziella orientalis in that its involucral filaments subtend rather than surround the lower portion of the gonimoblast mass (= Izziella type) and a fusion cell is formed from cells of the carpogonial branch, but it can be separated by differences in the cell numbers and branching pattern of the involucral filaments, as well as thallus morphology. In contrast to other species that also bear short lateral branchlets, I. hommersandii is unique in possessing a mixture of short and long involucral filaments, a phenomenon not reported before. The length of the involucral filaments is species specific among species of Izziella and contrasts to the behavior of the involucral filaments after fertilization in species such as “Liagora”setchellii [= Titanophycus setchellii comb. nov.], in which the filaments completely envelop the gonimoblast. In addition, the cells of the carpogonial branch in Titanophycus do not fuse after fertilization to form a fusion cell. Thus, a combination of characters with respect to the behavior of the carpogonial branch and the involucral filaments after fertilization is very useful for delineating species boundaries in Izziella and for separating Titanophycus from Izziella and Liagora.     

Coccidian Parasites (Apicomplexa: Eimeriidae) of Nerodia spp. (Serpentes: Colubridae), with a Description of a New Species of Eimeria

Eimeria conanli n. sp. (Apicomplexa: Eimeriidae) is described from intestinal contents and feces of Nerodia erythrogaster transversa and N harteri harteri from northcentral Texas. Oocysts of the new species are ellipsoid in shape. 17.9 × 13.0(15–21 × 12–15) μm, with a smooth, thin, single-layered wall; shape index 1.4 (1.2–1.5). One to several (usually 2) polar granule(s) and an oocyst residuum are present, but a micropyie is absent. Sporocysts are elongate, 12.9 × 5.2 (13–15 × 5–6) -m, apparently without a true Stieda body structure. Each sporoeyst contains an ellipsoid residuum, 3.9 × 3.2 (3–6 × 2–4) μm, and elongate sporozoites, 11.4 × 2.5 (10–14 × 2–3) μm in situ, each with a spherical or subspherical anterior refractile body and spherical to ellipsoid posterior refractile body. In addition to the new species, oocysts of 4 previously described eimerians from colubrid snakes were found in these hosts.     

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu²⁺ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO₄. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO₄. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU g^–1 total protein) when the strain was grown in the absence of CuSO₄. However, the activity was stimulated (25.8 mU g^–1 total protein) when cells were grown with 1 mM CuSO₄ and further enhanced to 110 mU g^–1 total protein with 10 mM CuSO₄. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO₄ in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO₄ in strain N6.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessions including Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions. Received: 5 April 2001 / Accepted: 13 July 2001     

3-羟基丙酸高产菌株的筛选及诱变选育

【目的】3-羟基丙酸是一种重要的化学平台化合物,期望得到一株能够高产3-羟基丙酸的菌株。【方法】从土壤及粪便筛选并对得到的菌株进行鉴定和复合诱变。【结果】得到了一株能够利用丙酸发酵生产3-羟基丙酸的酵母Y-11,经生理生化鉴定及18S rDNA序列分析确定其为Candida sp.(假丝酵母)。以Y-11为出发菌株,经紫外-亚硝基胍-60Coγ复合诱变得到了突变性状稳定且可遗传的高产菌株5-13B,其3-羟基丙酸的产量为11.78 g/L,是出发菌株的2.46倍。【结论】对出发菌株和突变株的发酵特性进行了比较,结果表明突变株的3-羟基丙酸产量、对底物丙酸的转化率、产物3-羟基丙酸的积累性能及丙酸的耐受性均优于出发菌株。     

Production of chitinolytic enzymes from a novel species ofAeromonas

A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source. The isolated bacterium, designated asAeromonas sp No. 16 exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml^–1. A slightly higher enzymatic activity of 1.5 U ml^–1 was obtained when cultivation was carried out in a 5-liter jar fermentor using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of -N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.