黄杆菌菌株RC2-3基因组生物信息学分析及高效降解岩藻多糖功能基因的挖掘

以从海带中筛选获得的一株具有降解岩藻多糖能力的黄杆菌菌株RC2-3为研究对象，该菌株产的岩藻多糖酶可以高效降解不同来源的岩藻多糖。为进一步探究菌株RC2-3降解岩藻多糖的机制，推动岩藻寡糖的酶法生产，采用Illumina测序技术对菌株RC2-3进行基因组测序、基因功能注释和碳水化合物活性酶注释以及岩藻多糖降解相关基因的生物信息学分析。结果表明，黄杆菌菌株RC2-3基因组全长3 414 532 bp,共编码2 967个基因，GC含量为30.92%。经碳水化合物活性酶数据库注释获得213个基因，与岩藻多糖降解有关的包括7个岩藻糖结合结构域的基因；2个β-D-岩藻糖苷酶(EC 3.2.1.38)基因；2个属于GH141家族的α-L-岩藻糖苷酶(EC 3.2.1.51)基因；12个属于GH29家族的α-1, 3/1, 4-L-岩藻糖苷酶(EC 3.2.1.111)基因；9个属于GH95家族的α-1, 2-L-岩藻糖苷酶(EC 3.2.1.63)基因。此外，通过与已报道的蛋白序列比对发现，岩藻多糖酶基因RC2.3＿GM001247编码的蛋白序列与FunA蛋白序列同源性达到70.98%,岩藻多糖酶...     

海洋真菌Dendryphiella Arenaria TM94产岩藻多糖酶发酵及酶学性质

通过对海洋真菌Dendryphiella Arenaria TM94产岩藻多糖酶发酵培养基组成及工艺条件的研究,并经正交实验,得到较优培养基配方为：麸皮3％,海带1％,葡萄糖0．3％,复合氮源为NaNO33％,(NH4)2SO40．5％,蛋白胨3％和酵母浸膏0．6％时,培养基的起始pH为6．5。在恒温振荡器中以120rpm的速度振荡培养,岩藻多糖酶活可达24．3IU／ml。     

Effect of Enzyme Preparation from the Marine Mollusk Littorina kurila on Fucoidan from the Brown Alga Fucus distichus

A fucoidanase preparation from the marine mollusk Littorina kurila cleaved some glycosidic bonds in fucoidan from the brown alga Fucus distichus, but neither fucose nor lower oligosaccharides were produced. The main product isolated from the incubation mixture was a polysaccharide built up of disaccharide repeating units -->3)-alpha-L-Fucp-(2,4-di-SO3(-))-(1-->4)-alpha-L-Fucp-(2SO3(-))-(1-->, the structure coinciding with the idealized formula proposed for the initial substance. A polymer fraction with the same carbohydrate chain but sulfated only at positions 2 and nonstoichiometrically acetylated at positions 3 and 4 of fucose residues was isolated as a minor component. It is suggested that the native polysaccharide should contain small amounts of non-sulfated and non-acetylated fucose residues, and only their glycosidic bonds are cleaved by the enzyme. The enzymatic hydrolysis showed that irregular regions of the native polysaccharide containing acetylated and partially sulfated repeating units were assembled in blocks.     

A comparative study of specificity of fucoidanases from marine microorganisms and invertebrates

Specificities of actions of fucoidanases from the marine microorganism Pseudoalteromonas citrea KMM 3296 and the marine mollusk Littorina kurila were studied. The enzymes possess similar specificities and catalyze the cleavage of accessible α-(1→3)-fucoside bonds in fucoidans with highly sulfated α-(1→4; 1→3)-L-fucooligosaccharides. A high degree of sulfation of the fucose residues in fucoidans makes α-(1→3)-L-fucoside bonds inaccessible for the action of the studied enzymes. The maximum degree of cleavage of fucoidan was achieved by the fucoidanase from the marine bacterium Pseudoalteromonas citrea KMM 3296.     

Isolation and Culture of a Marine Bacterium Degrading the Sulfated Fucans from Marine Brown Algae

Fucoidans are matrix polysaccharides from marine brown algae, consisting of an α-l-fucose backbone substituted by sulfate-ester groups and masked with ramifications containing other monosaccharide residues. In spite of their interest as biologically active compounds in a number of homologous and heterologous systems, no convenient sources with fucanase activity are available yet for the degradation of the fucalean algae. We here report on the isolation, characterization, and culture conditions of a bacterial strain capable of degrading various brown algal fucoidans. This bacterium, a member of the family Flavobacteriaceae, was shown to secrete fucoidan endo-hydrolase activity. An extracellular enzyme preparation was used to degrade the fucoidan from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of α-1→3-l-fucopyranose-2-sulfate-α-1→4-l-fucopyranose-2,3-disulfate, with the 3-linked residues at the nonreducing end.     

Isolation and Characterization of a Fucoidan-Degrading Marine Bacterial Strain and Its Fucoidanase

A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in our laboratory. The strain was gram-negative, ubiquinone 8 was the predominant respiratory quinone, and the GC-content of its genomic DNA was 36%. The cells of the strain were rod-shaped (2.0 m long × 1.0 m wide), and each cell was motile by means of one polar flagellum. Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Alteromonadaceae. It produced a type of extracellular fucoidanase, an endosulfated fucan-digesting enzyme. The enzyme was purified with 3500-fold purity at 12.0% yield. Optimum conditions for the enzyme reaction were approximately pH 6.5 to 8.0 and temperature 30° to 35°C. The enzyme was activated by calcium ions, and maximum activity was observed in the presence of greater than 30 mM calcium ion.     

海洋真菌岩藻多糖酶的固态发酵条件研究*

本文对海洋真菌Dendryphiella arenaria(TM94)岩藻多糖酶的固态发酵条件进行了研究,主要内容包括碳源、氮源、添加物、起始pH、接种量及温度等.固态发酵最佳培养基组成麸皮7.5g,葡萄糖0.5g,海带粉0.6g,NaNO3为4g/L最佳培养条件为培养温度28℃,起始pH6,接种量3ml(孢子浓度为106个/ml).在28℃培养24h,酶活力可达35.5IU/g干培养基,比活力为1.39IU/mg蛋白质.对岩藻多糖酶酶学性质也进行了研究.