1,3-β-Glucanases fromStreptomyces sp.1228 lytic enzyme system

Summary Four 1,3--glucanases GI, GII, GIV and GVIII from a culture filtrate ofStreptomyces sp. 1228 were purified by anion exchange chromatography using DEAE-Sepharose Cl-6B or DEAE-Cellulose, gel filtration on Bio-Gel P-200 or Sephacryl S-200, Amicon ultrafiltration and preparative PAGE. The M_r of these enzymes were 19000, 74000, 78000 and 56000 respectively. The glucanase GVIII consisted of two subunits. The optimal catalytic activity of the purified preparations was at 50–55°C and pH 5.5–6.0. The enzymes were also most stable at this pH. Both glucanases GI and GVIII were characterized by high thermostability. The glucanases showed different affinities towards laminarin with Km values of 6.65 x 10^–5 mol/l for GI, 2.35 x 10^–4 mol/l for GII, 8.1 x 10^–5mol/l for GIV and 8.1 x 10^–4mol/l for GVIII. The presence of metal ions was not required for activity of these enzymes but thiol groups increased their activity. D-glucono--lactone did not inhibit the enzymes.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Co-Localization of β-1,3-Glucanases and Callose During Somatic Embryogenesis in Cichorium

During direct somatic embryogenesis in leaves of Cichorium hybrid clone ‘474’, 38 kDa β-1,3-glucanases are accumulated in the culture medium of the embryogenic hybrid to a higher level when compared with a non-embryogenic cultivar. In the same time, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. This callosic deposition disappeared as embryos grew. Callose consisted of β-1,3-glucan linkages and so represented a possible substrate for β-1,3-glucanases. Using immunolocalization experiments, we demonstrated that from the three types of callose deposits observed during the culturing of Cichorium leaf explants, only the callose present in the walls surrounding reactivated cells seemed specifically related to somatic embryogenesis. Moreover, callose and the 38-kDa β-1,3-glucanases were co-localized dispersed throughout the thick and swelled walls of reactivated cells and embryo cell walls. This suggests that callose and β-1,3-glucanases are implicated in the process of somatic embryogenesis since they were always detected in or quite near embryogenic and embryo cell. This also suggested that β-1,3-glucanases could be involved in the degradation of this callose.Key Words: β-1,3-glucanases, callose, Cichorium, immunolocalizations, somatic embryogenesis     

Inbreeding Alters Activities of the Stress-Related Enzymes Chitinases and β-1,3-Glucanases

Pathogenesis-related proteins, chitinases (CHT) and β-1,3-glucanases (GLU), are stress proteins up-regulated as response to extrinsic environmental stress in plants. It is unknown whether these PR proteins are also influenced by inbreeding, which has been suggested to constitute intrinsic genetic stress, and which is also known to affect the ability of plants to cope with environmental stress. We investigated activities of CHT and GLU in response to inbreeding in plants from 13 Ragged Robin (Lychnis flos-cuculi) populations. We also studied whether activities of these enzymes were associated with levels of herbivore damage and pathogen infection in the populations from which the plants originated. We found an increase in pathogenesis-related protein activity in inbred plants from five out of the 13 investigated populations, which suggests that these proteins may play a role in how plants respond to intrinsic genetic stress brought about by inbreeding in some populations depending on the allele frequencies of loci affecting the expression of CHT and the past levels of inbreeding. More importantly, we found that CHT activities were higher in plants from populations with higher levels of herbivore or pathogen damage, but inbreeding reduced CHT activity in these populations disrupting the increased activities of this resistance-related enzyme in populations where high resistance is beneficial. These results provide novel information on the effects of plant inbreeding on plant-enemy interactions on a biochemical level.     

The Physiology and Molecular Biology of Plant 1,3-β-D-Glucanases and 1,3;1,4-β-D-Glucanases

This review covers the physiology and molecular biology of the plant β-glucanases possessing either endo-1,3-β-D-glucanase (EC 3.2.1.39) or endo-1,3;1,4-β-D-glucanase (EC 3.2.1.73) activity. These β-glucanases are structurally related enzymes that are believed to be involved in many important aspects of plant physiology and development, such as germination, growth, defense against pathogens, flowering, cellular and tissue development and differentiation, and probably other roles. They also are regulated by numerous plant hormones, biotic and abiotic elicitors and stresses, and they exhibit complex tissue- and developmental-specific gene expression.     

β-1,3-葡聚糖酶的研究和应用

β-1,3-葡聚精酶的生产菌种由冻土毛霉经紫外线诱变获得,据正交试验确定培养条件。经硫酸铵盐析和SephadexG-100柱层析,酶得到纯化,并对酶的性质进行了研究。酶对酵母自溶有明显促进作用,使自溶产物的游离氨基氮含量、还原糖含量、总蛋白质得率、干物质得率均得以提高。     

β-1,3-Glucanase and dimorphism in Paracoccidioides brasiliensis

Mycelial and yeast forms of P. brasiliensis were tested for several glucohydrolases. In addition to high levels of -blucanases, low amounts of -glucanase, chitinase and maltase were found. Tests for invertase, amylase and lactase were negative. The levels of -1,3-glucanase were higher in the mycelial form. The shift to the mycelial phase correlated with an increase in the levels of -1,3-glucanase. The enzyme was present in the cytoplasm, cell wall and culture medium. The extracellular enzyme was purified 42 fold by ammonium sulphate precipitation and gel filtration. Maximal activity was obtained at 60°C and pH of 5.0 acetate buffer or pH 6.0 (phosphate buffer). Its K _m was 0.205 mg/ml. The cell wall-bound enzyme showed a higher temperature optimum. Optimum pH and K _m were also slightly different. Following treatment of the cell walls with chitinase, -1,3-glucanase was released into the medium.     

混合真菌发酵制备支化β-1,3-葡寡糖

真菌来源的β-1,3-葡聚糖被证实具有很好的抗肿瘤、免疫调节功能,但其过大的分子量和水不溶性影响其功能和应用,研究表明低分子量结构的葡寡糖具有更好的水溶性和生物活性。因此,本研究将能产这种结构多糖的真菌与能产内切β-1,3-葡聚糖酶的哈茨木霉混合发酵,从而得到低分子量的葡寡糖,并保留支链结构。本研究建立了齐整小核菌-哈茨木霉与裂褶菌-哈茨木霉混合发酵体系,制得了2种支化β-1,3-葡寡糖,分别为小核寡糖和裂褶寡糖。通过发酵条件优化,小核寡糖摇瓶产量最高为4.53 g/L,在7 L发酵罐中的产量最高为9.94 g/L,其中小核寡糖聚合度(DP)为5～12,裂褶寡糖DP为5～15。抗氧化实验表明,小核寡糖和裂褶寡糖都有较好的活性,且裂褶寡糖优于小核寡糖。本研究成功制得2种支化β-1,3-葡寡糖,并证明具有良好的水溶性和抗氧化活性。     

几丁质酶和β-1,3-葡聚糖酶基因研究进展

几丁质酶和β-1,3-葡聚糖酶是重要的水解酶,实践证明,转几丁质酶和β-1,3-葡聚糖酶基因植物能比较有效地抵抗真菌侵染。本综述了几丁质酶和β-1,3-葡聚糖酶结构分类、抗真菌机理．及其近年来在抗黄曲霉病研究中的应嗣研究,并对今后的研究及应用进行了预测。     

生防木霉菌β-1,3-葡聚糖酶活性研究

通过采取还原糖法对生防木霉菌真菌2号和真菌4号菌株在不同发酵时间产β-1,3-葡聚糖酶活性进行测定,对其产β-1,3-葡聚糖酶特性进行初步研究。结果表明,同一菌株在不同发酵时间β-1,3-葡聚糖酶活性大小变化的趋势大致相同,并均在培养72 h时β-1,3-葡聚糖酶活性达到最大,在相同发酵时间,真菌2号菌株比真菌4号菌株的β-1,3-葡聚糖酶活性要高。     

木霉β-1,3-葡聚糖酶的分离纯化

目的:对木霉菌株LE02所产β-1,3-葡聚糖酶的分离纯化方法进行研究。方法:粗酶液分别用硫酸铵、乙醇和丙酮进行沉淀,再用DEAE-Sepharose CL-6B离子交换层析进一步分离纯化,并用SDS-PAGE法测其分子量。结果:硫酸铵分段盐析法沉淀酶蛋白的效果优于乙醇和丙酮沉淀;盐析得到的酶蛋白经透析浓缩后,再经DEAE-Sepharose CL-6B层析分离,可得到单一酶蛋白,总酶活回收率达78.71%,比酶活达到689.9U/mg,提高了53.74倍,经SDS-PAGE法测得该β-1,3-葡聚糖酶的分子量为80.137kDa。结论:采用硫酸铵分段盐析和离子交换层析法可获得电泳纯的β-1,3-葡聚糖酶,且酶活回收率高。     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

细菌编码β-1,3-1,4-葡聚糖酶催化活性优化方法

β-1,3-1,4-葡聚糖酶是一类专一降解β-葡聚糖的内切水解酶。高效β-葡聚糖酶在啤酒酿造工业上具有十分重要的应用价值。目前,研究较多的β-1,3-1,4-葡聚糖酶主要来源于细菌。文中概述了细菌编码β-1,3-1,4-葡聚糖酶的分子生物学性质,并且从蛋白分子改造、表达调控和发酵条件优化三方面阐述了其催化活性提高的方法和成果。     

1,3-β-Glucan synthase from Citrus phloem

1,3-β-Glucan synthase activity has been demonstrated in particulate fractions of bark extracts from Mexican lime. With respect to substrate, the enzyme kinetics did not conform to the Michaelis-Menten equation. The value of the Hill coefficient was 1.2 and S_0.5 is 1.1 mM. The enzyme had an optimum pH of 7.5. Maltose, sucrose, and especially cellobiose and glucose, were enzyme activators when tested at physiological concentrations. In the presence of 15 mM MgCl₂ the enzymic activity was stimulated at 10 μM UDP-glucose but decreased at 1 mM UDP-glucose, suggesting a minor 1,4-β-glucan synthase activity.     

连续温度变化对β-1,3-葡聚糖酶酶解酵母β-葡聚糖的影响

为了探索反应温度对产物组分的影响,利用自制连续变化的温度梯度实验装置,研究了22 ℃~60 ℃ (±0.1 ℃) 区间内温度对一内切β-1,3-葡聚糖酶酶解酵母β-葡聚糖的影响,获得了酶解过程多点温度特性数据。分析表明：该酶酶解酵母β-葡聚糖的活化能为84.17 kJ/mol;以产物积累表示的最适酶解温度随时间延长呈指数下降;酶解产物组分受温度的影响,低温较高温获得的寡糖链长,高温区大于46 ℃可以获得以昆布二糖、昆布三糖为主的组分,而低温区小于30 ℃可以获得昆布五糖及更大分子量的产物。研究结果可为寡糖     

Two Pine Endo-β-1,4-Glucanases Are Associated with Rapidly Growing Reproductive Structures

Two cDNA clones encoding endo-β-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (Pinus radiata cellulase 1) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.The growth and development of floral organs involves many physiological processes, including modifications to the cell wall. EGases (cellulases) may play roles in cell wall loosening, which is required for expansion or major cell wall disruption. Cell expansion has been reviewed by Cosgrove (1993), who demonstrated that acid-induced extension of cell walls appears to require the activity of expansins. Wall-modifying enzymes such as endoxyloglucan transferase, and wall-degrading enzymes such as glucanases, are also likely to be involved, but there is no evidence that they can cause extension of isolated walls. Major cell wall disruption also occurs at several steps in the development of flower reproductive organs (del Campillo and Lewis, 1992). The callose wall that protects the meiotic cells is broken down during early pollen differentiation, releasing the microspores into the anther locule. Later, the tapetum begins to break down and the cytoplasm is released into the locule. Finally, the release of the mature pollen grains from the anthers is facilitated by the formation of a fissure, the stomium. Similarly, during pollen-stigma interactions, cell wall loosening of the papillary cells at the surface of the stigma has been reported. EGases have been shown to accumulate in anthers of beans and sweet peas in a developmentally regulated manner and may be involved in the cell wall disruption required for pollen differentiation.Plant EGases typically lack the ability to degrade microcrystalline cellulose in vitro. Bacterial EGases, however, are able to degrade cellulose. Therefore, all EGases are sometimes referred to as cellulases. Genes encoding EGases have been isolated from many different plant species, including tomato (Lycopersicon esculentum) (Lashbrook et al., 1994; Milligan and Gasser, 1995), elder (Sambucus nigra) (Taylor et al., 1994), pea (Pisum sativum) (Wu et al., 1996), soybean (Glycine max) (Kemmerer and Tucker, 1994), Arabidopsis (Ferl, 1995), poplar (Populus alba) (Nakamura et al., 1995), kidney bean (Phaeseolus vulgaris) (Tucker and Milligan, 1991), and avocado (Persea americana) (Tucker et al., 1987). Some of these enzymes, including TomCel2 (Lashbrook et al., 1994), EGL1 (Wu et al., 1996), and AvoCel1 (Tucker et al., 1987), are primarily associated with fruit ripening. Another group, including BAC (Tucker and Milligan, 1991), SAC1 (Kemmerer and Tucker, 1994), TomCel1 (Lashbrook et al., 1994), and JET1 (Taylor et al., 1994), are associated with abscission. Yet another group of enzymes appears to be expressed predominantly in rapidly expanding tissues. Expression of TPP18 (Milligan and Gasser, 1995), which is identical to Cel4 (Brummell et al., 1997), occurs in growing pistils of tomato flowers, and to a lesser extent in stamens, but not in fully expanded flower parts. Expression is also high in the growing zones of etiolated hypocotyls and in expanding leaves. Here we report on the expression of two EGases cloned from reproductive structures of radiata (Monterey) pine (Pinus radiata).As in angiosperms, the “flowering” of radiata pine starts with the transition of an undetermined axillary apex into a determinant reproductive apex, which develops into the strobili (cones). Reproductive buds are simple because they normally contain a single strobilus and no leaves. Mature male (pollen) cones are small (1–2 cm in length) and are made up of spirally arranged microsporophylls, each bearing two microsporangia (pollen sacs). The microspores develop into four-celled pollen grains. Female (seed) cones consist of an axis, which bears a specially arranged series of small appendages termed bracts. In the axil of each bract is a thick scale upon which two ovules are borne, attached to the adaxial surface of the cone scale near the base. Because the ovuliferous scales are lateral structures subtended by a bract, the entire cone is a “compound” strobilus, and may be compared in this respect with an inflorescence. Such female axes generally are located at the top of the adult tree, whereas male cones are located farther down the stem and contain only microsporophylls.A few genes have previously been cloned from various parts or stages of developing radiata pine cones, including the cDNAs encoding genes preferentially expressed in immature female and male cone buds. Homologs of the angiosperm late-flowering, meristem-identity, and organ-identity genes regulate development of unisexual cones in the conifer radiata pine (Mouradov et al., 1996, 1997a, 1997b). MADS box genes have also been cloned from another conifer, Norway spruce (Picea abies) (Tandre et al., 1995). Two different cDNAs with homology to legumins have been isolated from fertilized ovules of white pine (Pinus strobus), but are not expressed in unfertilized ovules (Baker et al., 1996). Several cDNA clones encoding seed-storage proteins have also been isolated from Douglas fir and interior spruce megagametophytes (Newton et al., 1992; Leal and Misra, 1993). Mature megagametophytes have been used for many years to study isozyme variation, and are commonly used as sources of DNA for genome mapping because of their haploid condition.To gain a greater understanding of the genes involved in the formation of pine reproductive structures, we constructed a cDNA library from immature female cones and differentially screened against vegetative buds. Here we report on the cloning, sequencing, and characterization of two of those genes, PrCel1 and PrCel2, that have very high homology to each other and to previously cloned EGases.     

细菌β-1,3-1,4-葡聚糖酶分子改造和表达策略

β-1,3-1,4-葡聚糖酶作为一种饲料和食品添加剂有着广泛用途。迄今在杆菌、梭菌、瘤胃细菌、真菌、高等植物中都发现了β-1,3-1,4-葡聚糖酶。综述了细菌来源β-1,3-1,4-葡聚糖酶的性质、结构、分子改造与表达研究进展。