Evaluation of an endo-β-mannanase produced by Streptomyces ipomoea CECT 3341 for the biobleaching of pine kraft pulps

An endo-beta-mannanase (EC 3.2.1.78) from Streptomyces ipomoea CECT 3341 was purified and applied to the biobleaching of pine kraft pulps. The maximum level of endo-beta-mannanase activity (0.6 units ml(-1)) was achieved after 4 days of growth in a medium containing locust bean gum and yeast extract. Zymograms revealed mannanase bands (Man) with high and low electrophoretic mobility on the second and seventh days of incubation (Man1, Man3) and three bands of high, medium and low mobility from the third to sixth days of growth (Man1, Man2, Man3). Although these exhibited different molecular masses, their amino-terminal sequences were identical. The action of proteases detected in the culture supernatant could be responsible for such events, suggesting that only one endo-beta-mannanase is produced by S. ipomoea. The purified Man3 exhibited a molecular mass of 40 kDa, an isoelectric point of 4.0 and an optimal temperature and pH reaction of 55 degrees C and 7.5, respectively. It was strongly inhibited by Ag+, Hg2+, Al3+ and Fe3+, and was strongly activated by Mn2+. The ability of the purified endo-beta-mannanase to improve the bleachability of pine kraft pulp, when applied with alkaline extraction, was demonstrated by an increase in the pulp brightness (1.7%, using the International Standards Organisation's test) and an absence of variations in the viscosity values. A relationship between the increase in pulp brightness and the presence of manganese in the pulps could be established.     

Properties of an α-1,3-Glucanase from Streptomyces sp. KI-8

Srome properties were examined of purified α-l,3-glucanase isolated from the culture supernatant of the soil microorganism Streptomyces KI-8.

The optimum pH and temperature were pH 5.4 and 60°C, respectively. The α-1,3-glucanase was stable up to 50°C on heating for 10 min. This enzyme hydrolyzed the substrate α-l,3-glucan into glucose and nigerose by an endo-type of action. Nigerotriose, nigerotetraose and nigeropentaose were hydrolyzed into glucose and nigerose, whereas nigerose was not attacked. The degree of hydrolysis of pseudonigeran, Lentinus α-1,3-glucan, mutan IG-1 (less soluble fraction) and IG-2 (more soluble fraction) by the α-1,3-glucanase were 28.5%, 14.3%, 8.8% and 10.0%, respectively. Km values (mg/ml) for pseudonigeran, Lentinus α-l,3-glucan, mutan IG-1 and IG-2 were 1.12, 1.98, 8.00 and 5.00. The enzyme solubilized 50 to 80% of mutan by concerted action with dextranase.     

Biochemical characteristics of two endo-β-1,4-xylanases produced byPenicillium capsulatum

Summary Two endo--1,4-xylan xylanohydrolases (EC 3.2.1.8), XynA and XynB, from solid-state cultures ofPenicillium capsulatum, were purified to apparent homogeneity as judged by electrophoresis and isoelectric focusing. Each is a single subunit glycoprotein. XynA containing 97 mol carbohydrate·mol^–1 protein, while XynB contains 63 mol·mol^–1.M _r and pI values are 28 500, 5.0–5.2 (XynA) and 29 500, 5.0–5.2 (XynB), respectively. Both enzymes are most active at pH 4 and 47–48°C, and have half-lives of 32 min (XynA) and 13 min (XynB) at pH 4, 60°C. Each form catalyzed the hydrolysis of a variety of xylans, albeit with different degrees of efficiency. In addition, XynB catalyzed extensive degradation of barley -glucan, CM-cellulose and, to a lesser extent, lichenan, but kinetic parameters indicate that it is primarily a xylanase. The products of hydrolysis of various xylans and xylopentaose differed for each enzyme and ranged from xylose to xyloheptaose depending on the substrate used. Each enzyme is endo-acting and has transferase as well as direct hydrolase activity. Inactivation byN-bromosuccinimide indicated the possible involvement of tryptophan in binding and/or catalysis.     

Bacteriolytic enzymes from Staphylococcus aureus. Properties of the endo-β-N-acetylglucosaminidase

An extracellular bacteriolytic endo-β-N-acetylglucosaminidase has been purified and its specificity of action has been investigated (Wadström & Hisatsune, 1970a,b). Some enzymic properties, such as optimum pH for enzyme activity on whole cells and cell walls of Micrococcus lysodeikticus and Staphylococcus aureus and optimum pH for stability, have been studied. The activity was maximum in 0.05m-tris–hydrochloric acid buffer, pH7.0. A higher ionic strength inhibited cell-wall hydrolysis. Since the crude and purified enzymes were found to be unstable on storage, the stabilizing and inhibiting effects of several compounds were investigated. Several heavy metal ions inactivated the enzyme at very low concentrations. Thiol compounds stabilized and thiol-reacting compounds partly inhibited the activity. Crude and purified glucosaminidase was found to be heat-stable at acidic pH and unstable at alkaline pH as previously found for several lysozymes (endo-β-N-acetylmuramidases). Other properties of the staphylococcal enzyme and hen''s-egg-white lysozyme have been compared, since the modes of action of the two are quite similar (Wadström & Hisatsune, 1970b).     

Cloning, characterization, and antifungal activity of an endo-1,3-β-d-glucanase from Streptomyces sp. S27

An endo-1,3-β-d-glucanase gene, designated as bglS27, was cloned from Streptomyces sp. S27 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene contains 1,362 bp and encodes a protein of 453 amino acids with a calculated molecular mass of 42.7 kDa. The encoded protein comprises a catalytic module of glycosyl hydrolase family 16, a short glycine linker region, and a family 13 carbohydrate-binding module. The purified recombinant enzyme (BglS27) showed optimal activity at 65°C and pH 5.5 and preferentially catalyzed the hydrolysis of glucans with a β-1,3-linkage using an endolytic mode of action. The specific activity and K _m value of BglS27 for laminarin were 236.0 U mg^–1 and 1.89 mg ml^–1, respectively. In antifungal assay, BglS27 had the ability to inhibit the growth of phytopathogenic fungi Rhizoctonic solani and Fusarium oxysporum and some mycotoxin-producing fungi Fusarium crookwellense and Paecilomyces variotii. These favorable properties make BglS27 a good candidate for utilization in biotechnological applications such as plant protection, feed, and food preservation.     

Cloning and enzymatic characterization of four thermostable fungal endo-1,4-β-xylanases

Endo-1,4-β-xylanases (EC 3.2.1.8) hydrolyze the 1,4-β-D-xylosidic linkages in xylans, the most abundant hemicellulose in plant cell walls. Xylanase enzymes have numerous industrial applications, including the manufacturing of animal feed, bread, juice and wine, pulp and paper, and biofuels. In this study, two glycosyl hydrolase family 10 members designated GtXyn10A and GtXyn10B and two glycosyl hydrolase family 11 members, OpXyn11A and CcXyn11C, were functionally expressed and subjected to biochemical characterization. The K _M , V _max, and k _cat values of the four xylanases, determined using birchwood xylan, ranged from 0.27 to 1.1 mg/mL, 130 to 980 μmol/min/mg, and 109 to 344 s^?1, respectively, where OpXyn11A gave the highest and GtXyn10B the lowest values for all three parameters. Substrate specificity studies and analysis of the products released during the degradation of xylo-oligosaccharides and three types of xylan revealed significant differences in catalytic properties, particularly between OpXyn11A and the other xylanases and between the family 10 and the family 11 xylanases. Molecular modeling suggests that the unique substrate specificity of OpXyn11A can be attributed to the presence of a serine rather that an asparagine or aspartate residue at the +1 substrate binding site. Additionally, all four xylanases exhibited biochemical characteristics of interest for various commercial applications.     

Purification and Some Properties of β-Glucosidase from Streptomyces sp

β-Glucosidases I, II, and III were isolated from the culture filtrate of a Streptomyces sp. by ammonium sulfate fractionation, hydroxylapatite column chromatography, filtration on Bio-Gel P-100, and DE-52 column chromatography. β-Glucosidase III had a single active band on disc-gel electrophoresis. Its optimum pH and temperature for activity were 6.0 and 60°C, respectively. The isoelectric point and molecular weight of the enzyme were pH 4.5 and 45,000, respectively. From an experiment using ¹⁴C-labeled glucose, gentiobiose seemed to be formed from laminaribiose as isomaltose is formed from maltose by fungal α-glucosidase. The enzyme showed transglucosylation and produced gentiobiose from β-gluco-disaccharides and 4-O-β-d-glucopyranosyl-d-manno-pyranose (epicellobiose). The enzyme acted on phenolic β-d-glucosides to produce unknown transfer products.     

The release of oligosaccharides from glycoproteins by endo-β-N-acetylglucosaminidase of Flavobacterium sp.

Endo-β-N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.     

Purification and Properties of Three Types of Xylanases Induced by Methyl β-Xyloside from Streptomyces sp.

When mycelia of Streptomyces sp. No. 3137 were cultivated in a medium containing methyl β-xyloside, xylanases (EC 3.2.1.8) were inductively produced into the medium. Three types of enzyme from the culture filtrate have been purified by ultrafiltration with DIAFLO UM-10, chromatography on DEAE-Sephadex A-25, gel filtration on Bio Gel P-100, and isoelectric focusing with Servalyt 6~8 or 9~11. The three purified enzymes, tentatively named X-I, X-II-A, and X-II-B, were homogeneous by Polyacrylamide gel electrophoresis at pH 4.3. The molecular weight of X-I was about 50,000 by SDS-polyacrylamide gel electrophoresis or gel filtration on Bio Gel P-100. The molecular weight of X-II-A and X-II-B were both approximately 25,000 by SDS-polyacrylamide gel electrophoresis and that of X-II-B was 25,680 by the sedimentation-equilibrium method. X-I had an isoelectric point at 7.10, and X-II-A and X-II-B had different isoelectric points, 10.06 and 10.26, respectively. The three enzymes were optimally active at 60~65°C and stable to 55°C. The optimal pH of X-I, X-II-A, and X-II-B were pH 5.5~6.5, 5.0~6.0, and 5.0~6.0, respectively. The ranges of two X-I’s pH stability (pH 1.5 ~ 11.5) were wider than that of X-I’s (pH 3.0 ~ 10.5). These purified preparations hydrolyzed xylotriose, xylotetraose, and xylan but not xylobiose, cellobiose, maltose, carboxymethyl cellulose, or soluble starch. Their actions were inhibited by Hg²⁺ and Fe³⁺ ions, sodium dodecyl sulfate, and N-bromosuccinimide.     

Properties of the deglycosylatedβ-fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524

Summary The carbohydrate moiety of -fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524 was largely removed by exposure of the enzyme to endo--N-acetylglucosaminidase F; the total carbohydrate content of the enzyme was decreased from 53% (w/w) to 15% (w/w). The stability of the deglycosylated enzyme at pH 4 to 7 and 40 to 50°C was decreased and theK _m value for sucrose was increased from 0.65 to 1.43 M. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is concluded that the carbohydrate moiety of -fructofuranosidaseP-2 contributes to the stability of the enzyme as well as its affinity for sucrose.     

Purification and properties of Acinetobacter sp. endo-β-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins

A novel endo-β-N-acetylglucosaminidase capable of acting on complex type sugar chains of glycoproteins was found in the culture broth of a bacterium which was isolated from soil and identified as Acinetobacter sp. The enzyme was purified to homogeneity on polyacrylamide gel electrophoresis by successive purification procedures involving ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G-150. Its molecular weight was about 35,000 on gel filtration. The optimum pH was 3.0–3.5, and the enzyme was stable in the pH range from 6–8. The enzyme had high activity on dansyl ovalbumin glycopeptide, and also could hydrolyze dansyl asialotransferrin glycopeptide and dansyl transferrin glycopeptide containing complex type sugar chains. The K_m value for dansyl asialotransferrin glycopeptide as the substrate of enzyme assay was 0.68 mM. The enzyme could release complex type sugar chains from intact asialotransferrin without the addition of any detergent.     

Genome sequence of Streptomyces sp. strain Tü6071

Streptomyces sp. Tü6071 is a soil-dwelling bacterium which has a highly active isoprenoid biosynthesis. Isoprenoids are important precursors for biopharmaceutical molecules such as antibiotics or anticancer agents, e.g., landomycin. Streptomyces sp. Tü6071 produces the industrially important terpene glycosides phenalinolactones, which have antibacterial activity against several Gram-positive bacteria. The availability of the genome sequence of Streptomyces sp. Tü6071 allows for understanding the biosynthesis of these pharmaceutical molecules and will facilitate rational genome modification to improve industrial use.     

Characterization of the bagremycin biosynthetic gene cluster in Streptomyces sp. Tü 4128

Bagremycin A and bagremycin B isolated from Streptomyces sp. Tü 4128 have activities against Gram-positive bacteria, fungi and also have a weak antitumor activity, which make them have great potential for development of novel antibiotics. Here, we report a draft genome 8,424,112 bp in length of S. sp. Tü 4128 by Illumina Hiseq2000, and identify the bagremycins biosynthetic gene cluster (BGC) by bioinformatics analysis. The putative bagremycins BGC includes 16 open reading frames (ORFs) with the functions of biosynthesis, resistance and regulation. Disruptions of relative genes and HPLC analysis of bagremycins production demonstrated that not all the genes within the BGC are responsible for the biosynthesis of bagremycins. In addition, the biosynthetic pathways of bagremycins are proposed for deeper inquiries into their intriguing biosynthetic mechanism.     

Isolation and properties of an endo-β-mannanase-producing Bacillus sp. LX114 capable of degrading guar gum

Endo-β-mannanase, catalyzing the random hydrolysis of β-1,4-mannosidic linkage in the backbone of (hetero) mannan, can increase feed conversion efficiency of animal feed or form functional mannanooligosaccharides. In this study, a gram-positive, straight-rod, facultative anaeorobic bacterium producing endo-β-mannanase was isolated from soil sample. The isolate only fermented glucose, galactose, sorbose, and raffinose to acid. The test in hydrogen sulfide production was positive. Combining the data acquired from phenotypic analysis and phylogenetic analysis based on 16S rRNA gene sequences, this strain presumably represented a novel species of the genus Bacillus and was designated as LX114. The strain LX114 could break down guar gum molecules, leading to a rapid decrease of the viscosity of guar gum solutions. Endo-β-mannanase activity was also detected in the culture supernatant. The isolate LX114 would be useful for potential application in degrading plant cell walls for increasing feed conversion efficiency and formation of functional oligosaccharides.     

Molecular cloning and expression in Escherichia coli of a thermophilic Bacillus sp. PDV endo-β-1,4-glucanase gene

The gene encoding endoglucanase in thermophilic Bacillus sp. PDV was cloned in Escherichia coli strain TB1 using pUC 8 as vector. The cloned 3.1 kb PstI DNA fragment was found to express the endoglucanase activity in either orientation. The deletion analysis of pSD 81 suggested that the Bacillus endoglucanase gene expressed in E. coli under the control of its own natural promoter, contained putatively in the 0.2 kb HindIII fragment at the 5′ end of the insert. The relative level of endoglucanase expression in E. coli was about three times higher than that in parent Bacillus sp. PDV. The cloned organism secreted about 84% of the total synthesized CMCase into the culture medium. The CMCase was stable up to 60°C and in the pH range of 4–10.     

Biochemical Properties and Atomic Resolution Structure of a Proteolytically Processed β-Mannanase from Cellulolytic Streptomyces sp. SirexAA-E

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.     

Mycelial β-N-Acetylglucosaminidase of Streptomyces sp. Having β-N-Acetylgalactosaminidase Activity

Formation of transfer products from soybean arabinogalactan and glycerol by endo-1,4-β-d-galactanase from Penicillium citrinum was described. The amount of transfer products depended on the glycerol concentration. About 50% of the galactose residues which could be liberated from the polysaccharide by the enzyme were transferred to glycerol at an acceptor concentration of 2.5% (w/v). Transfer products with various polymerization degrees were accumulated at the beginning of the reaction and then those with higher polymerization degrees were degraded gradually. At a final stage of the reaction, two transfer products in addition to two hydrolysis products (galactose and galactobiose) were mainly accumulated. The two transfer products were isolated and their structures were examined. They were 2-O-β-d-galactosyl glycerol and O-β-d-galactosyl-(1 → 4)-O-β-d-galactosyl-(1 → 2)glycerol.     

Structures of Conversion Products Formed from 18β-Glycyrrhetinic Acid by Streptomyces sp. G-20

Dynamic profiles of the rate of O₂ generation from press-injured and inoculated rice leaf slices, versus the time after inoculation, discriminated between the incompatible and compatible combination of blast fungus races with a cultivar. The application of sodium saccharin to rice seedlings via the root system for 6 days changed the compatible to incompatible profile. Even after press- injury and inoculation with the compatible conidia, the leaf application of sodium saccharin enhanced superoxide generation. The application of N-methylsaccharin in a similar manner, however, did not enhance the superoxide generation. Inoculation of press-injured leaves with incompatible conidia in the presence of an aqueous diffusate of the germinating compatible conidia changed the incompatible to compatible profile. The application to press-injured of concanavalin A or a lyophylized preparation from 5 m ammonia extracts of rice leaf homogenate prior to stimulating with a resistance-inducing factor (RIF) from the fungus also enhanced the superoxide generation. The RIF, either from the incompatible or compatible race, gave a quite similar profile of activation upon the generation of the superoxide anion.     

Biosynthesis of poly(ɛ- l-lysine)s in two newly isolated strains of Streptomyces sp.

The biosynthesis of poly(ɛ-l-lysine) (ɛ-PL) in the two newly isolated strains of Streptomyces lydicus USE-11 (USE-11) and Streptomyces sp. USE-51 (USE-51) was studied by a newly developed two-stage culture method of cell growth at pH 6.8 and ɛ-PL production at pH 4.5. USE-11 synthesized ɛ-PL consisting of about 28 residues at a high production level, whereas USE-51 did the polymer with 15 ones at a low level. The secreted ɛ-PLs in culture media were digested in a neutral pH range with a peptide hydrolase(s) produced by the ɛ-PL producers. The optimum production levels were presumed to be dependent upon the inherent ɛ-PL synthesis machinery of each producer. The production in USE-51 was sharply dependent upon cell density as was often observed in the production of antibiotics, whereas that in USE-11 was scarcely affected by the density. The was found to be essential for the ɛ-PL production in both strains. This might suggest the involvement of a thiol group in the polymerization reactions including the activation of l-lysine. This study indicates that USE-11 is a most suitable strain for the exploration of the ɛ-PL biosynthesis at the molecular level as well as for the technical applications.Electronic supplementary material Electronic supplementary material is available if you access this article at and is accesible for authorized users.An erratum to this article can be found at