An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.     

Pyrolysis mass spectrometry characterisation and numerical taxonomy ofAeromonas spp.

Reference strains (2) and 29 isolates ofAeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of typical isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate betweenA. caviae andA. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis forA. caviae andA. sobria, but the main group ofA. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate betweenA. hydrophila andA. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains ofA. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable nonpathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.Abbreviations CTRP conventional test reaction pattern - PyMS pyrolysis mass spectrometry     

Production of chitinase and its optimization from a novel isolate Alcaligenes xylosoxydans: potential in antifungal biocontrol

A novel, highly chitinolytic strain of Alcaligenes xylosoxydans was isolated which showed potential for use as an antifungal biocontrol agent for the control of two fungal plant pathogens. It could degrade and utilize dead mycelia of Rhizoctonia bataticola and Fusarium sp. (fungal plant pathogens of Cajanus cajan). In vitro it could inhibit the growth of Fusarium sp. and R. bataticola. Chitin at 10–15 g/l was found to be good carbon and nitrogen source. Alcaligenes xylosoxydans showed optimum chitinase production at 72 h, pH optima at 8 and growth peak at 120 h. Yeast extract, arabinose, Tween 20 and several other surfactants enhanced chitinase production.     

Production of cellulolytic and xylanolytic enzymes by an Arthrographis species

A fungal isolate, Arthrographis sp. strain F4, when grown in shake-flask culture, produced cellulolytic and xylanolytic enzymes optimally at 30°C with an initial pH of 5.0 to 6.0. Coarsely-ground filter paper was the most suitable carbon substrate for production of the enzymes. Inorganic nitrogen sources gave higher activities of the enzymes than organic nitrogen sources: NH₄NO₃ and yeast extract was the most effective combination. Significant stimulation (P<0.05) of enzyme production was achieved with 0.1% (v/v) Tween 80.B.C. Okeke was and S.K.C. Obi is with the Department of Microbiology, University of Nigeria, Nsukka, Nigeria. B.C. Okeke is now with the Department of Bioscience and Biotechnology, Royal College Building, University of Strathclyde, Glasgow G1 1XW, UK     

Comparison of different media for the identification and quantification ofAeromonas spp. in water

In order to assess the suitability of the Starch glutamate ampicillin penicillin-10C agar for the isolation ofAeromonas spp. from waters it was necessary to compare the properties of this medium with those of three others, Starch ampicillin agar, Ampicillin dextrin agar and m-Aeromonas medium, and to monitor different kinds of waters. A selection of forty eight samples were taken from moderately polluted river water, highly polluted river water, polluted sea water (littoral) and treatment & distribution water and monitored using these media. The results were similar with Ampicillin dextrin agar, m-Aeromonas medium and Starch glutamate ampicillin penicillin-10C, but the simplicity of composition and use and its selectivity recommends the last medium as the most adequate for the isolation ofAeromonas spp.Abbreviations ADA ampicillin dextrin agar - mA m-aeromonas medium - SA starch ampicillin agar - SGAP-10C starch glutamate ampicillin penicillin-10C     

Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501

The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA–DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of l-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities (>97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85^T and CDC 2555-87 were low (0.6–1.1%). The DNA G+C content of the type strain was 62.2 mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85^T (=CECT 4254^T=ATCC 43946^T=LMG 17321^T).     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.     

Activities of chitinolytic enzymes during primary and secondary colonization of wood by basidiomycetous fungi

The nitrogen (N) content of wood is usually suboptimal for fungal colonization. During decomposition of wood, an increasing fraction of the N becomes incorporated into fungal mycelium. Between 5 and 50% of the N in wood-degrading mycelium may be incorporated into chitin. Chitinolytic enzymes render this N available for re-utilization. Here, the activities of chitinolytic enzymes produced by wood-rotting fungi during degradation of spruce (Picea abies) wood were quantified in situ using fluorogenic 4-methylumbelliferyl substrates. A new method was developed that enables spatial quantification of enzyme activities on solid surfaces. All of the three tested fungi produced endochitinases, chitobiosidases and N-acetylhexosaminidases during colonization of wood. N-acetylhexosaminidase activity, and in some cases also chitobiosidase and endochitinase activities, were higher during secondary overgrowth of another fungus than during primary colonization of noncolonized wood. The results suggest that wood-degrading fungi degrade their own cell walls as well as the hyphae of earlier colonizers. Recycling of cell wall material within single mycelia and between fungal individuals during succession may lead to retention of N within woody debris.     

Plant growth and development influenced by transgenic insertion of bacterial chitinolytic enzymes

The role of the chitinolytic enzymes in plants is not necessarilyrestricted to plant defense. Tomato plants transformed with an endochitinaseand a chitobiosidase gene from Streptomyces albidoflavus andgrowth under greenhouse conditions showed a significant reduction in plantheight, and reduced time to flowering compared with the control(non-transformed) plants. The levels of chitobiosidase and endochitinaseactivity in the transgenic tomato plants were positively correlated with earlyflowering, and negatively correlated with plant height. We have not determinedwhether these effects are exclusively due to the expression of the transgenesof endochitinase and chitobiosidase from S. albidoflavus orthe additive effect of these 2 enzymes combined with the endogenouschitinolytic enzymes produced by the plants. However, when control plants were trimmed,early flowering was observed compared with the controls that were not trimmed, whichindicates that wound induced proteins such as chitinolytic enzymes affect thetime of flowering. In addition, the expression of the endochitinase andchitobiosidase genes significantly increased the number of flowers and fruit onthe plants, resulting in an increase in yield of fruit. One of the primarygoals of crop breeding programs is to increase the productivity of plants. These twogenes were directly associated with plant productivity, and should be studied further.     

Bensingtonia pseudonaganoensis sp. nov., a novel ballistoconidium-forming yeast species isolated from plant leaves

Among the basidiomycetous yeasts isolated from plant leaves collected in different regions of China, two ballistoconidium-forming strains were revealed to represent an undescribed species of the genus Bensingtonia by conventional, chemotaxonomic and molecular phylogenetic characterization. Sequence analysis of the 26S rDNA D1/D2 domains and the internal transcribed spacer (ITS) region indicated that the novel species was located in the Agaricostilbum lineage and closely related to Bensingtonia naganoensis and Bensingtonia ciliata, with the former as its closest relative. The name Bensingtonia pseudonaganoensis sp. nov. is proposed (type strain: AS 2.2601^T = CBS 10121^T)     

Branchiibius cervicis sp. nov., a novel species isolated from patients with atopic dermatitis

Novel actinobacterial strains, PAGU 1247^T, PAGU 1251 and PAGU 1252, were isolated from the skin of atopic dermatitis patients and were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that these isolates were located within the family Dermacoccaceae. The most closely related species of PAGU 1247^T in phylogenetic terms was Branchiibius hedensis Mer 29717^T, with 16S rRNA gene sequence similarity of 99.6%, although the DNA–DNA relatedness value was less than 43.9%. Some biochemical traits, such as lipase (C14) and α-galactosidase activity, could distinguish these isolates from B. hedensis. Strain PAGU 1247^T contained iso-C_16:0 and brC_18:0 as the major fatty acids. The quinone system consisted of menaquinone MK-8(H₆ and H₄). The G + C content of the genomic DNA was 67.6 mol%. On the basis of its phenotypic properties and genetic distinctiveness, strains PAGU 1247^T, PAGU 1251 and PAGU 1252 represents a novel species of the genus Branchiibius, for which the name Branchiibius cervicis sp. nov. is proposed. The type strain is PAGU 1247^T (=NBRC 106593^T = DSM 24166^T).     

Isolation of a chitin-binding lectin, with insecticidal activity in chemically-defined synthetic diets, from two wild brassica species with resistance to cabbage aphidBrevicoryne brassicae

A lectin, with a chitin-binding domain and chitinase activity, is present in significant quantities in the wild brassica speciesB. fruticulosa andB. spinescens but at low levels in cultivated cabbage cv. Offenham Compacta. The lectin, purified >1000 fold after binding to chitin, migrated on SDS-PAGE gels as a single band with a M _r of 14.500. The amino acid composition of the lectin fromB. spinescens indicated high concentrations of asparagine/aspartic acid, glycine, leucine and serine in common with other chitin-binding lectins with insecticidal and antifungal activities. Brassica lectin and the closely related agglutinin from wheatgerm and nettle show significant insecticidal activity when presented toBrevicoryne brassicae in chemically-defined synthetic diets.     

Properties of an extracellular amylase purified from a Bacillus species

A strain of Bacillus produced an amylase with properties characteristically different from known bacterial amylases. The purified 80 kDa protein of pI 5.1 dextrinized starch, glycogen and pullulan. The temperature and pH optima of the enzyme were 60 °C and 6.6 respectively. In the presence of 0.05 M CaCl₂, the enzyme retained stability for 15 min at 80 °C. Antibodies raised to the amylase protein showed no reaction with -amylases of Bacillus sp. and B. licheniformis. In culture, proteolytic degradation of the enzyme was observed.     

Four new species of the genusSporobolomyces isolated from leaves in Thailand

Thirteen undescribed strains of ballistoconidium-forming yeasts, isolated from leaves collected in the suburbs and along the southeast seacoast of Bangkok, Thailand, were divided into four different groups in the genusSporobolomyces on the basis of morphological, physiological, biochemical, and chemotaxonomical characteristics, and analyses of the sequences of 18S rDNA and internal transcribed spacer regions. DNA-DNA reassociation experiments with related species revealed that these four groups were four new distinct species.Sporobolomyces nylandii sp. nov.,S. poonsookiae sp. nov.,S. blumeae sp. nov. andS. vermiculatus sp. nov. are proposed for these strains.     

一种来自乳酸乳球菌的新型氨肽酶A的制备及特性分析

氨肽酶A(aminopeptidase A,Pep A)能特异性地水解N末端为谷氨酸(glutamic acid,Glu)或天冬氨酸(asparticacid,Asp)的肽链,提高蛋白质的水溶性和食物的风味,在食品工业和肉类加工中具有一定的应用前景。本研究采用全基因合成的方式获得了乳酸乳球菌(Lactococcus lactis ssp.lactis)IL1403氨肽酶A(Lactococcus lactis-Pep A,Lc-Pep A)的编码基因,将该基因克隆并导入毕赤酵母(Pichia pastoris)GS115(His4),在毕赤酵母中实现了Lc-Pep A的高效分泌表达,表达产物经鉴定和纯化制备后,进行了生物学特性的分析。结果表明,Lc-Pep A具有较强的底物特异性,对2种底物谷氨酸对硝基苯胺(glutamicacid-p-nitroaniline,Glu-pNA)和天冬氨酸对硝基苯胺(aspartic acid-p-nitroaniline,Asp-pNA)具有相似的催化活力和酶动力学参数。Lc-Pep A是一种金属蛋白酶,最适反应温度为60℃,最适pH为8.0,具有较宽的热稳定性和酸碱稳定性。金属离子Co^(2+)、Mn^(2+)及Zn^(2+)等对酶活力具有不同程度的激活作用,而Ni^(2+)和Cu^(2+)对酶活力具有强烈的抑制作用。Lc-Pep A对常规蛋白酶抑制剂不敏感,但能被金属蛋白酶抑制剂、EDTA及二硫键还原剂抑制。这些研究为Lc-Pep A的生产和指导该酶的应用打下了坚实的基础。     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

Production of a cellulase-free xylanase from agricultural waste materials by a thermotolerant Streptomyces sp.

1444 microorganisms were isolated from soil samples from the northern Thai and screened at 55 °C by using basal medium supplemented with 1% carboxymethyl cellulose as a sole carbon source. One isolate, Streptomyces Ab106, had a high activity of a cellulase-free xylanase also without mannanase activity. The maximum cellulase-free xylanase activities of 3.5, 3.3, 3.1 and 2.7 IU were after growth of the organism with 1% (w/v) corn hull, corncob, bagasse and oat spelt xylan, respectively, at 55 °C for 6 days, respectively. The activity was more than 5 times higher than that at 35 °C.     

Acceptor reactions of a novel transfructosylating enzyme from Bacillus sp.

Many different oligosaccharides were produced by transferring the fructose residue of sucrose to maltose, cellobiose, lactose and sucrose (self-transfer), where their yields of fructosylated acceptor products accounted for 26–30% (w/w). The maximum conversion yield (30%) was obtained in fructosyl cellobioside formation with 500 g sucrose l^–1 (substrate) and 200 g cellobiose l^–1 (acceptor). These four acceptors gave various products having DP (degree of polymerization) 2–7 by successive transfer reactions.     

Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants

In a taxonomic study on the ascomycetous yeasts isolated from plant materials collected in tropical forests in Yunnan and Hainan Provinces, southern China, four strains isolated from tree sap (YJ2E(T)) and flowers (YF9E(T), YWZH3C(T) and YYF2A(T)) were revealed to represent four undescribed yeast species. Molecular phylogenetic analysis based on the large subunit (26S) rRNA gene D1/D2 domain sequences showed that strain YJ2E(T) was located in a clade together with Candida haemulonii and C. pseudohaemulonii. Strain YF9E(T) was most closely related to C. azyma and strain YWZH3C(T) to C. sorbophila and C. spandovensis. Strain YYF2A(T) was clustered in a clade containing small-spored Metschnikowia species and related anamorphic Candida species. The new strains differed from their closely related described species by more than 10% mismatches in the D1/D2 domain. No sexual states were observed for the four strains on various sporulation media. The new species are therefore assigned to the genus Candida and described as Candida alocasiicola sp. nov. (type strain, YF9E(T) = AS 2.3484(T) = CBS 10702(T)), Candida hainanensis sp. nov. (type strain, YYF2A(T) = AS 2.3478(T) = CBS 10696(T)), Candida heveicola sp. nov. (type strain, YJ2E(T) = AS 2.3483(T) = CBS 10701(T)) and Candida musiphila sp. nov. (type strain, YWZH3C(T) = AS 2.3479(T) = CBS 10697(T)).     

Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli

Summary A DNA fragment encoding for 598 amino acids of chitinase protein from Bacillus circulans No. 4.1 was subcloned into pQE-30 expression vector and transformed into Escherichia coli M15 (pREP4). The molecular weight of the expressed protein was approximately 66 kDa. Enzymatic activity of the recombinant protein was assayed after purification using affinity chromatography on a nickel chelating resin. The enzyme hydrolyzed N-acetylchitooligosaccharides mainly to N-acetylchitobiose, and was active toward chitin, carboxymethyl-chitin, colloidal chitin, glycol chitin and 4-methylumbelliferyl-β-d-N, N′-diacetylchitobiose. The pH and temperature optima of the chitinase enzyme were 7.0 and 45 °C, respectively. This enzyme was stable in the pH range of 5.0–9.0 and at temperatures up to 50 °C. In addition, when cleaved by a proteolytic enzyme, the 20-kDa product could retain high chitinolytic activity.