Amino acid regions of family 45 endoglucanases involved in cotton defibrillation and in resistance to anionic surfactants and oxidizing agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

Determination of silver nanoparticle species released from textiles into artificial sweat and laundry wash for a risk assessment

Increasing use of nanomaterials for consumer products has induced growing concerns on their adverse effects on human health and the environment. To assess the environmental and human health risks of these nanoproducts, it is essential to identify physicochemical forms and quantify the amount of nanomaterials released from nanoproducts upon exposure to various environments. In this study, we have investigated the release assessment of nano-Ag textile products in terms of the total Ag content, and the distribution of Ag materials in the textile and the surrounding environment. The results suggest that the release of Ag nanomaterials from consumer products is less dependent on the total Ag content in the consumer product and depends significantly on the manufacturing processes of the consumer products as well as exposure environment. Based on these experimental results and a simple exposure model, the highest total exposure to particulate/dissolved Ag per use during sweating (1 h) was estimated to be 0.81/2.03 μg Ag/kg body weight with a standard body weight of 77 kg for a male. These results are fairly minimal human exposure and suggest that textiles containing Ag nanomaterials may be less of a concern in terms of human exposure to Ag nanomaterials.     

Purification and characterisation of two endoglucanases from Arthrobotrys oligospora

Two endoglucanases, designated Endo I and Endo II, were purified from the culture filtrates of a nematode trapping fungus, Arthrobotrys oligospora. The purification procedure entailed ammonium sulphate precipitation, gel filtration and preparative PAGE. Both the preparations (Endo I and Endo II) were homogeneous by PAGE, had molecular weights of 24,300 and 44,500 respectively as determined by non-denaturing PAGE, and yielded only cellobiose as the main product of CM-cellulose hydrolysis. The optimum pH and temperature for Endo I were 6.0 and 50 degrees C, and for Endo II, pH 5.6-6.4 and 50 degrees C. The two enzymes differed with respect to their Km (Endo I, 5.04 mg/ml; Endo II, 3.2 mg/ml) and energy of activation values (Endo I, 10.7 kCal; Endo II, 9.5 k Cal). Both enzymes were completely inhibited by 1.25 mH Hg2+ and partially by Pb2+, DTNB and p-HMB while DTT and GSH enhanced their activities.     

Structural characteristics of DNA from sarcoma 45

It is shown that thermodynamical parameters of thermal melting and the content of 5-methylcytosine for tumor DNA of sarcoma 45 differ from DNA in the norm. The reason of such difference is the presence of regions with changed DNA structure in sarcoma 45, which occurs apparently owing to hypermethylation of cytosine in tumor DNA.     

A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.     

Polyamines and the Phorogenesis of Mucorales

Reyna-lopez, G. and Ruiz-Herrera, J. 1993. Polyamines and the phorogenesis of mucorales. Experimental Mycology 17, 79-89. Appearance of sporangiophore primordia of Mucor rouxii and Phycomyces blakesleeanus was preceded by a transitory elevation in the levels of ornithine decarboxylase (ODC) and the polyamines putrescine and spermidine. If the ODC competitive inhibitor 1,4-diamino-2-butanone (DAB) was added before this time, it blocked the appearance of sporangiophores as well as the elevation of ODC and putrescine levels, but not those of spermidine, nor did it affect the growth of vegetative mycelium. If added later on, the drug did not prevent photogenesis, nor the differentiation of primordia into macro- or microphores. The effect of DAB on phorogenesis could be counteracted by putrescine (but not by spermidine) and by 5-azacytidine. It is suggested that polyamines are necessary to trigger the differentiation of some vegetative hyphae into sporangiophore primordia, probably affecting the expression of specific genes through a process involving DNA hypomethylation.     

Characterization of three bacterial glycoside hydrolase family 9 endoglucanases with different modular architectures isolated from a compost metagenome

BackgroundEnvironmental bacteria express a wide diversity of glycoside hydrolases (GH). Screening and characterization of GH from metagenomic sources provides an insight into biomass degradation strategies of non-cultivated prokaryotes.MethodsIn the present report, we screened a compost metagenome for lignocellulolytic activities and identified six genes encoding enzymes belonging to family GH9 (GH9a-f). Three of these enzymes (GH9b, GH9d and GH9e) were successfully expressed and characterized.ResultsA phylogenetic analysis of the catalytic domain of pro- and eukaryotic GH9 enzymes suggested the existence of two major subgroups. Bacterial GH9s displayed a wide variety of modular architectures and those harboring an N-terminal Ig-like domain, such as GH9b and GH9d, segregated from the remainder. We purified and characterized GH9 endoglucanases from both subgroups and examined their stabilities, substrate specificities and product profiles. GH9e exhibited an original hydrolysis pattern, liberating an elevated proportion of oligosaccharides longer than cellobiose. All of the enzymes exhibited processive behavior and a synergistic action on crystalline cellulose. Synergy was also evidenced between GH9d and a GH48 enzyme identified from the same metagenome.ConclusionsThe characterized GH9 enzymes displayed different modular architectures and distinct substrate and product profiles. The presence of a cellulose binding domain was shown to be necessary for binding and digestion of insoluble cellulosic substrates, but not for processivity.General significanceThe identification of six GH9 enzymes from a compost metagenome and the functional variety of three characterized members highlight the importance of this enzyme family in bacterial biomass deconstruction.     

Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes

The enzymatic properties of two endoglucanases from Fibrobacter succinogenes, EGB and EGC, were analysed. EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E. coli as well as its detection in F. succinogenes cultures. Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies. One of the polyclonal antipeptide antisera was used to purify EGB. EGC was purified by affinity chromatography with Ni-NTA resin. The endo mode of action of the two enzymes on carboxymethyl-cellulose was different. The values of K(m) and V(max) were respectively 13.6 mg/ml and 46 micromol/min mg protein for EGB, and 7 mg/ml and 110 micromol/min mg protein for EGC. The reactivity of the antipeptide and the anti-EGC sera with F. succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational modification of the two enzymes in F. succinogenes cultures. Expression of endB and endC genes in F. succinogenes was confirmed by RT-PCR.     

Syncephalastrum contaminatum,a new species in the Mucorales from Australia

A new species is described in the Mucorales family Syncephalastraceae: Syncephalastrum contaminatum, isolated as an in vitro culture from a laboratory contaminant. The species has variable copies of the internal transcribed spacer (ITS) regions, requiring cloning of these regions prior to Sanger sequencing before subsequent use in phylogenetic comparisons with other fungi. The genome of the strain was sequenced using short paired-reads to yield a draft genome of 28.6 Mb. Syncephalastrum contaminatum is distinguished by diverse DNA sequences at several loci from the other species of Syncephalastrum, including only 81% sequence identity with its ITS regions to that of S. racemosum. Its merosporangium produces four or more asexual spores and the genome sequencing information suggests that the species is heterothallic. The identification of this species highlights the limited knowledge about the early lineages of fungi both in Australia and globally.     

Outbreaks of Mucorales and the Species Involved

The order Mucorales is an ancient group of fungi classified in the subphylum Mucoromycotina. Mucorales are mainly fast-growing saprotrophs that belong to the first colonizers of diverse organic materials and represent a permanent part of the human environment. Several species are able to cause human infections (mucormycoses) predominantly in patients with impaired immune system, diabetes, or deep trauma. In this review, we compiled 32 reports on community- and hospital-acquired outbreaks caused by Mucorales. The most common source of mucoralean outbreaks was contaminated medical devices that are responsible for 40.7% of the outbreaks followed by contaminated air (31.3%), traumatic inoculation of soil or foreign bodies (9.4%), and the contact (6.2%) or the ingestion (6.2%) of contaminated plant material. The most prevalent species were Rhizopus arrhizus and R. microsporus causing 57% of the outbreaks. The genus Rhizomucor was dominating in outbreaks related to contaminated air while outbreaks of Lichtheimia species and Mucor circinelloides were transmitted by direct contact. Outbreaks with the involvement of several species are reported. Subtyping of strains revealed clonality in two outbreaks and no close relation in two other outbreaks. Based on the existing data, outbreaks of Mucorales can be caused by heterogeneous sources consisting of different strains or different species. Person-to-person transmission cannot be excluded because Mucorales can sporulate on wounds. For a better understanding and prevention of outbreaks, we need to increase our knowledge on the physiology, ecology, and population structure of outbreak causing species and more subtyping data.

     

Liberation of asexual propagules in the Mucorales

The liberation of asexual propagules in the Mucorales was investigated by means of wind-tunnel experiments. Some species do not liberate propagules into an air stream, others liberate single spores or groups of spores. The effect of the relative humidity of the air stream upon propagule liberation has also been considered.     

Specific image characteristics influence attitudes about chimpanzee conservation and use as pets

Chimpanzees are endangered in their native Africa but in the United States, they are housed not only in zoos and research centers but owned privately as pets and performers. In 2008, survey data revealed that the public is less likely to think that chimpanzees are endangered compared to other great apes, and that this is likely the result of media misportrayals in movies, television and advertisements. Here, we use an experimental survey paradigm with composite images of chimpanzees to determine the effects of specific image characteristics. We found that those viewing a photograph of a chimpanzee with a human standing nearby were 35.5% more likely to consider wild populations to be stable/healthy compared to those seeing the exact same picture without a human. Likewise, the presence of a human in the photograph increases the likelihood that they consider chimpanzees as appealing as a pet. We also found that respondents seeing images in which chimpanzees are shown in typically human settings (such as an office space) were more likely to perceive wild populations as being stable and healthy compared to those seeing chimpanzees in other contexts. These findings shed light on the way that media portrayals of chimpanzees influence public attitudes about this important and endangered species.     

Adsorption and kinetic behavior of purified endoglucanases and exoglucanases from Trichoderma viride

Adsorption on crystalline cellulose of six endoglucanases (Endo I, II, III, IV, V and VI; 1, 4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) and two exoglucanases (Exo II and III; 1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.92), purified from a commercial cellulase preparation of Trichoderma viride origin, was studied. Endo I, III, and V adsorbed strongly on Avicel cellulose, while adsorption of Endo II, IV, and VI was much lower. Also, the two exoglucanases could be divided into one enzyme (Exo III) that had a high adsorption affinity and another enzyme (Exo II) that adsorbed only moderately. Adsorption data fitted the Langmuir-type adsorption isotherm. However, adsorption was only partially reversible with respect to dilution. No relation could be found between adsorption affinity and degree of randomness in cellulose hydrolysis, measured as the diversity of released hydrolytic products. Kinetic measurements indicated that only part of the adsorbed enzyme molecules are hydrolytically active.     

Purification and biochemical characteristics of actin from the rat malignancy sarcoma-45]

Actin was purified from rat sarcoma-45 by using affinity chromatography on DNase I agarose. Actin was detected in the soluble and cytoskeletal fractions. The molecular mass of the protein was found to be equal to 45 kDa. The tumour actin specifically reacted with the antibody against skeletal muscle actin, inhibited the DNAase I activity and activated in the fibrillar state Mg(2+)-ATPases of sarcoma-45 and skeletal muscle myosins. The activating effect of the tumour protein was lower than that of its skeletal muscle counterpart. V8-protease peptide mapping revealed a similarity between tumour and brain actins. Sarcoma-45 actin was found to contain beta- and gamma-actin isoforms and an unusual isoform which appeared to be more acidic than the alpha-actin isoform.     

Purification and biochemical characteristics of myosin from rat malignant sarcoma-45

Myosin was purified from rat tumour sarcoma-45 whose properties (effects of cations on ATPase activity, substrate specificity, temperature- and pH-optima, thermal stability, sensitivity of Mg2(+)-ATPase to F-actin, molecular mass, subunit composition) are similar to those of fast skeletal muscle myosin. Some parameters of the protein, namely, the levels of Ca2(+)- and K+, EDTA-ATPase activity, relative content of myosin light chains with Mr 16500 and the degree of tumoural myosin Mg2(+)-ATPase activation by F-actin, were significantly lower than those of skeletal muscle myosin.     

Purification and characterization of recombinant endoglucanases from the pine wood nematode Bursaphelenchus xylophilus

A family of endoglucanases belonging to glycoside hydrolase family (GHF) 45 have been isolated from the pine wood nematode Bursaphelenchus xylophilus. Here we describe the purification and characterization of the recombinant enzymes, named Bx-ENG-1, 2, and 3, expressed in Pichia pastoris. The respective molecular masses of purified Bx-ENG-1, 2, and 3 were estimated to be 18, 33-39, and 100-140 kDa by SDS-PAGE, and 18, 67, and 252 kDa by gel filtration, suggesting that Bx-ENG-1 existed in an unglycosylated monomeric form and Bx-ENG-2 and Bx-ENG-3 in a glycosylated dimeric form. The enzymatic properties of the recombinant enzymes were similar to each other: optimal activity at 60 degrees C at about pH 6.0, like other endoglucanases of GHF45. The recombinant enzymes displayed the highest activity toward lichenan, and lower activities were observed on carboxymethyl cellulose and amorphous cellulose. Nematode enzymes also hydrolyzed glucomannan, the most abundant hemicellulose in the cell walls of softwood. These substrate specificities suggest that B. xylophilus endoglucanases acted on the cellulose-hemicellulose complex in the cell walls, resulting in a weakening of the mechanical strength of the cell walls to facilitate the nematode's feeding on plant cells.     

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.     

Purification and partial characterization of two extracellular endoglucanases from Cellulomonas fermentans

Avicelase assay of gel slices after non-denaturing polyacrylamide gel electrophoresis of concentrated supernatants from Cellulomonas fermentans revealed four active bands. One of them corresponded to the principal active band on CM-cellulose. Among the three others, at least one did not correspond to any active band on CM-cellulose and might reflect the presence of an exoglucanase (EC 3.2.1.91). The active band on CM-cellulose was composed of two endoglucanases (EC 3.2.1.4), called CFA and CFB, which we purified by the means of DEAE-Trisacryl chromatography and high performance liquid chromatography (anion exchange chromatography and gel chromatography). These two monomeric enzymes differ in their molecular weights (40,000 and 57,000 for CFA and CFB, respectively) and in their catalytic constants in the reaction with CM-cellulose (Km were 1.5 g/l and 59 g/l for CFA and CFB, respectively), but have similar modes of action on this substrate and similar substrate specificities.     

Stabilization of chitin synthetase and purification of chitosomes from several mycelial Mucorales

Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.