Deletion mutants in COP9/signalosome subunits in fission yeast Schizosaccharomyces pombe display distinct phenotypes

The COP9/signalosome complex is highly conserved in evolution and possesses significant structural similarity to the 19S regulatory lid complex of the proteasome. It also shares limited similarity to the translation initiation factor eIF3. The signalosome interacts with multiple cullins in mammalian cells. In the fission yeast Schizosaccharomyces pombe, the Csn1 subunit is required for the removal of covalently attached Nedd8 from Pcu1, one of three S. pombe cullins. It remains unclear whether this activity is required for all the functions ascribed to the signalosome. We previously identified Csn1 and Csn2 as signalosome subunits in S. pombe. csn1 and csn2 null mutants are DNA damage sensitive and exhibit slow DNA replication. Two further putative subunits, Csn4 and Csn5, were identified from the S. pombe genome database. Herein, we characterize null mutations of csn4 and csn5 and demonstrate that both genes are required for removal of Nedd8 from the S. pombe cullin Pcu1 and that their protein products associate with Csn1 and Csn2. However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes. Our data suggest that the subunits of the signalosome cannot be considered as a distinct functional unit and imply that different subunits of the signalosome mediate distinct functions.     

Identification and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies

The Gram-positive spore-forming bacterium, Bacillus thuringiensis, a member of the Bacillus cereus group, produces chitosanases that catalyze the hydrolysis of chitosan to chitosan-oligosaccharides (COS). Although fungal and bacterial chitosanases belonging to other glycoside hydrolase (GH) families have been characterized in a variety of microorganisms, knowledge on the genetics and phylogeny of the GH-8 chitosanases remains limited. Nine genes encoding chitosanases were cloned from 29 different serovar strains of B. thuringiensis and they were expressed in Escherichia coli. The ORFs of the chitosanases contained 1,359 nucleotides and the protein products had high levels of sequence identity (>96%) to other Bacillus species GH-8 chitosanases. Thin-layer chromatography and HPLC analyses demonstrated that these enzymes hydrolyzed chitosan to a chitosan-trimer and a chitosan-tetramer as major products, and this could be useful in the production of COS. In addition, a simple plate assay was developed, involving a soluble chitosan, for high-throughput screening of chitosanases. This system allowed screening for mutant enzymes with higher enzyme activity generated by error-prone PCR, indicating that it can be used for directed chitosanase evolution.     

Modification of genetic regulation of a heterologous chitosanase gene in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> TK24 leads to chitosanase production in the absence of chitosan

Background  

Chitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous chitosanase gene (csnN106) in Streptomyces lividans.     

Production, purification and characterization of chitosanase produced by Gongronella sp. JG

AIMS: To optimize the production condition of chitosanases of Gongronella sp. JG and to characterize the major chitosanase. METHODS AND RESULTS: In the optimized medium and culturing condition, strain JG produced 800 micromol min(-1) l(-1) chitosanase activity at 72 h. The major chitosanase - csn1 was purified through three chromatography steps: CM (carboxymethyl)-Sepharose fast flow (FF), Sephacryl S200, SP (sulfopropyl)-Sepharose FF. The molecular weight and the pI value of csn1 were about 90,000 Da and 5 x 8, respectively. Its specific activity was 82 micromol min(-1) mg(-1). The optimal reaction pH for csn1 was between 4 x 6 and 4 x 8. The optimal reaction temperature was 50 degrees C. The half-life of csn1 at 50 degrees C was estimated to be about 65 min. Mn(2+) was a strong stimulator of csn1 activity, both at 1 and 10 mmol l(-1). csn1 showed its highest activity with chitosan of 85% degree of deacetylation, but did not hydrolyse colloidal chitin and carboxylmethyl cellulose. In 20 mmol l(-1) sodium acetate buffer (pH 4 x 8) and at 50 degrees C, the K(m) of csn1 was calculated to be 4 x 5 mg ml(-1). CONCLUSIONS: The production condition of chitosanases by Gongronella JG was optimized and the major chitosanase, csn1, was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work for the first time reported the production, purification and characterization of chitosanases produced by fungus of Gongronella sp. These results provided us more information on fungal chitosanases.     

Xyloglucan breakdown by endo-xyloglucanase family 74 from Aspergillus fumigatus

Xyloglucan is the most abundant hemicellulose in primary walls of spermatophytes except for grasses. Xyloglucan-degrading enzymes are important in lignocellulosic biomass hydrolysis because they remove xyloglucan, which is abundant in monocot-derived biomass. Fungal genomes encode numerous xyloglucanase genes, belonging to at least six glycoside hydrolase (GH) families. GH74 endo-xyloglucanases cleave xyloglucan backbones with unsubstituted glucose at the −1 subsite or prefer xylosyl-substituted residues in the −1 subsite. In this work, 137 GH74-related genes were detected by examining 293 Eurotiomycete genomes and Ascomycete fungi contained one or no GH74 xyloglucanase gene per genome. Another interesting feature is that the triad of tryptophan residues along the catalytic cleft was found to be widely conserved among Ascomycetes. The GH74 from Aspergillus fumigatus (AfXEG74) was chosen as an example to conduct comprehensive biochemical studies to determine the catalytic mechanism. AfXEG74 has no CBM and cleaves the xyloglucan backbone between the unsubstituted glucose and xylose-substituted glucose at specific positions, along the XX motif when linked to regions deprived of galactosyl branches. It resembles an endo-processive activity, which after initial random hydrolysis releases xyloglucan-oligosaccharides as major reaction products. This work provides insights on phylogenetic diversity and catalytic mechanism of GH74 xyloglucanases from Ascomycete fungi.

     

Chitosanase from Streptomyces coelicolor A3(2): biochemical properties and role in protection against antibacterial effect of chitosan

Chitosan, an N-deacetylated derivative of chitin, has attracted much attention as an antimicrobial agent against fungi, bacteria, and viruses. Chitosanases, the glycoside hydrolases responsible for chitosan depolymerisation, are intensively studied as tools for biotechnological transformation of chitosan. The chitosanase CsnA (SCO0677) from Streptomyces coelicolor A3(2) was purified and characterized. CsnA belongs to the GH46 family of glycoside hydrolases. However, it is secreted efficiently by the Tat translocation pathway despite its similarity to the well-studied chitosanase from Streptomyces sp. N174 (CsnN174), which is preferentially secreted through the Sec pathway. Melting point determination, however, revealed substantial differences between these chitosanases, both in the absence and in the presence of chitosan. We further assessed the role of CsnA as a potential protective enzyme against the antimicrobial effect of chitosan. A Streptomyces lividans TK24 strain in which the csnA gene was inactivated by gene disruption was more sensitive to chitosan than the wild-type strain or a chitosanase-overproducing strain. This is the first genetic evidence for the involvement of chitosanases in the protection of bacteria against the antimicrobial effect of chitosan.     

Broad-specificity GH131 β-glucanases are a hallmark of fungi and oomycetes that colonize plants

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β-1,4, β-1,3 and mixed β-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.     

Characterization of a novel fungal chitosanase Csn2 from Gongronella sp. JG

A 28kDa chitosanase designated as Csn2 was purified from the culture broth of the fungus Gongronella sp. JG through three chromatography steps: CM-Sepharose FF, Superdex 200 and SP-Sepharose FF. Its optimal reaction pH and temperature were pH 5.6 and between 55 degrees C and 60 degrees C. The half-lives of Csn2 at 50 degrees C and 55 degrees C were estimated to be 30min and 11min, respectively. The K(m) value of Csn2 in sodium acetate buffer (pH 5.6) at 55 degrees C was 8.86mg/mL. Mn(2+), Ca(2+) and Sr(2+) were activators of Csn2; ETDA was an inhibitor. Cu(2+) stimulated Csn2 at 1mM, but inhibited Csn2 activity at 10mM. Csn2 displayed strong activity on colloidal chitosan, but did not hydrolyze colloidal chitin and carboxylmethyl cellulose. Thin layer chromatography analysis showed the end products of colloidal chitosan hydrolyzed by Csn2 were chitobiose, chitotriose and chitotetraose with chitotriose as the major product. The N terminus of Csn2 was determined to be YQLPANLKKIYDSHKSGTC. Part of the genomic DNA sequence corresponding to Csn2 was cloned. Sequence alignment showed DNA sequence of Csn2 was partly identical to chitosanase genes from Metarhizium anisopliae var. acridum, Hypocrea lixii and Aspergillus fumigatus. Based on sequence similarity, Csn2 was classified as a GH-75 chitosanase.     

Purification and characterization of two novel β-glucosidases from Penicillium oxalicum and their application in bioactive ginsenoside production

Abstract

The fungus Penicillium oxalicum is able to selectively metabolize the 20(S)-protopanaxadiol ginsenosides Rb1, Rb2 and Rc to the bioactive ginsenoside compound K using extracellular glycosidases. In this study, two novel extracellular ginsenoside-hydrolyzing enzymes GH3-1 and GH3-2 were purified and characterized from P. oxalicum culture. Using ginsenosides as substrates, GH3-1 and GH3-2 synergistically catalyzed the hydrolysis of Rb1, Rb2 and Rc to yield the final product Compound K (C-K). The hydrolysis pathways were determined to be: Rb1→Rd→F2→C-K, Rb2→CO→CY→C-K and Rc→Mb→Mc→C-K for GH3-1 and GH3-2, respectively. The two enzymes differ, especially in composition, molecular weight, stability and substrate specificity, from GH1, a glycosidase previously purified from the same fungus. These enzymes could be of interest in glycoside degradation, especially in the production of minor ginsenosides.     

Uncoupling chitosanase production from chitosan

There is a growing interest in chitosanases as enzymatic tools to hydrolyze chitosan into bioactive forms: low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS). However chitosanases are still expensive and methods of large-scale production of these enzymes are not yet established. The article reviews the approaches used for chitosanase production in various bacterial hosts, pointing out the difficulties resulting from the necessity to include chitosan into the medium composition. A mutated Streptomyces host allows for the efficient production of several chitosanases originating from actinobacteria in the absence of chitosan as inducer.     

Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein‐engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N‐acetyl‐D‐glucosamine units at their subsite (?2), which is impossible for the wildtype enzyme.     

Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent K _m and the catalytic constant k_cat were 0.042 mg/ml and 7,588 min^?1, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way (“3?+?3”) but hydrolysis rate was much faster for (GlcN)₆ than (Glc)₆. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.     

Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57

The glycoside hydrolase family 57 (GH57) contains five well-established enzyme specificities: α-amylase, amylopullulanase, branching enzyme, 4-α-glucanotransferase and α-galactosidase. Around 700 GH57 members originate from Bacteria and Archaea, a substantial number being produced by thermophiles. An intriguing feature of family GH57 is that only slightly more than 2 % of its members (i.e., less than 20 enzymes) have already been biochemically characterized. The main goal of the present bioinformatics study was to retrieve from databases, and analyze in detail, sequences having clear features of the five GH57 enzyme specificities mentioned above. Of the 367 GH57 sequences, 56 were evaluated as α-amylases, 99 as amylopullulanases, 158 as branching enzymes, 46 as 4-α-glucanotransferases and 8 as α-galactosidases. Based on the analysis of collected sequences, sequence logos were created for each specificity and unique sequence features were identified within the logos. These features were proposed to define the so-called sequence fingerprints of GH57 enzyme specificities. Domain arrangements characteristic of the individual enzyme specificities as well as evolutionary relationships within the family GH57 are also discussed. The results of this study could find use in rational protein design of family GH57 amylolytic enzymes and also in the possibility of assigning a GH57 specificity to a hypothetical GH57 member prior to its biochemical characterization.     

Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation

Abstract From autolysed cultures of Mucor rouxii , two chitosanases, A and B, were purified to electrophoretic homogeneity. Apparent M _r values of 76 000 and 58 000 and p I values of 4.9 and 4.7 were determined for A and B, respectively. Both chitosanases showed a high specificity for chitosan and chitosan derivatives. They had optimum activities at pH 5.0 and at temperatures of 55°C and 50°C for A and B, respectively. Enzyme A was inhibited by acetate ions and enzyme B by high substrate concentration. Both enzymes showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of dimer, trimer and higher molecular mass oligomers of glucosamine. Glucosamine oligosaccharides were poorly hydrolysed by these enzymes. Both enzymes extensively degraded the chitosan extracted from M. rouxii cell walls.     

Production of Two Chitosanases from a Chitosan-Assimilating Bacterium, Acinetobacter sp. Strain CHB101

A bacterial strain capable of utilizing chitosan as a sole carbon source was isolated from soil and was identified as a member of the genus Acinetobacter. This strain, designated CHB101, produced extracellular chitosan-degrading enzymes in the absence of chitosan. The chitosan-degrading activity in the culture fluid increased when cultures reached the early stationary phase, although the level of activity was low in the exponential growth phase. Two chitosanases, chitosanases I and II, which had molecular weights of 37,000 and 30,000, respectively, were purified from the culture fluid. Chitosanase I exhibited substrate specificity for chitosan that had a low degree of acetylation (10 to 30%), while chitosanase II degraded colloidal chitin and glycol chitin, as well as chitosan that had a degree of acetylation of 30%. Rapid decreases in the viscosities of chitosan solutions suggested that both chitosanases catalyzed an endo type of cleavage reaction; however, chitosan oligomers (molecules smaller than pentamers) were not produced after a prolonged reaction.     

The crystal structure of a family GH25 lysozyme from Bacillus anthracis implies a neighboring-group catalytic mechanism with retention of anomeric configuration

Lysozymes are found in many of the sequence-based families of glycoside hydrolases (www.cazy.org) where they show considerable structural and mechanistic diversity. Lysozymes from glycoside hydrolase family GH25 adopt a (α/β)₅(β)₃-barrel-like fold with a proposal in the literature that these enzymes act with inversion of anomeric configuration; the lack of a suitable substrate, however, means that no group has successfully demonstrated the configuration of the product. Here we report the 3-D structure of the GH25 enzyme from Bacillus anthracis at 1.4 Å resolution. We show that the active center is extremely similar to those from glycoside hydrolase families GH18, GH20, GH56, GH84, and GH85 implying that, in the absence of evidence to the contrary, GH25 enzymes also act with net retention of anomeric configuration using the neighboring-group catalytic mechanism that is common to this ‘super-family’ of enzymes.     

Genome-wide identification,characterization, and expression profiles of auxin responsive GH3 gene family in Salvia miltiorrhiza involved in MeJA treatment

GH3 (Gretchen Hagen 3) is a gene family involved in the response to auxin and plays a role in regulation of plant growth, development and stress responses. The GH3 gene family has been well investigated in genome wide in various plants containing Arabidopsis, rice, maize, etc. However, the study on the GH3 family and its roles involved in JA-signal pathway in Salvia miltiorrhiza is lacking. In this study, we performed a systematic identification of the SmGH3 gene family in genome wide and detected 11 members on 8 S. miltiorrhiza scaffolds. Phylogenetic analyses revealed that SmGH3 proteins could be clustered in two major categories with groups 1 and 2 of GH3 family of Arabidopsis. Diversified cis-elements in the promoter of SmGH3 were predicted as essential players in regulating SmGH3 expression patterns by using PlantCARE database. Gene structure and motif analyses indicated that most SmGH3 genes had relatively conserved exon/intron arrangements and motif compositions. RNA-seq analysis and RT-qPCR showed that 3 SmGH3s (SmGH3.2, SmGH3.6, and SmGH3.10) were up-regulated in S. miltiorrhiza treated by MeJA. Moreover, tissue-specific expression patterns of each SmGH3 genes in different tissues suggested that the various members of GH3 genes conducted their role in the different tissues of S. miltiorrhiza. These results would provide a comprehensive understanding of the GH3 gene family in S. miltiorrhiza and lay a foundation for exploration of their functional divergence and genetic manipulation.

     

A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan

A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. ¹H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.     

Identification of new GH 10 and GH 11 xylanase genes from Aspergillus versicolor MKU3 by genome-walking PCR

Xylanases randomly clear the backbone of xylans, which are hemicelluloses representing a considerable source of fixed carbon in nature. Consequently, these enzymes have important industrial applications. To characterize the genes responsible for producing these enzymes, we cloned xylanase genes belonging to the GH11 and GH10 families from Aspergillus versicolor MKU3 using a 2-step polymerase chain reaction (PCR) protocol involving degenerate PCR and genome-walking PCR (GWPCR). We amplified a family 10 xylanase consensus fragment using degenerate PCR primers exhibiting specificity for conserved motifs within fungal family 10 xylanase genes. We identified a single family 10 xylanase gene (xynv10) and determined its entire gene sequence during the second step of GWPCR, which was used to amplify genomic DNA fragments upstream and downstream of xynv10. The xynv10 sequence contains a 1,378-bp open reading frame separated by 8 introns with an average size of 49 bp. We also amplified a partial GH11 xylanase gene sequence (xynv11) using degenerate PCR and genome-walking methods. Amplification of the C-terminal region of xynv11 using a degenerate primer designed from sequences revealed strong homology with the partial GH11 xylanase gene of A. versicolor MKU3. The structural region in xynv11 was approximately 680 bp and has one intron that is approximately 64 bp in length. Further expression and characterization of these genes will give better understanding of the role of these genes in xylan degradation by A. versicolor.