Molecular cloning and expression analysis of chymotrypsin-like serine protease from the redclaw crayfish (Cherax quadricarinatus): A possible role in the junior and adult innate immune systems

A novel chymotrypsin-like serine protease (CLSP) was isolated from the hepatopancreas of the redclaw crayfish Cherax quadricarinatus (Cq-chy). The full-length cDNA of Cq-chy contains 951 nucleotides encodes a peptide of 270 amino acids. The mature peptide comprising 223 amino acids contains the conserved catalytic triad (H, D, and S). Similarity analysis showed that Cq-chy shares high identity with chymotrypsins from the fiddler crab; Uca pugilator. Cq-chy mRNA expression in C. quadricarinatus was shown to be: (a) tissue-related with the highest expression in the hepatotpancreas and widely distributed, (b) highly responsive in the hepatopancreas to White Spot Syndrome Virus (WSSV) challenge, and (c) differently regulated in immature and adult crayfish. In this study we successfully isolated Cq-chy. Our observations indicate that Cq-chy is differently involved in the immature and adult innate immune reactions, thus suggesting a role for CLSPs in the invertebrate innate immune system.     

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.     

Characterization of Cdc2 kinase in the red claw crayfish (Cherax quadricarinatus): Evidence for its role in regulating oogenesis

Cdc2 kinase is a catalytic subunit of the maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocytes. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized. In this study, a complete cDNA sequence of Cdc2 kinase was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. The Cdc2 cDNA (1769 bp) encodes for a 299 amino acid protein with a calculated molecular weight of 34.7 kDa. Quantitative real-time PCR demonstrated that Cdc2 mRNA was expressed mainly in the ovary tissue and the expression decreased as the ovaries developed. Immunohistochemistry analysis revealed that the Cdc2 protein relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that Cdc2 kinase may play an important role in the gametogenesis and gonad development in C. quadricarinatus.     

Unwrapping the hydrolytic system of the phytopathogenic fungus Phoma exigua by secretome analysis

The present work describes the secretome profiling of a phytopathogenic fungus, Phoma exigua by liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) based proteomics approach to highlight the suites of enzymes responsible for biomass hydrolysis. Mass spectrometry identified 33 proteins in the Phoma secretome when grown on α-cellulose as the sole carbon source. The functional classification revealed a unique extracellular enzyme system mainly belonging to the family of glycosyl hydrolase proteins (52%). This hydrolytic system consisted of cellulases (endo-1,4-β-glucanase, cellobiohydrolase I, exoglucanase, and β-glucosidase), hemicellulases (1,4-β-xylosidase and endo-1,4-β-xylanase) and other hypothetical proteins including GH3, GH5, GH6, GH7, GH11, GH20, GH32 and GH54. The synergistic action of this enzyme cocktail was assessed by the saccharification of alkali treated wheat straw. Since the Phoma secretome has limited β-glucosidase activity, it was supplemented with commercial β-glucosidase. After supplementation, this enzyme complex resulted in high yields of glucose (177.2 ± 1.0 mg/gds), xylose (209.2 ± 1.5 mg/gds) and arabinose (25.2 ± 0.3 mg/gds). The secretome analysis and biomass hydrolysis by P. exigua revealed its unique potential as a source of hydrolytic enzymes for lignocellulosic biomass hydrolysis.     

Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae)

The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356 bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5 kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.     

Pectinase and cellulase activity in the digestive system of the elm leaf beetle,Xanthogaleruca luteola Muller (Coleoptera: Chrysomelidae)

The elm leaf beetle, Xanthogaleruca luteola, is a serious pest of elm trees in urban areas. Partial biochemical characterization of pectinases and cellulases was conducted using the larval digestive system of the pest. Midgut extracts from larvae showed optimum activity for pectinase and cellulase against pectin and carboxymethyl cellulose, respectively, under acidic conditions (pH 6). Pectinases and cellulases were respectively more stable under acidic conditions (pH 4–7) and slightly acidic conditions (pH 5–7) than under highly acidic and alkaline conditions. However, the enzymes were more stable in slightly acidic conditions (pH 6) when incubation time was increased. Maximum activity for the pectinases and cellulases incubated at different temperatures was observed at 45 and 50 °C, respectively. Mg²⁺ remarkably increased pectinase activity, and cellulase activity increased significantly in the presence of Ca²⁺ and Mg²⁺. Sodium dodecyl sulfate significantly decreased pectinase and cellulase activity. The Michaelis–Menten constant (K_M) and the maximal reaction velocity (V_max) values for pectinase were 2 mg·mL^? 1 and 0.017 mmol·min^? 1·mg^? 1 protein toward pectin, respectively. Zymogram analyses revealed the presence of one and five bands of pectinase and cellulase activity, respectively, in the larval midgut extract.     

The role of PopCel1 and PopCel2 in poplar leaf growth and cellulose biosynthesis

Poplar calli transcribed two cellulase (endo-1,4-beta-glucanase) genes, PopCel1 and PopCel2, whose mRNAs were differentially located in the growing leaves of poplar during cell wall synthesis. Histochemical and RT-PCR analyses of promoter-GUS fusion gene activities in transgenic poplar demonstrated that PopCel1 promoter-derived GUS activity was localized in the petiole and leaf veins, whereas PopCel2 was confined to mesophyll cells and disappeared from the tip during the development of leaves. Autoradiography of the leaf showed that the radioactivity of [14C]sucrose incorporated into cellulose corresponded to the combination of the sucrose-induced tissue-specific patterns of PopCel1 and PopCel2. Interestingly, 2,6-dichlorobenzonitrile (DCB) not only inhibited the incorporation of the radioactivity into cellulose, but also repressed the induction of both cellulase genes. Suppression of cellulases by expression of PopCel1 antisense cDNA or co-suppression of PopCel1 mRNA by overexpression of PopCel1 sense cDNA reduced leaf growth. Therefore, we came to the conclusion that PopCel1 and PopCel2 probably function to promote leaf growth in poplar by the endohydrolysis of 1,4-beta-glucan.     

Molecular and biochemical characterizations of a novel arthropod endo-β-1,3-glucanase from the Antarctic springtail,Cryptopygus antarcticus,horizontally acquired from bacteria

Collembolan species have been known to have β-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-β-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813 bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E–I–D–I–T–E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (K_m ～ 9.98 mg/mL) with an optimal temperature 50 °C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-β-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial β-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63 bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer.     

Catalytic properties of a GH10 endo-β-1,4-xylanase from Streptomyces thermocarboxydus HY-15 isolated from the gut of Eisenia fetida

A novel GH10 endo-β-1,4-xylanase (XylG) gene from Streptomyces thermocarboxydus HY-15, which was isolated from the gut of Eisenia fetida, was cloned, over-expressed, and characterized. The XylG gene (1182 bp) encoded a polypeptide of 393 amino acids with a deduced molecular mass of 43,962 Da and a calculated pI of 6.74. The primary structure of XylG was 69% similar to that of Thermobifida fusca YX endo-β-1,4-xylanase. It was most active at pH 6.0 and 55 °C. The susceptibilities of xylans to XylG were as follows: oat spelt xylan > birchwood xylan > beechwood xylan. The XylG also showed high activity (474 IU/mg) toward p-nitrophenylcellobioside. Moreover, at pH 6.0 and 50 °C, the V_max and K_m values of the XylG were 127 IU/mg and 2.51 mg/ml, respectively, for oat spelt xylan and 782 IU/mg and 5.26 mM, respectively, for p-nitrophenylcellobioside. A homology model indicated that XylG folded to form a (β/α)₈-barrel with two catalytic residues of an acid/base (Glu181) and a nucleophile (Glu289). The formation of a disulfide bond between Cys321 and Cys327 were predicted by homology modeling.     

In vitro effect of methyl farnesoate on the vitellogenin content of ovary and hepatopancreas,in the crayfish Cherax quadricarinatus

Vitellogenin levels were determined in pieces of either ovary or hepatopancreas taken from females of the crayfish Cherax quadricarinatus during both early pre-reproductive and reproductive periods, and exposed in vitro to 0.15, 1.5, or 15?µM of the juvenoid methyl farnesoate (MF). A significant accumulation of vitellogenin was seen in ovaries treated with 15?µM of MF during the early pre-reproductive period. Besides, protein synthesis, measured as tritiated leucine incorporation, was correspondingly increased in ovaries taken during both pre- and post-reproductive periods and exposed to 15?µM of MF. On the other hand, no stimulating effect of MF on the vitellogenin content of the hepatopancreas was seen in any period. These results, taken together, support the hypothesis of the endogenous vitellogenin synthesis stimulated by MF during the early ovarian maturation. However, this hormone was not able to stimulate vitellogenin synthesis in the ovary during the reproductive period.     

Haemolymph constituents and osmolality as functions of moult stage,body weight,and feeding status in marron,Cherax cainii () and yabbies,Cherax destructor (Clark, 1936)

The study investigates the change in osmolality and haemolymph constituents in marron Cherax cainii and yabbies Cherax destructor associated with moult stages, body weights and their feeding status. A total of 582 haemolymph samples from 5 moult stages (postmoult-AB, intermoult-C, and premoult stages – D₀, D₁, D₂), two body weight classes (2–15 g and 61–75 g) and nutritional status were used for analysis of osmolality, protein, glucose, and ionic concentrations of potassium and chloride following the standard biochemical procedures. The haemolymph protein, glucose, potassium and chloride levels were highest at intermoult and early premoult stages, and lowest at postmoult in both crayfish species. Except protein, no significant differences were seen in analyzed parameters between various weight classes and two species. Haemolymph osmolality, protein and glucose were significantly higher in fed crayfish, whereas no variations in haemolymph potassium and chloride concentrations were observed between the fed and unfed crayfish. Maximum osmolality was recorded at 7–8 h after feeding in both crayfish species. The results showed that the biochemical changes in the haemolymph of marron and yabbies are related to moult stages, body weight and feeding and thus can be used as tools for determining suitable diets.     

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.     

Sequence analysis and comparison of avocado fruit and bean abscission cellulases

A 1700 nucleotide cDNA clone for a bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase (endo-(1,4)-β-d-glucanase) has been identified and sequenced. This cDNA clone contains a 1485 nucleotide open reading frame which includes coding sequences for a putative signal peptide and mature protein. The nucleotide and deduced amino acid sequences for the bean abscission cellulase are compared to the previously reported sequences of an avocado fruit ripening cellulase. Optimal alignment of these sequences shows 64% and 50% identically matched nucleotides and amino acids, respectively. Analysis of the deduced amino acid sequences for the mature bean and avocado cellulases indicates that these two proteins share similar molecular weights, position of cysteine residues, and hydropathic character, but have very different isoelectric points and glycosylation. Genomic blot data suggest that the avocado fruit cellulase belongs to a small gene family, whereas the bean abscission cellulase appears to be encoded by a single gene or a few very closely related genes.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.