Band In SDS-PAGE Research Articles

Extração e caracterização de pectinametilesterase (PME) de resíduos agroindustrais de abacaxi pérola

The objective was to obtain and characterize pectinmethylesterase (PME) of industrial residue from the Pearl Pineapple. Specific activities, the optimum conditions of pH and temperature, and also the thermal stability partially purified and concentrated PME were determined. The PME specific activity was 6.36 U/mg. The pH optimum of 7.5 PME was extracted, also considering alkaline. During the trial time, the partially purified PME maintained itself stable upon exposure to the temperatures of 25 and 60ºC. It obtained an activity elevation between 40 and 50ºC. In temperatures above 70ºC, there was a substantial reduction in a specific activity. The partially purified PME showed three gel polyacrylamide SDS-PAGE bands of apparent molar mass (MM) of 25.4 and 23.1 kDa and 16.4 kDa. These results demonstrate the feasibility of PME extraction from the Pearl Pineapple’s processed residue and its potential for use in manufacturing pulp and fruit juices, since the currently applied enzymes are imported and expensive.

Insights into the fermentation biochemistry of Kombucha teas and potential impacts of Kombucha drinking on starch digestion

The biochemistry of Kombucha fermentation was analyzed using green and black teas (GTK and BTK). Sucrose disappeared linearly in Kombucha mediums during the two week fermentation period for GTK (2.3g.day−1.L−1) but only during the first week for BTK (5.0g.d−1.L−1). The produced glucose and fructose formed disappeared faster in BTK than in GTK and in both fermentation mediums glucose was preferred to fructose as the carbon source. Ethanol, acetate equivalents and cellulose linearly increased during the fermentation (0.17, 0.35 and 0.50g.d−1.L−1 in GTK, and 0.15, 0.46 and 0.63g.d−1.L−1 in BTK, respectively). Likewise, the disappearance of glucose+fructose in Kombucha first linearly increased during the first stages of fermentation before it dropped at day 9 for GTK and at day 12 for BTK, suggesting a change of the carbon source. The protein fraction (<0.5g/L) transiently increased during the fermentation of teas and so did a 60-kDa protein band in SDS-PAGE. In both GTK and BTK, only slight changes were observed in total phenolics and in the main tea flavanols. Nevertheless, the theaflavins moderately increased (more than 50% at day 15) and the thearubigins markedly decreased (more than 2-fold at day 15). The Kombucha beverage was able to strongly inhibit starch hydrolysis by porcine pancreatic alpha-amylase and the inhibition potency increased during fermentation progress. The active compounds were suspected to be monomeric and/or oligomeric phenolic compounds. After drinking a bowl of Kombucha beverage, the concentration of these phenolics could be sufficient for inhibiting, to a certain extent, pancreatic alpha-amylase in the small intestine, with a possible impact on starch digestion and on net absorbed glucose.

Effectiveness of egg yolk immunoglobulin (IgY) against periodontal disease-causing Fusobacterium nucleatum

To evaluate the in vitro and in vivo effectiveness of egg yolk immunoglobulin (IgY) against periodontal disease-causing Fusobacterium nucleatum. Four White Leghorn hens (120days old) were immunized with whole Fus.nucleatum cells killed with 1% formaldehyde using three injections provided at 2-week intervals. IgY was produced from egg yolks obtained from these immunized hens using water dilution, two-step salt precipitation and ultrafiltration. This IgY was shown to have a purity of 86·8% based on its optical intensity in the stained SDS-PAGE bands. An enzyme-linked immunosorbent assay indicated a high specificity for the IgY against Fus.nucleatum with a maximum antibody titre of 80000. The IgY had only weak cross-reactivity with Porphyromonas gingivalis, Prevotella intermedia and Solobacterium moorei. Growth and biofilm formation by Fus.nucleatum were inhibited by IgY at concentrations of 10 and 20mgml(-1) . Immunofluorescence and immunoelectron microscope assays revealed a high binding ability of specific IgY, which may explain the in vitro effectiveness of IgY. In an in vivo study, IgY treatment resulted in a marked decrease in alveolar bone loss after Fus.nucleatum infection in a mouse model confirming the effectiveness of IgY against periodontal disease-causing Fus.nucleatum. IgY effectively inhibited growth and biofilm formation by Fus.nucleatum and prevented the progression of periodontal disease by decreasing alveolar bone loss. Specific IgY may have potential for the treatment of periodontal disease.

Chemical Composition and Bioactivity of Polypeptide from Velvet Antlers of Cervus nippon Temminck

In order to study the isolation and purification of an active peptide component from velvet antler of sika deer(Cervus nippon Temminck),confirm the primary structure of the novel peptide and compare the sequence and the activity with those of velvet antler of red deer(Cervus elaphus Linaeus),the velvet antler polypeptide was isolated and purified by ion exchange chromatography,gel filtration and RP-HPLC,which showed a single peak in HPLC chromatography and a single band in SDS-PAGE.The molecular weight was measured by MALDI-TOF MS.This polypeptide consists of a single linear chain of 32 amino acid residues VLSATDKTNVLAAWGKVGGNAPAFGAEALERM.The experiment results of bioactivity showed that the polypeptide had stimulant effects on the growth of rat epithelial cells,rabbit costal chondrocytes and rat NIH3T3 cell line,respectively in vitro.The straight chain of 32 amino acid residues of the velvet antler polypeptides of Cervus nippon Temminck was consisted with that of Cervus elaphus Linaeus,but they make a difference of 5,8,11 and 30 amino acid.There was no effect on stimulating proliferation of cells.

Pseudoalteromonas strains are potent immunomodulators owing to low-stimulatory LPS

Many species of marine bacteria elicit a weak immune response. In this study, the aim was to assess the immunomodulatory properties of Gram-negative Pseudoalteromonas strains compared with other marine Gram-negative bacteria and to identify the molecular cause of the immunomodulation. Using murine bone-marrow derived dendritic cells (DCs), it was found that Pseudoalteromonas strains induced low cytokine production and modest up-regulation of surface markers CD40 and CD86 compared with other marine bacteria and Escherichia coli LPS. Two strains, Ps. luteoviolacea and Ps. ruthenica, were further investigated with respect to their immunomodulatory properties in DCs. Both inhibited IL-12 and increased IL-10 production induced by E. coli LPS. LPS isolated from the two Pseudoalteromonas strains had characteristic lipid A bands in SDS-PAGE. Stimulation of HEK293 TLR4/MD2 cells with the isolated LPS confirmed the involvement of LPS and TLR4 and established Pseudoalteromonas LPS as TLR4 antagonists. The isolated LPS was active in the endotoxin limulus amoebocyte lysate assay and capable of inducing increased endocytosis in DCs. This study highlights that antagonistic LPS from Pseudoalteromonas strains has potential as a new candidate of therapeutic agent capable of modulating immune responses.

Cloning and expression of a novel antifreeze protein AFP72 from the beetle Tenebrio molitor

A novel antifreeze protein AFP72 cDNA (GenBbank accession No. AY929389) was obtained by RT-PCR from Tenebrio molitor. The 216 bp fragment encodes a protein of 72 amino acid residues. Sequence analysis revealed that the cDNA displays a high degree of homology with T. molitor antifreeze proteins, ranging up to 90.78%. Recombinant plasmids pMAL-p2X-afp72 and pMAL-c2X-afp72 were transferred into E. coil TBI to induce a MBP fusion protein by IPTG. The target fusion protein was released from the periplasm and cytoplasm by the cold osmotic shock procedure and sonication respectively. The content of the fusion protein came up to 38.9 and 41.5% of the total dissolved protein, respectively. The fusion protein was purified through an amylose affinity column, and incised by factor Xa. Molecular sieve chromatography was used to achieve a high state of purity of the target protein. The purified target protein displayed a single band in SDS-PAGE. The fusion protein was shown to increase resistance to low temperatures in bacteria. This finding could help in further investigations of the properties and function of antifreeze proteins.

Isolation of a Glucosamine Binding Leguminous Lectin with Mitogenic Activity towards Splenocytes and Anti-Proliferative Activity towards Tumor Cells

A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. “brown kidney bean.” The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20°C–60°C. However, drastic reduction of the activity occurred at temperatures above 65°C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0–2, and only residual activity was observed at pH 13–14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC50 of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

Novel HMW glutenin genes from Aegilops tauschii and their unique structures

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx5.3t and 1Dy12.1**t were revealed and characterized from Ae. tauschii accession PI554324. SDS-PAGE band of 1Dx5.3t was between those of 1Bx6 and 1Bx7, while 1Dy12.1**t with slightly faster migration rate than that of 1Dy12. The lengths of 1Dx5.3t and 1Dy12.1**t were 2115 bp and 1986 bp, encoding 703 and 660 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. 1Dx5.3t is the shortest of the known Dx-type alleles. 1Dy12.1**t is also a special subunit since it has an additional cysteine in the front of the central repetitive domain. This cysteine that is not existed in previously reported Dy-type genes may be useful for improving bread wheat quality. Median-joining Network analysis indicated that 1Dy12.1**t may be a key site in the genealogy of the Glu-Dy.

Competitive Binding of the SecA ATPase and Ribosomes to the SecYEG Translocon

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.

가압가열 및 microwave에 의한 중력분 반죽 gliadin의 항원성 변화

본 연구에서는 가압가열 및 microwave 처리에 의한 중력분 반죽 추출물 내의 gliadin 단백질의 항원성 변화에 대해 살펴보았다. 중력분 반죽에 가압가열과 microwave를 단독 또는 병행으로 처리하여 ci-ELISA, SDS-PAGE 및 immunoblotting을 실시하였으며, 가압가열 처리에 의해서 anti-gliadin IgG 항체와 gliadin과의 결합력이 다소 감소한 것을 확인하였다. 특히 30 min 이상 처리시 더욱 감소한 것으로 나타났으며, SDS-PAGE와 immunoblotting 결과에서도 gliadin band의 강도가 약해지고 항체와의 반응도 나타나지 않았다. Microwave 처리의 경우, 5 min 이상 처리시 일부gliadin 단백질의 소실이 관찰되었으나, 항원성에는 큰 변화가 없었다. 또한 가압가열 및 microwave 병행 처리에 의해 항원-항체 결합력이 더욱 감소되었으며, 특히 가압가열 50 min, microwave 10 min 처리시 약 35.0%로 감소되었다. 이상의 결과를 통해 가압 가열 처리에 의해 중력분 반죽 추출물 내 gliadin의 항원성이 감소되는 것을 확인하였으며, microwave와 병행 처리하는 경우, 더욱 감소하는 것을 확인하였다. The aim of this study was to determine the optimal physical treatment to reduce the antigenicity of gliadin in wheat dough. Medium wheat dough was treated with an autoclave (5, 10, 30, and 50 min at <TEX>$121^{\circ}C$</TEX>, 1 atm), a microwave (1, 5, and 10 min) or both (10, 30, and 50 min/5, 10 min). The proteins in the dough extracts were analyzed by SDSPAGE and the binding ability of anti-gliadin IgG to gliadin was examined by ci-ELISA and immunoblotting. Results showed that the ability of anti-gliadin IgG to bind to gliadin in wheat dough treated with an autoclave alone or in combination with a microwave was decreased. Especially, it declined to ~77% after autoclaving for 30 min and 35% after both autoclaving for 50 min and microwaving for 5 min. In addition, the intensity of gliadin bands in SDS-PAGE were weakened and anti-gliadin IgG did not recognize gliadin in immunoblotting. However, microwaving alone did not affect the antigenicity of gliadin in wheat dough. These results indicate that autoclaving may affect the reduction of the antigenicity of gliadin in medium wheat dough. Moreover, autoclaving in combination with microwaving is more effective for reducing the antigenicity of wheat dough.

Constitutive expression, purification and characterization of bovine prochymosin in Pichia pastoris GS115

Chymosin can specifically break down the Phe105-Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS-PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25-50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn(2+), Fe(3+), Mg(2+) and Na(+), but inhibited by K(+), Co(2+), Zn(2+), Ni(2+), and to a lesser extent by Cu(2+). These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.

Novel and ancient HMW glutenin genes from Aegilops tauschii and their phylogenetic positions

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx3.1t and 1Dy11*t were revealed and characterized from Aegilops tauschii Coss. subspecies tauschii accession AS60. SDS-PAGE band of 1Dx3.1t was between those of 1Dx2 and 1Dx3, while 1Dy11*t was between 1Dy11 and 1Dy12. The lengths of 1Dx3.1t and 1Dy11*t were 2,514 bp and 1,968 bp, encoding 836 and 654 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. Network analysis indicated that 1Dx3.1t together with other five rare alleles only detected in Asia common wheat populations represented the ancestral sequences in Glu-D1 locus. Neighbor-joining tree analysis of previously cloned x-type and y-type alleles in the Glu-D1 locus supported the hypothesis that more than one Ae. tauschii genotypes were involved in the origin of hexaploid wheat and that different Ae. tauschii accessions contributed the D genome to common wheat and Ae. cylindrical Host, respectively. An Ae. tauschii accession with 1Dx3.1t or a closely related allele probably have involved in the origin of common wheat. Since accession AS60 used in this study belonged to typical ssp. tauschii, present results suggested the possibility that ssp. tauschii was involved in the evolution of common wheat.

Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application

The use of pediocins as food additives or drugs requires a simple and rapid method by which large quantities of homogeneous pediocin are produced at industrial level. Two centrifugation steps required during initial stages of purification i.e. separation of cells from fermentation broth and collection of precipitates after ammonium sulphate precipitation are the major bottlenecks for their large scale purification. In the present work, pediocin production by a new a dairy strain, Pediococcus pentosaceous NCDC 273 (identical to pediocin PA-1 at nucleotide sequence level), was found to be optimum at initial pH of 6.0 and 7.0 of basal MRS supplemented with 20g/l of glucose or lactose at 20 and 24h, respectively. Immobilization of cells through entrapment in alginate-xanthan gum gel beads with chitosan coating resulted in negligible cell release during fermentation. Thus, the cell free extract was directly collected through decantation, avoiding the need of centrifugation step at this stage. Subsequent ammonium sulphate precipitation at isoelectric point of pediocin PA-1 (8.85), using magnetic stirrer at high speed (approx. 1200rpm), resulted in forceful deposition of precipitates on the wall of precipitation beaker allowing their collection using a spatula, avoiding centrifugation step at this stage also. Further purification using cation-exchange chromatography resulted in yield of 134.4% with more than 320 fold purification with the specific activity of 19×105AU/mg. The collection of single peak of pediocin at 41.9min in RP-HPLC, overlapping with standard pediocin PA-1, resulted in yield of 1.15μg from 20μl of sample applied. The overlapping of RP-HPLC peak and SDS-PAGE band corresponding to 4.6kDa, confirmed the purity and identity of pediocin 273 as pediocin PA-1.

Identification of Cashmere Fiber by Using SDS-PAGE and Maldi-Tof Mass Spectrometry

Cashmere, wool and yak were reduced with dithiothreitol in 0.1 M phosphate buffer (pH 7.8) containing 4% sodium dodecyl sulfate at 95℃. Keratin proteins were extracted with around 80% yield. Higher SDS concentration and temperature were important factor for effective extraction of keratin proteins from wool and animal fiber. More than 8 bands were observed in SDS-PAGE analysis of extracted proteins. Maldi-Tof mass analysis of tryptic digests of major bands in SDS-PAGE and data base search showed that those bands are assigned to be keratin I and II. Typical peaks for keratin I of cashmere, wool and yak were observed in the region between 2500 and 2700 m/z in mass spectra and these peaks were shown to be very useful for identification of cashmere, wool and yak.

Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50°C for 16h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299u/mg) with 300mM Ca(2+) and was stable up to 60°C with 150mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.

Expression, Purification and Characterization of the Recombinant Hirudin Variant iii in the Bacillus Subtilis

Hirudin is the most potent natural inhibitor of thrombin and a powerful anticoagulant. Large-scale production of recombinant hirudin is desirable for therapy. In this study, the gene encoding hirudin variant III was redesigned and synthesized by usingBacillus subtilispreferred codons, and the recombinant hirudin variant III (rHV3) was overexpressed inB. subtilisDB403 with strong anticoagulation activity for the first time. The hirudin activity from the supernatant of culture with optimized expression conditions could reach 210 ATU/ml. The protein in culture supernatant was precipitated by trichloroacetic acid, then desalted by ultrafiltration and purified by anion exchange chromatography. Strong anion Q F.F. performed better than weak anion DEAE F.F. The proper pH and conductivity was determined at pH 8 and 6 ms/cm, respectively. The maximum applied sample was 240 ATU/ml to medium of strong anion Q F.F. This optimized procedure was employed in strong anion exchange HiPrep 16/10Q with the 90% recovery rate and 70.2% purity. After gel filtration, the purity of rHV3 checked by HPLC could reach 95.1%, and the recovery rate was 93% for this step. The purified recombinant rHV3 showed a single band in SDS-PAGE. The rHV3 was stable at 100 °C and acidity condition, but was unstable under the condition of both heating and alkalinity. In conclusion, theses studies suggests thatB.subtilismight be useful for the production of biologically active medicine peptides in secretion facilitating purification procedures, and that this isolation method was suitable for scale-up purification process at a low cost.

Correlating crosslink formation with enzymatic activity in cysteine dioxygenase

Cysteine dioxygenase (CDO) from rat and other mammals exhibits a covalent post-translational modification between the residues C93 and Y157 that is in close proximity to the active site, and whose presence enhances the enzyme's activity. Protein with and without C93-Y157 crosslink migrates as distinct bands in SDS-PAGE, allowing quantification of the relative ratios between the two forms by densitometry of the respective bands. Expression of recombinant rat wild type CDO in Escherichia coli typically produces 40–50% with the C93-Y157 crosslink. A strategy was developed to increase the ratio of the non-crosslinked form in an enzyme preparation of reasonable quantity and purity, allowing direct assessment of the activity of non-crosslinked CDO and mechanism of formation of the crosslink. The presence of ferrous iron and oxygen is a prerequisite for C93-Y157 crosslink formation. Absence of oxygen during protein expression increased the fraction of non-crosslinked CDO, while presence of the metal chelator EDTA had little effect. Metal affinity chromatography was used to enrich non-crosslinked content. Both the enzymatic rate of cysteine oxidation and the amount of cross-linking between C93 and Y157 increased significantly upon exposure of CDO to air/oxygen and substrate cysteine in the presence of iron in a hitherto unreported two-phase process. The instantaneous activity was proportional to the amount of crosslinked enzyme present, demonstrating that the non-crosslinked form has negligible enzymatic activity. The biphasic kinetics suggest the existence of an as yet uncharacterised intermediate in crosslink formation and enzyme activation.

Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.

Cross-linking of collagen with laccases and tyrosinases

Oxidation of acid soluble collagen (ASC), collagen suspension and BrCN-peptides (BrCN-P) with tyrosinases from B. obtusa (BoT1, BoT2) and A. bisporus (AbT) and laccases from T. versicolor (TvL) and T. hirsuta (ThL) resulted in UV/VIS peaks at 475 nm and 305 nm indicating formation of reactive o-quinones and cross-linked components. Concomitant oxygen consumption was higher for the low molecular weight enzymes (TvL and BoT2) indicating limited accessibility. SDS-PAGE and SEC bands at higher MW demonstrated the formation of cross-linked material. LC-MS/MS analysis suggested the involvement of tyrosine residues in cross-linking without major changes of sequence similarities to untreated collagen. However, an increase of the SEC α-peak together with a decrease of β-peak and the 1235/1450 cm − 1 ratio (FTIR) indicated partial degradation. Crosslinking was enhanced by phenolic molecules such as catechine which lead to increased denaturation temperature and reduced degradation by microbial collagenase. The tensile strength was increased whereas resistance to compressive forces was not influenced.

A Cysteine Endopeptidase (“Dionain”) Is Involved in the Digestive Fluid ofDionaea muscipula(Venus’s Fly-trap)

The carnivorous plant Dionaea muscipula (Venus's flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS-PAGE band protein. The name "dionain" is proposed for the enzyme.