Chitinase Chit62J4 Essential for Chitin Processing by Human Microbiome Bacterium Clostridium paraputrificum J4.

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-ß-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.

Rheological study of chitosan acetate solutions containing chitin nanofibrils.

Rheological properties of chitosan acetate solutions containing chitin nanofibrils (n-chitin) and biocompatible plasticizers intended for preparation of biodegradable films are reported in the steady, oscillatory and transient shear flow. The experiments were performed on slurries with an optimum proportion of 65/35 wt.% between chitosan and n-chitin in the films which was determined from our results of mechanical properties and absorption of water vapor. The time-dependent dynamic experiments revealed the chitin nanofibrils as an effective "gelling agent" of chitosan phase. The phenomenon is explained by a chitosan-like surface of n-chitin and by the interactions inducing orientational cooperativity of chitosan molecules dissolved in close neighborhood of the anisotropic chitin nanofibrils. Additions of glycerol or poly(ethylene glycol), improving mechanical properties of the films, delay significantly the onset of gelation of chitosan/n-chitin slurries. The effect is induced by an increase in viscosity of the slurries and by their enhanced chaotropic character.

Sample preparation in separation of the extracellular chitinolytic enzymes of the human intestinal bacterium Clostridium paraputrificum J4 from the culture fluids.

Membrane ultrafiltration (UF) was used in sample preparation of the culture fluids of the human intestinal bacterium Clostridium paraputrificum strain J4 containing seven extracellular chitinolytic isoenzymes (38-90 kDa). The subsequent filtration of the bacteria-free supernatants was carried out through Millipore membranes with cut-off 100 and 30 kDa for separation of undigested components of the culture medium and bacterial metabolites with molecular weight higher and lower than that of the target enzymes. The chitinolytic enzymes, which were the minor components in the culture fluids, were concentrated at UF as well. The aim of the research consisted in evaluation of the effect of component composition of bacteria-free supernatants and the chemical nature of membrane active layer on partial fractionation of the chitinolytic enzymes, their recovery in retentates and purification degree. On the basis of the obtained experimental results, the sample preparation procedure of the culture fluids of C. paraputrificum J4 was established to be used further in chromatographic separations of the chitinolytic enzymes.

Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract -- critical parameters of protein isolation from anaerobic culture.

The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum J4 isolated from the gastrointestinal tract of a healthy human. In particular, we focus on the development of purification protocols, determination of properties of the enzymes and their activity profiles. The process of bacteria cultivation and isolation of chitinolytic complex of enzymes showing specific activities of endo-, exo-chitinase and N-acetyl-ß-glucosaminidase was optimized. A range of various purification procedures were used such as ultrafiltration, precipitation, chromatographic separations (ion-exchange, size exclusion, chromatofocusing) in altered combinations. The optimal purification protocol comprises two or three steps. Individual samples were analyzed by SDS/PAGE electrophoresis and after renaturation their activity could be detected using zymograms. Mass spectroscopy peptide fragment analysis and MALDI analysis of the purest samples indicate presence of endochitinase B (molecular mass about 85 kDa) and of 60-kDa endo- and exochitinases.

Surface-deposited acid/base on glass microfibers in formation of (3-aminopropyl)triethoxysilane-[2-(3,4-epoxycyclohexyl)ethyl]heptaisobutyloctasilsesquioxane bioverlay.

Silanization of macroporous glass microfiber filters with (3-aminopropyl)triethoxysilane (APTES) and subsequent binding of [2-(3,4-epoxycyclohexyl)ethyl]heptaisobutyloctasilsesquioxane (E-POSS) to the amine-terminated surface of microfibers was studied. Prior to the silanization, minute quantities of concentrated aqueous solutions of hydrochloric acid or ammonia were adsorbed in the filters while attachment of E-POSS molecules to APTES overlay was not specially catalyzed. Analysis of DRIFT, XPS, and 13C CP/MAS NMR spectra has shown that the formation of APTES overlay is affected differently by the surface-deposited acid or base. It was proved by XPS that microfibers with the adsorbed acid take up higher amounts of covalently attached APTES by 42% and, subsequently, of E-POSS by 65% than microfibers with the adsorbed ammonia. The molecular mechanics model calculations, which were made using silica as a template, have shown that approximately two-layered APTES coating can be built on the model surface if complete hydrolysis of ethoxy groups and vertical condensation of APTES species are assumed.

Effect of the matrix structure and concentration of polymer-immobilized Ni2+-iminodiacetic acid complexes on retention of IgG1.

Terpolymer bead particles (100-350 microm in diameter) were prepared by suspension radical polymerization from methacrylate esters [2,3-epoxypropyl methacrylate (GMA), 2-(2-hydroxyethoxy)ethyl methacrylate (DEGMA) and ethylene dimethacrylate (EDMA)] and subsequently derivatized affording iminodiacetic acid (IDA) chelating sorbents. The sorbents differed in pore volumes (0-0.7 cm3/g) and specific surface areas (0.03-9.8 m2/g) of their matrices as well as in the amounts of immobilized Ni2+-IDA complexes (0.03-1.58 mmol/g). The binding of imidazole was studied by frontal chromatography to evaluate the accessibility of Ni2+-IDA complexes. It was found that an increase in the bonded imidazole content with increasing immobilized Ni2+-IDA concentration was strongly dependent on the matrix morphology. A higher pore volume of the matrix significantly improved the utilizability of Ni2+-IDA complexes for imidazole binding. The performance of the sorbents based on two porous matrices with immobilized Ni2+-IDA concentration (0.1-1.58 mmol/g) differing in pore size distributions was compared in immobilized metal affinity chromatography (IMAC) of monoclonal mouse immunoglobulin IgG1 specific against human choriogonadotropic hormone (GTH-spec IgG1). The results have shown that sorbents based on matrix with large pores (up to 20 microm in diameter) exhibited high protein binding capacities. The GTH-spec IgG1 (Mw=158,000) was eluted from all the sorbents in its native form as was confirmed by MALDI-TOF.

Nickel and copper complexes of a chelating methacrylate sorbent in the purification of chitinases and specific immunoglobulin G1 by immobilized metal ion affinity chromatography.

The isolation of the isoforms of endo- and exochitinases of Clostridium aminovalericum T1 and of the horseradish peroxidase (HRP)-specific immunoglobulin G1 from natural sources by immobilized metal ion affinity chromatography was studied. The effect of Cu2+ and Ni2+ complexes of iminodiacetic acid incorporated in porous glycidyl methacrylate-co-ethylene dimethacrylate and in agarose (Sepharose Fast Flow) beads on separation of the target polypeptides was analyzed. It was found that the Cu2+ complexes bound both the HRP-specific IgG1 and some isoforms of chitinases more strongly than the Ni2+ complexes. From the former complexes, both target polypeptides were eluted by a stepwise imidazole concentration gradient of 5-100 mM. The lower strength of Ni2+ complex binding with the HRP-specific IgG1 resulted in its easy elution with a pH gradient of 5.5-5 while some isoforms of chitinases required imidazole for their elution. The "fraction elution degree" of a target polypeptide (i.e., the ratio of its amounts in each eluate fraction and in the combined fractions) was used for the evaluation of the sorption selectivity and binding affinity of the separating components to the studied metal complexes.

Purification of the specific immunoglobulin G1 by immobilized metal ion affinity chromatography using nickel complexes of chelating porous and nonporous polymeric sorbents based on poly(methacrylic esters). Effect of polymer structure.

Ni2+ complexes of the chelating nonporous and porous bead sorbents based on methacrylic esters crosslinked with ethylene dimethacrylate were used in isolation of the horseradish peroxidase-specific immunoglobulin IgG1 from the crude mouse ascitic fluid by immobilized metal ion affinity chromatography (IMAC). Iminodiacetic and aspartic acids were attached to porous poly(glycidyl methacrylate) beads differing in size, morphology and chemical composition. Ethylenediaminetriacetic acid and quinolin-8-ol chelating groups were attached mainly to the surface hydroxyl groups in nonporous poly(diethylene glycol methacrylate) beads through spacers. The latter sorbents exhibited better kinetic characteristics than the former but a very low IgG1 sorption capacity. In a single-step IMAC procedure, the best efficiency in the specific IgG1 purification was obtained with porous sorbents (recovery 92%, purity 73%). Differences in IMAC separations are discussed from the point of view of morphology of polymer beads as well as of the type and concentration of chelating ligands.