Kenzo Koike

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like cyrstals.

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237

Abstract Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H3PO4-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely hydrolyzed at all.

Purification of alkaline proteases from a Bacillus strain and their possible interrelationship

Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55°C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60°C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5°C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.

Purification and characterization of alkaline endo-1,4-β-glucanases from alkalophilic Bacillus sp. KSM-635

Summary: Two carboxymethylcellulases (CMCase, 1,4-1,4-β-d-glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic Bacillus sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0.5m. The purified CMCases both contained approximately 2–3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9.5, and their optimum temperatures were around 40°C. Activities of both enzymes were inhibited by Hg2+, Cu2+, Fe2+ and Fe3+, but sulphydryl inhibitors, such as N-ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. N-Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from Bacillus. In addition, the activities of both E-H and E-L were stimulated by Co2+, and they required Mg2+, Ca2+, Mn2+ or Co2+ for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (β-1,4-linkage) and lichenan (β-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (β-1,3-linkage), laminarin (β-1,3; 1,6-linkage) and 4-nitrophenyl-β-d-glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl-β-d-cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.

ENZYMATIC PROPERTIES AND DEDUCED AMINO ACID SEQUENCE OF A HIGH-ALKALINE PECTATE LYASE FROM AN ALKALIPHILIC BACILLUS ISOLATE

A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from alkaliphilic Bacillus sp. strain KSM-P7, designated Pel-7, was purified to homogeneity. The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was close to or higher than pH 10.5. In the presence of Ca2+ ions, Pel-7 trans-eliminated polygalacturonate in random manner to generate oligogalacturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 65 degrees C in glycine-NaOH buffer. Mn2+ and Sr2+ ions can serve as cofactors at almost the same level of Ca2+ ions. It also exhibited a protopectinase-like activity, liberating soluble pectin and/or oligogalacturonides from cotton fibers. The pel gene was cloned and sequenced, and the deduced amino acid sequence of mature Pel-7 (302 amino acids, 33, 355 Da) showed some conserved regions in Pel superfamily, although homology to amino acid sequences of known Pels with 27 to 32% identity. Furthermore, Pel-7 appears to have similar core structure of parallel beta-helix and active site topology with other Pels as revealed by secondary structure prediction in the Pel proteins. These results suggest that Pel-7 is basically grouped into Pel superfamily although the enzymatic and molecular properties are different.

Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an alkaliphilic Bacillus strain

Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-β-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-amino-acid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.

Cortical cell types and intermediate filament arrangements correlate with fiber curvature in Japanese human hair

Naturally straight and curved human scalp hairs were examined using fluorescence and electron microscopy techniques to determine morphological and ultrastructural features contributing to single fiber curvature. The study excluded cuticle and medulla, which lack known bilateral structural asymmetry and therefore potential to form curved fibers. The cortex contained four classifiable cell types, two of which were always present in much greater abundance than the remaining two types. In straight hair, these cell types were arranged annularly and evenly within the cortex, implying that the averaging of differing structural features would maintain a straight fiber conformation. In curved fibers, the cell types were bilaterally distributed approximately perpendicular to fiber curvature direction with one dominant cell type predominantly located closest to the convex fiber side and the other, closest to the concave side. Electron tomography confirmed that the dominant cell type closest to the convex fiber side contained discrete macrofibrils composed of helically arranged intermediate filaments, while the dominant cell type closest to the concave side contained larger fused macrofibrils composed of intermediate filament arrangements varying from helical to hexagonal arrays approximately parallel to the longitudinal fiber axis. These findings concur with the current hypothesis of hair curvature formation and behavior.

Laser capture microscopy in a study of expression of structural proteins in the cuticle cells of human hair

Abstract:  The cuticle of human hair consists of several layers of flat cells that are hardened through their content of cross‐linked proteins and protect the hair structure from the environment. Known proteins in the cuticle are the sulphur‐rich KAP 5 and KAP10 proteins located in the exocuticle and cross‐linked by disulphide bonds. Isopeptide bonds are also present and led to a proposal from amino acid analysis that the surface of cuticle cells also contains keratinocyte cell envelope proteins, loricrin, involucrin and small proline‐rich proteins that contribute to the stability of the hair cuticle. Confirmation of that proposal by protein chemical methods is difficult because of the insolubility of the surface membranes. In the previous studies by other authors, involucrin was not detected in the cuticle by in situ hybridization or by immunoelectron microscopy with specific antibodies. An alternative approach was undertaken to determine whether mRNAs encoding keratinocyte envelope proteins are expressed in cuticle cells in the human hair follicle. The study utilized dissection of the cuticle, cortex and inner root sheath layers from follicles by laser capture microscopy. RNA was isolated and subjected to PCR analysis with specific primers to detect expression of mRNAs encoding cell envelope proteins. Their presence in the cuticle was not detected, and it was concluded that the proteins they encode are not produced. The structural consequences including the possibility that KAPs 5 and 10 are the prime components cross‐linked by both disulphide and isopeptide bonds are discussed.

Production of cis-unsaturated hydrocarbons by a strain of Rhodococcus in repeated batch culture with a phase-inversion, hollow-fiber system

Abstract A mutant strain of Rhodococcus was found to cis -desaturate a variety of hydrocarbons and acyl fatty acids at central positions of the chains. Application of phase inversion of the reaction medium, from an oil-water to a water-oil emulsion, to the desaturation reaction contributed to an improvement in the productivity of repeated batch operations with a hydrophobic hollow fiber.

Eumelanin broadband absorption develops from aggregation-modulated chromophore interactions under structural and redox control

Eumelanins, the chief photoprotective pigments in man and mammals, owe their black color to an unusual broadband absorption spectrum whose origin is still a conundrum. Excitonic effects from the interplay of geometric order and disorder in 5,6-dihydroxyindole (DHI)-based oligomeric/polymeric structures play a central role, however the contributions of structural (scaffold-controlled) and redox (π-electron-controlled) disorder have remained uncharted. Herein, we report an integrated experimental-theoretical entry to eumelanin chromophore dynamics based on poly(vinyl alcohol)-controlled polymerization of a large set of 5,6-dihydroxyindoles and related dimers. The results a) uncover the impact of the structural scaffold on eumelanin optical properties, disproving the widespread assumption of a universal monotonic chromophore; b) delineate eumelanin chromophore buildup as a three-step dynamic process involving the rapid generation of oxidized oligomers, termed melanochromes (phase I), followed by a slow oxidant-independent band broadening (phase II) leading eventually to scattering (phase III); c) point to a slow reorganization-stabilization of melanochromes via intermolecular redox interactions as the main determinant of visible broadband absorption.