Kinetic study of catalytic CO(2) hydration by water-soluble model compound of carbonic anhydrase and anion inhibition effect on CO(2) hydration

A kinetic study of CO(2) hydration was carried out using the water-soluble zinc model complex with water-soluble nitrilotris(2-benzimidazolylmethyl-6-sulfonate) L1S, [L1SZn(OH(2))](-), mimicking the active site of carbonic anhydrase, in the presence and absence of anion inhibitors NCS(-) and Cl(-). The obtained rate constants k(cat) for CO(2) hydration were 5.9x10(2), 1. 7x10(3), and 3.1x10(3) M(-1) s(-1) at 5, 10, and 15 degrees C, respectively: the k(cat)=ca. 10(4) M(-1) s(-1) extrapolated towards 25 degrees C has been the largest among the reported k(cat) using zinc model complexes for carbonic anhydrase. It was also revealed that NCS(-), Cl(-) and acetazolamide play a role of inhibitors by the decrease of k(cat): 7x10(2) and 2x10(3) M(-1) s(-1) for NCS(-) and Cl(-) at 15 degrees C, respectively. The sequence of their magnitudes in k(cat) is Cl(-) approximately acetazolamide>NCS(-), where the sequence Cl(-)>NCS(-) is confirmed for native carbonic anhydrase. The difference of k(cat) or k(obs) between NCS(-) and Cl(-) resulted from that between the stability constants K(st)=2x10(3) for [L1SZn(NCS)](2-) and 1x10(2) M(-1) for [L1SZnCl](2-) in D(2)O: for water-insoluble tris(2-benzimidazolylmethyl)amine L1, K(st)=1.8x10(4) for [L1Zn(NCS)](2-) and 1.5x10(3) M(-1) for [L1ZnCl](2-)in CD(3)CN/D(2)O (50% v/v). The crystal structure of anion-binding zinc model complexes [L1Zn(OH(2))](0.5)[L1ZnCl](0.5) (ClO(4))(1.5) 1(0.5)2(0.5)(ClO(4))(1.5) was revealed by X-ray crystallography. The geometry around Zn(2+) in 1 and 2 was tetrahedrally coordinated by three benzimidazolyl nitrogen atoms and one oxygen atom of H(2)O, or Cl(-).     

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.     

Substrate specificity of thePenicillium lilacinum enzyme lytic to the cell wall ofRhodotorula glutinis and the structure of theRhodotorula cell wall glucomannan

The lytic enzyme active on cell walls ofRhodotorula, which was produced byPenicillium lilacinum ATCC 36010, decomposed an extracellular mannan fromRhodotorula glutinis IFO 0695 and was confined to the β-1,3-mannoside bond on the reducing side of the β-1,4-linkedd-mannopyranose. Based on these results, the structure of the glucomannan, which was a major component ofRhodotorula cell walls, was proposed.     

Brasilicardins B-D, new tricyclic terpenoids [correction of terpernoids] from actinomycete Nocardia brasiliensis

Three new tricyclic terpenoids, brasilicardins B-D (2-4), were isolated together with brasilicardin A (1), a potent immunosuppressive compound, from the cultured broth of a pathogenic actinomycete Nocardia brasiliensis IFM0406, and the structures and stereochemistry were determined by spectroscopic data and a single crystal X-ray diffraction analysis. The immunosuppressive and cytotoxic activities of 2-4 were examined in the comparison with 1.     

Evaluation of apolipoprotein B-100 fragmentation and cross-linkage in serum as an index of atherosclerosis

It is well established that radical reaction of low density lipoprotein (LDL) causes fragmentation and cross-linkage of apolipoprotein B-100 (apoB). Our previous studies demonstrated that fragmented and cross-linked apoB proteins are present in normal human serum and tended to increase with age based on immunoblot analysis. These observations suggest that the fragmentation and cross-linkage pattern of apoB reflects the oxidative stress in an individual and that this pattern is a good atherosclerotic index. In this study, a method was developed to evaluate the fragmentation and conjugation pattern of apoB. A parameter named B-ox was introduced for each serum sample to quantitate the staining bands of the immunoblotting analysis. B-ox represents the relative abundance of radical reaction products (a sum of fragmented and conjugated apoB proteins) based on one control subject. If this value increases, it indicates that radical reaction products have increased, i.e., the oxidative stress has increased in the subject. Based on measurements of subjects in a rural area of Japan, B-ox showed significant positive correlation with intima-media thickness (IMT) of the carotid artery, LDL cholesterol, and age, while it showed significant negative correlation with high density lipoprotein (HDL) cholesterol and vitamin C. These results suggest that B-ox is a reliable indicator of atherosclerosis.     

High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens

Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO.     

Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis

Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca²⁺ or Mg²⁺ at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).     

Acid-Stable α-Amylase of Black Aspergilli

The acid-stable α-amylase or the acid-unstable α-amylase from Aspergillus niger contained 24 moles or 7 moles mannose and 4 moles or 1 mole hexosamine per mole of protein, respectively.

The acid-stable α-amylase and the acid-unstable α-amylase contained calcium only, but not detectable amounts of other metals. Calcium contents of the both enzymes were converged to at least one gram atom per mole of enzyme by dialysis against acetate buffer. The last calcium could be removed under the suitable conditions by EDTA. Calcium removal by EDTA was accompanied by the loss of activity and by the little change of UV absorption spectra. The phenomenon caused by calcium removal were partially reversible. This last one atom of calcium seemed to be essential for the maintenance of active structure of α-amylase.     

Cell Aggregation Factor Produced by Streptomyces sp. Strain No. A-3315

We searched for a new aggregation factor, and found one we named 3315-AF in the culture filtrate of Streptomyces sp. strain No. A-3315. 3315-AF was purified by active carbon treatment, ethanol precipitation, gel filtration on Sepharose 2B, ether extraction, silica gel chromatography and gel filtration on Sephadex LH-20. 3315-AF was found to be a triglyceride which consists of myristic acid, pentadecanoic acid, and palmitic acid. The aggregation activity of 3315-AF was maximum around pH 8.0 at 30°C and the activity increased by addition of metallic ions such as calcium and cobalt. Hyaluronic acid, ovalbumin, BSA, and casein inhibited the aggregation activity. 3315-AF aggregated Proteus vulgaris and HeLa cells as well as Serratia marcescens and weakly aggregated Saccharomyces cerevisiae, Candida albicans, C. neoformans, and Leukemia P388, but it was inert to human erythrocytes and Sarcoma 180.     

Red Yeast Cell Lysis by Red Yeast Cell Wall Lytic Enzyme and Protease

The purified red yeast cell wall lytic enzyme of Penicillium lilacinum No. 2093 has a potent saccharifying activity against cell walls, but the living cell lytic activity of it is considerably lower than that of the culture filtrate. Therefore, the living cell lytic factors in the culture filtrate were examined. The alkaline protease of Pen. lilacinum played an important role for living cell lysis. The synergistic effect on living cell lysis was also detected, when acid proteases from various origins were combined with the cell wall lytic enzyme. These results indicated that the protein layers of red yeast cell surface inhibited the action of a glycanase,cell wall lytic enzyme, and the protein molecule contributed to retain the rigid structure of the wall.