Carboxy-terminus truncations of Bacillus licheniformis SK-1 CHI72 with distinct substrate specificity

Bacillus licheniformis SK-1 naturally produces chitinase 72 (CHI72) with two truncation derivatives at the C-terminus, one with deletion of the chitin binding domain (ChBD), and the other with deletions of both fibronectin type III domain (FnIIID) and ChBD. We constructed deletions mutants of CHI72 with deletion of ChBD (CHI72ΔChBD) and deletions of both FnIIID and ChBD (CHI72ΔFnIIIDΔChBD), and studied their activity on soluble, amorphous and crystalline substrates. Interestingly, when equivalent amount of specific activity of each enzyme on soluble substrate was used, the product yield from CHI72- ΔChBD and CHI72ΔFnIIIDΔChBD on colloidal chitin was 2.5 and 1.6 fold higher than CHI72, respectively. In contrast, the product yield from CHI72ΔChBD and CHI72ΔFnIIID- ΔChBD on Β-chitin reduced to 0.7 and 0.5 fold of CHI72, respectively. These results suggest that CHI72 can modulate its substrate specificities through truncations of the functional domains at the C-terminus, producing a mixture of enzymes with elevated efficiency of hydrolysis.     

A comparative investigation of various cellulase assay procedures

The cellulolytic activity of crude enzyme preparations from different cellulolytic fungi (namely Trichoderma viride, Trichoderma Koningii, Fusarium solani, Sporotrichum pulverulentum, Sporotrichum thermophile) was assayed comparatively with several common analytical procedures described in the literature. The investigation was carried out with the objective of evaluating, with raw culture filtrates, the different cellulase tests in relation to their specificity for endo- and exo-cellulase action as well as to allow comparisons to be made between results from different research groups using different methods. (1)Cellulase activity was tested viscometrically as well as chemically (determination of reducing end groups) with different carboxymethylcelluloses as substrates. Essentially constant ratios between both kinds of activities were obtained, indicating that they are directly related. Nevertheless, international units of activity, calculated from viscometric measurements (glycosidic bonds broken per unit time) were considerably lower than international units deduced from the increase in reducing power (glucose equivalents liberated per unit time), this discrepancy most likely accounted for by the predominant influence of the exo-cellulase component in cellulase tests based on the determination of reducing eng groups. (2) By estimating cellulase activity with insoluble cellulosic substrates no direct relationship could be established with the above-described activities except in the case where the cellulose was amorphous. The ratio profile between activities thus obtained and endo-cellulase activities determined viscometrically shows that some enzyme preparations (such as those from both Trichoderma sp.) are clearly more active than others against crystalline cellulose reflecting quantitative differences in enzyme composition. Nevertheless, for a biological understanding of cellulolysis. analytical procedures using crystalline celluloses are not adequate for specifically monitoring exo-cellulase activity in crude enzyme solutions for essentially two reasons: (a) they are not sufficiently sensitive to detect small changes in enzyme activity during the early phase of growth, and (b) exo-cellulase activity in crude enzyme solutions also depends on the endo-cellulase activity present.     

Substrate binding modes and anomer selectivity of chitinase A from Vibrio harveyi

High-performance liquid chromatography mass spectrometry (HPLC MS) was employed to assess the binding behaviors of various substrates to Vibrio harveyi chitinase A. Quantitative analysis revealed that hexaNAG preferred subsites −2 to +2 over subsites −3 to +2 and pentaNAG only required subsites −2 to +2, while subsites −4 to +2 were not used at all by both substrates. The results suggested that binding of the chitooligosaccharides to the enzyme essentially occurred in compulsory fashion. The symmetrical binding mode (−2 to +2) was favored presumably to allow the natural form of sugars to be utilized effectively. Crystalline α chitin was initially hydrolyzed into a diverse ensemble of chitin oligomers, providing a clear sign of random attacks that took place within chitin chains. However, the progressive degradation was shown to occur in greater extent at later time to complete hydrolysis. The effect of the reducing-end residues were also investigated by means of HPLC MS. Substitutions of Trp275 to Gly and Trp397 to Phe significantly shifted the anomer selectivity of the enzyme toward β substrates. The Trp275 mutation modulated the kinetic property of the enzyme by decreasing the catalytic constant (k _cat) and the substrate specificity (k _cat/K _m) toward all substrates by five- to tenfold. In contrast, the Trp397 mutation weakened the binding strength at subsite (+2), thereby speeding up the rate of the enzymatic cleavage toward soluble substrates but slowing down the rate of the progressive degradation toward insoluble chitin.     

Cloning, heterologous gene expression and biochemical characterization of the alpha-1,3-glucanase from the filamentous fungus Penicillium purpurogenum

There has been much recent interest in α-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for α-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 °C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.     

Production of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

The pharmaceutically important compound N-acetylglucosamine (NAG), is used in various therapeutic formulations, skin care products and dietary supplements. Currently, NAG is being produced by an environment-unfriendly chemical process using chitin, a polysaccharide present in abundance in the exoskeleton of crustaceans, as a substrate. In the present study, we report the potential of an eco-friendly biological process for the production of NAG using recombinant bacterial enzymes, chitinase (CHI) and chitobiase (CHB). The treatment of chitin with recombinant CHI alone produced 8% NAG and 72% chitobiose, a homodimer of NAG. However, supplementation of the reaction mixture with another recombinant enzyme, CHB, resulted in approximately six fold increase in NAG production. The product, NAG, was confirmed by HPLC, TLC and ESI-MS studies. Conditions are being optimized for increased production of NAG from chitin.     

Potential Interaction of Plasmodium falciparum Hsp60 and Calpain

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]₂ and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K_m and k_cat values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4–G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2–G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1–G5, and then the high-M_r intermediates (G3–G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

Synthesis of 2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes via LiAlH4-induced reductive cyclization of 2-(4-chloro-2-cyano-2-phenylbutyl)aziridines and evaluation of their antimalarial activity

2-(4-Chloro-2-cyano-2-phenylbutyl)aziridines were employed for the one-step stereoselective construction of both endo- and exo-2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes as new azaheterobicyclic scaffolds via a double LiAlH₄-induced reductive cyclization protocol. Antiplasmodial assessment of these 1-azabicyclo[2.2.1]heptanes revealed moderate to good activities in the micromolar range, with the exo-isomers being the most promising structures. Furthermore, the proposed mode of action was supported by ligand docking studies, pointing to a strong binding interaction with the enzyme plasmepsin II.     

Tropane alkaloid metabolism by Pseudomonas AT3 cell cultures: Interchange between the nortropine and norpseudotropine catabolic pathways

Selected strains of Pseudomonas bacteria can degrade tropane alkaloids to obtain both nitrogen and carbon for growth. In order to probe the mechanisms of the catabolic enzymes involved, the metabolic process responsible for the opening of the 8-azabicyclo[3.2.1]octan-3-ol ring of nortropane alkaloids has been explored. It is found that the bacteria contain considerable flexibility in their enzyme complement and can convert (3-endo)-8-azabicyclo[3.2.1]octan-3-ol) (nortropine (2) to (3-exo)-8-azabicyclo[3.2.1]octan-3-ol) (norpseudotropine). Both of these compounds can serve as substrates for the catalytic cascade. In order to establish the proportionation between direct and indirect pathways, metabolism has been probed by competitive substrate availability and by incorporation of stable heavy labels into substrate pools. The results indicate that, while norpseudotropine is almost entirely metabolized directly, nortropine is partitioned c. 4:1 between direct and indirect catabolism.     

Influence of organic co-solvents on the activity and substrate specificity of feruloyl esterases

Organic co-solvents can expand the use of enzymes in lignocellulose deconstruction through making substrates more soluble and thus more accessible. In choosing the most adequate co-solvent for feruloyl esterases, hydrolysis of methyl p-hydroxycinnamates by three pure enzymes (and a multi-enzyme preparation) was evaluated. Low concentrations of dimethylsulfoxide (DMSO) enhanced hydrolysis by two of the enzymes while at levels >20%, activity was reduced. DMSO also enhanced acetyl esterase-type activity of the enzymes. The co-solvent effect was different for each enzyme-substrate couple, indicating that other factors are also involved. Kinetic studies with a Talaromyces stipitatus feruloyl esterase showed low concentrations of dimethylsulfoxide enhanced the hydrolytic rate while K_m also increased. Moreover, long-term incubation (96 h) of an Aspergillus niger feruloyl esterase in dimethylsulfoxide:water provided to the enzyme the ability to hydrolyze methyl p-coumarate, suggesting an active-site re-arrangement. Dimethylsulfoxide (10-30%) is proposed as an adequate co-solvent for feruloyl esterase treatment of water-insoluble substrates.     

Substrate-mediated increased reactivity of a critical sulfhydryl group of crystals of cytoplasmic aspartate transaminase

The extramitochondrial isozyme of aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1) contains a cysteinyl residue (cysteine-390) which, in the presence of substrate, displays enhanced reactivity toward sulfhydryl reagents. To gain insight into the structural similarity of the enzyme in solution compared to its crystalline state and into the type of structural change induced by substrates, the reactivity of Cys-390 in the crystalline enzyme has been studied. The flat yellow plates, crystallized from polyethylene glycol, form spectroscopically detectable enzyme-substrate complexes (C. M. Metzler, D. E. Metzler, D. S. Martin, R. Newman, A. Arnone, and P. Rodgers, 1978, J. Biol. Chem. 253, 5251–5254). The crystals, both in the presence and absence of the substrate pair, glutamate and α-ketoglutarate, were treated with N-ethylmaleimide or N-ethyl[1-¹⁴C]maleimide and the extent of the reaction was monitored by the colorimetric sulfhydryl reaction with 5,5′-dithiobis-2-nitrobenzoic acid, by amino acid analysis, by radioactivity incorporated, and by the measurement of enzyme activity. A cysteine residue was modified only in the presence of substrate; the crystals remained undamaged. Since, any large conformational change in the enzyme would either be prevented by the crystalline lattice or would disrupt its integrity, it is concluded that the enhanced reactivity of cysteine-390 in the presence of substrates must be due to only a small local conformational change in the substrate binding region.     

Discovery of human Golgi β-galactosidase with no identified glycosidase using a QMC substrate design platform for exo-glycosidase

Post-translational modifications (PTMs) of proteins play important roles in the physiology of eukaryotes. In the PTMs, non-reversible glycosylations are classified as N-glycosylations and O-glycosylations, and are catalyzed by various glycosidases and glycosyltransferases. However, β-glycosidases are not known to play a role in N- and O-glycan processing, although both glycans provide partial structures as substrates for β-galactosidase and β-N-acetylglucosaminidase in the Golgi apparatus of human cells. We explored human Golgi β-galactosidase using fluorescent substrates based on a quinone methide cleavage (QMC) substrate design platform that was previously developed to image exo-type glycosidases in living cells. As a result, we discovered a novel Golgi β-galactosidase in human cells. It is possible to predict a novel and important function in glycan processing of this β-galactosidase, because various β-galactosyl linkages in N- and O-glycans exist in Golgi apparatus. In addition, these results show that the QMC platform is excellent for imaging exo-type glycosidases.     

Patterns of oligosaccharide production by polygalacturonases

The products of hydrolytic action of 18 enzyme preparations at pH 3·5 and 5·5 on pectate were analyzed by gel-filtration chromatography early in the course of reaction (8–15% hydrolysis), and at a time 10 times that required for 10% hydrolysis. The degree of hydrolysis at the latter time varied from 25 to 74%. Three patterns of oligosaccharide production could be distinguished: endo-hydrolysis, exo-hydrolysis, and that due to S-polygalacturonase. The initial products of endo-hydrolysis were mixed oligosaccharides 5–30 units long; monomer and dimer appeared early but represented less than 2% of the products until late in the reaction. exo-Polygalacturonase (not entirely free of endo-) showed predominant production of the monomer and was clearly evident when mixed with four parts of endo-polygalacturonase. The time course of reducing group production by highly purified S-polygalacturonase could be reproduced by the above mixture of exo- and endo-polygalacturonases, but the pattern of products and the pH relations could not. The initial products of S-polygalacturonase were monomer, dimer and pentamer with lesser amounts of trimer and tetramer. After the hydolysis of the polymer and large oligomers, the pentamer was attacked by S-polygalacturonase, in the same way that the accumulated hexamer, etc. were finally hydrolysed by the endo-polygalacturonase.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

High production of cold-tolerant chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus Lecanicillium muscarium CCFEE 5003: Bioprocess optimization and characterization of two main enzymes

The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flasks to check its chitinase production on rough shrimp and crab wastes. Production on shrimp shells was much higher than that on crab shells (104.6 ± 9.3 and 48.6 ± 3.1 U/L, respectively). For possible industrial applications, bioprocess optimization was studied on shrimp shells in bioreactor using RSM to state best conditions of pH and substrate concentration. Optimization improved the production by 137% (243.6 ± 17.3). Two chitinolytic enzymes (CHI1 and CHI2) were purified and characterized. CHI1 (MW ca. 61 kDa) showed optima at pH 5.5 and 45 °C while CHI2 (MW ca. 25 kDa) optima were at pH 4.5 and 40 °C. Both enzymes maintained high activity levels at 5 °C and were inhibited by Fe⁺⁺, Hg⁺⁺ and Cu⁺⁺. CHI2 was strongly allosamidin-sensitive. Both proteins were N-acetyl-hexosaminidases (E.C. 3.2.1.52) but showed different roles in chitin hydrolysis: CHI1 could be defined as “chitobiase” while CHI2 revealed a main “eso-chitinase” activity.     

Roles of the exposed aromatic residues in crystalline chitin hydrolysis by chitinase A from Serratia marcescens 2170

Four exposed aromatic residues, two in the N-terminal domain (Trp-69 and Trp-33) and two in the catalytic domain (Trp-245 and Phe-232) of Serratia marcescens chitinase A, are linearly aligned with the deep catalytic cleft. To investigate the importance of these residues in the binding activity and hydrolyzing activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 significantly reduced the binding activity to both highly crystalline beta-chitin and colloidal chitin. The substitution of Phe-232, which is located closest to the catalytic cleft, did not affect the binding activity. On the other hand, the hydrolyzing activity against beta-chitin microfibrils was significantly reduced by the substitution of any one of the four aromatic residues including Phe-232. None of the mutations reduced the hydrolyzing activity against soluble substrates. These results clearly demonstrate that the four exposed aromatic residues are essential determinants for crystalline chitin hydrolysis. Three of them, two in the N-terminal domain and one in the catalytic domain, play vital roles in the chitin binding. Phe-232 appeared to be important for guiding the chitin chain into the catalytic cleft. Based on these observations, a model for processive hydrolysis of crystalline chitin by chitinase A is proposed.     

Irreversible kinetics of β-N-acetyl-d-glucosaminidase from prawn (Penaeus vannamei) inactivated by mercuric ion

Cysteine residues in prawn (Penaeus vannamei) β-N-acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) have been modified by p-chloromercuribenzoate (PCMB). The results show that sulfhydryl group is essential for the activity of the enzyme. Inactivation kinetics of the enzyme by mercuric chloride (HgCl₂) has been studied using the kinetic method of the substrate reaction during inactivation of enzyme previously described by Tsou. The kinetic results show that the inactivation of the enzyme is an irreversible reaction. The microscopic rate constants for the reaction of Hg²⁺ with free enzyme and with the enzyme-substrate complex are determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by Hg²⁺. The above results suggest that the cysteine residue is essential for activity.     

Study of the mode of action of endopolygalacturonase from Fusarium moniliforme

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis–Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V_max was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from −3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.     

Ab initio and DFT study on the electrophilic addition of bromine to endo-tricyclo[3.2.1.02,4]oct-6-ene

Full geometric optimization of endo-tricyclo[3.2.1.0^2,4]oct-6-ene (endo-TCO) by ab initio and DFT methods allowed us to investigate the structure of the molecule. The double bond is endo-pyramidalized and its two faces are no longer found to be equivalent. The exo face of the double bond has regions with far more electron density (q_i,HOMO) and more negative electrostatic potential. The endo-TCO-Br₂ system was investigated at the B3LYP/6-311+G** level and the endo-TCO···Br₂(exo) molecular complex was found to be relatively more stable than the endo-TCO···Br₂(endo) complex. The cationic intermediates of the reaction were studied by ab initio and DFT methods. The bridged exo-bromonium cation(I) is relatively more stable than the endo-bromonium cation(II). An absolute exo-facial selectivity should be observed in the addition reaction of Br₂ to endo-TCO, which is caused by steric and electronic factors. The nonclassical rearranged cation IV was found to be the most stable ion among the cationic intermediates and the ionic addition occurs via the formation of this cation. The mechanism of the addition reaction is also discussed.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).