Microsomal chitinase activity from Candida albicans

Chitinase (E.C. 3.2.1.14) was characterized in microsomal fractions from yeast cells of Candida albicans. Following six washes with buffer (50 mM Bis-Tris.Cl, pH 6.5), enzyme activity of microsomes fell markedly to 0.3% of total and 6% of the specific activity detected in the low-speed supernatant (9000 X g) of a cell lysate. An apparently zymogenic, microsomal chitinase activity became more readily detectable with washing and after six washes enzyme activity was activated 1.7-fold following pre-incubation with trypsin. The following properties of microsomal chitinase were closely comparable with those for cytosolic chitinase (indicated in parentheses): Km = 2.1 mg chitin per ml (2.9 mg chitin per ml); temperature optimum = 45 degrees C (45 degrees C); inhibition by allosamidin competitive, Ki = 0.29 microM (competitive, Ki = 0.23 microM). A range of detergents solubilized and activated microsomal chitinase in a highly specific manner. Following density gradient centrifugation of microsomes, chitinase was distributed approximately evenly throughout the gradient suggesting that microsomal chitinase is not associated exclusively with any one membrane component. The possible morphogenetic role of microsomal chitinase is discussed in relation to the potential of this enzyme as a target for highly specific antifungal agents.     

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.     

Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11

An alkalophilic, environmental micro-organism, Bacillus sp. BG-11, has been isolated and characterized. It produced 76 U ml-1 of chitinase in liquid batch fermentation after 72 h of incubation at 50 degrees C using chitin-enriched medium. The molecular weight of purified chitinase was estimated to be 41 kDa by SDS-PAGE. The pH and temperature optima of chitinase immobilized on chitosan and calcium alginate were 8.5 and 50 degrees C, respectively, which were same as that of free enzyme. The pH and thermostability of immobilized chitinase were enhanced significantly. The chitinase immobilized on chitosan was stable between pH 5.0 and 10.0, and the half-life of chitosan-immobilized enzyme at 70, 80 and 90 degrees C was 90, 70 and 60 min, respectively. The end-products formed during the enzyme-substrate reaction were identified by 13C-NMR, and N-acetyl-D-glucosamine was found to be the major end-product. GlcNAc (GlcNAc)2 and (GlcNAc)3 inhibited the chitinase activity by 32, 25 and 18%, respectively, at a concentration of 10 mmol l-1. The shelf-life of chitinase (retained 100% activity) at 4 degrees C was 8 weeks in the presence of either sodium azide (100 microgram ml-1), sodium metabisulphite (0.1% w/v) or KCl (15% w/v). The enzyme was resistant to the action of proteases and allosamidin.     

Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1

We describe the overexpression and characterization of a new 30 kDa family 18 chitinase (Ech30) from Trichoderma atroviride strain P1. Sequence alignments indicate that the active site architecture of Ech30 resembles that of endochitinases such as hevamine from the rubber tree (Hevea brasiliensis). The ech30 gene was overexpressed in Escherichia coli without its signal peptide and with an N-terminal His-tag. The enzyme was produced as inclusion bodies, from which active chitinase could be recovered using a simple refolding procedure. The enzyme displayed an acidic pH-optimum (pH 4.5-5.0), probably due to the presence of a conserved Asn residue near the catalytic glutamate, which is characteristic for acidic family 18 chitinases. Studies with oligomers of N-acetylglucosamine [(GlcNAc)(n)], 4-methylumbelliferyl (4-MU) labelled GlcNAc oligomers and beta-chitin reveal enzymatic properties typical of an endochitinase: 1) low activity towards short substrates (kinetic parameters for the hydrolysis of 4-MU-(GlcNAc)2 were K(m), 149+/-29 microM and k(cat), 0.0048+/-0.0005 s(-1)), and 2) production of relatively large amounts of trimers and tetramers during degradation of beta-chitin. Detailed studies with GlcNAc oligomers indicated that Ech30 has as many as seven subsites for sugar binding. As expected for a family 18 chitinase, catalysis proceeded with retention of the beta-anomeric configuration.     

Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain.

The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. The pH and temperature optima of the enzyme were 6.0 and 45 degrees C, respectively. The Km and Vmax values for 4-MU-(GlcNAc)2 were estimated to be 6.3 microM and 46 micromol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin.     

Chemical modification studies of the active centre of Candida albicans chitinase and its inhibition by allosamidin.

Allosamidin, a glycoside antibiotic, is shown to be a strong, competitive inhibitor of semi-purified chitinase from yeast cells of Candida albicans. The inhibitory potency of allosamidin was pH-dependent, with IC50 values of 280 nM at pH 5.0 and 21 nM at pH 7.5. At higher, micromolar, concentrations, allosamidin inactivated this chitinase in a time- and concentration-dependent manner. Kinetic studies of this inactivation provided evidence for the formation of a reversible complex between allosamidin and chitinase, characterized by Kinact = 5 microM, followed by irreversible modification of the enzyme with velocity constant k2 = 4.6 x 10(-3) s-1. Chemical modification studies with the use of group-specific reagents suggested the presence of Glu/Asp carboxyl group(s) at or near the active site, that were important for enzyme activity. The carboxyl-specific reagent, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, inactivated the chitinase in a single step process, with apparent second-order rate constant of 0.014 M-1 s-1.     

Molecular analysis of the gene encoding a novel transglycosylative enzyme from Alteromonas sp. strain O-7 and its physiological role in the chitinolytic system.

We purified from the culture supernatant of Alteromonas sp. strain O-7 and characterized a transglycosylating enzyme which synthesized beta-(1-->6)-(GlcNAc)2, 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2- deoxyglucopyranose from beta-(1-->4)-(GlcNAc)2. The gene encoding a novel transglycosylating enzyme was cloned into Escherichia coli, and its nucleotide sequence was determined. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 99,560 Da which corresponds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the cloned enzyme was much larger than that of enzyme (70 kDa) purified from the supernatant of this strain. These results suggest that the native enzyme was the result of partial proteolysis occurring in the N-terminal region. The enzyme showed significant sequence homology with several bacterial beta-N-acetylhexosaminidases which belong to family 20 glycosyl hydrolases. However, this novel enzyme differs from all reported beta-N-acetylhexosaminidases in its substrate specificity. To clarify the role of the enzyme in the chitinolytic system of the strain, the effect of beta-(1-->6)-(GlcNAc)2 on the induction of chitinase was investigated. beta-(1-->6)-(GlcNAc)2 induced a level of production of chitinase similar to that induced by the medium containing chitin. On the other hand, GlcNAc, (GlcNAc)2, and (GlcNAc)3 conversely repressed the production of chitinase to below the basal level of chitinase activity produced constitutively in medium without a carbon source.     

Kinetic evidence related to substrate-assisted catalysis of family 18 chitinases

The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N-acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAc)2-UMB (1). The kcat of 2 was 3.4% of 1, but the Km of 2 was 10-fold that of 1. In contrast, the kcat of 3 was only 0.3% of that of 1, and the two reactions had an identical Km. The drastic decreases in kcat were probably due to the weak nucleophilic activity of the C2-N-trifluoroacetamide and N-thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N-acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases.     

Expression, refolding, and purification of active diacetylchitobiose deacetylase from Pyrococcus horikoshii

A chitinase from the hyperthermophilic archaeon Pyrococcus furiosus degrades chitin to produce diacetylchitobiose [(GlcNAc)(2)] as the end product. To further investigate the degradation mechanism of (GlcNAc)(2) in Pyrococcus spp., we cloned the gene of PH0499 from Pyrococcus horikoshii, which encodes a protein homologous to the diacetylchitobiose deacetylase of Thermococcus kodakaraensis. The deacetylase (Ph-Dac) was overexpressed as inclusion bodies in Escherichia coli Rosetta (DE3) pLys. The insoluble inclusion body was solubilized and reactivated through a refolding procedure. After several purification steps, 40 mg of soluble, thermostable (up to 80°C) Ph-Dac was obtained from 1L of culture. The apparent molecular mass of the refolded Ph-Dac was 180 kDa, indicating Ph-Dac to be a homohexamer. The refolded Ph-Dac also exhibited deacetylase activity toward (GlcNAc)(2), and the deacetylation site was revealed to be specific to the nonreducing end residue of (GlcNAc)(2). These expression and purification systems are useful for further characterization of Ph-Dac.     

Purification and characterization of a novel isozyme of chitinase from Bombyx mori

75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc5) and 10 toward glycolchitin. The optimum temperature was 60 degrees C toward GlcNAc5 and 25 degrees C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40 degrees C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the beta anomeric product and that it prefers the longer N-acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.     

Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101

A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.     

Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS

The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of alpha and beta anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated beta-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher Mr chitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues - Glu315 and Asp392 - by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed approximately 20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme.     

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to β-lactamase inhibitors

Abstract A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50°C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occuring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans . Evidence is provided for the existence of a complex chitinolytic system in this organism.     

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)₂) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)_n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)_4–6 and produced (GlcNAc)_2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)₅ to produce (GlcNAc)₂ (79.2%) and (GlcNAc)₃ (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)_n (n = 2–4) was pNp-(GlcNAc)₂ >> pNp-(GlcNAc)₄ > pNp-(GlcNAc)₃. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)_n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.     

Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second β-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the β-anomer of (GlcNAc)?. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid β-chitin, producing only (GlcNAc)?. The AcMNPV chitinase processively hydrolyzes solid β-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.     

Biosynthesis of chitin by particulate fractions from Cunnighamella elegans.

The enzyme chitin synthetase (UDP-acetylaminodeoxyglucosyl transferase, EC 2.4.1.16) in Cunninghamella elegans has been investigated. The enzyme was present in the microsomal, cell wall, mitochondrial and the soluble cytoplasmic fraction of the mycelium, with the former having the highest specific activity. The properties of the enzyme in this fraction were investigated; the Km for UDP GlcNAc was 1.23 mM and 2.08 mM GlcNAc in the presence of 1 mM UDP GlcNAc. The temperature optimum was between 26 degrees and 29 degrees C and maximal activity was at pH 6.25. Mg++ ions had no effect on chitin synthesis, but soluble chitodextrins inhibited the enzyme. The production of chitin synthetase was correlated with the growth of the fungus, maximum activity being found during the late exponential phase of growth. Chitin was confirmed as the sole product of enzyme action, by digestion with chitinase.     

Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase.

An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.     

Goat liver beta-mannosidases: molecular properties, inhibition and inactivation of the lysosomal and nonlysosomal forms

The two caprine hepatic beta-mannosidases have been partially purified and their properties have been compared. The lysosomal beta-mannosidase A had an apparent molecular weight of 127,000 +/- 10,000 and an isoelectric point of pH 6-7. Its activity was unaffected by incubation with Triton X-100 (0.1%) and cysteine (20 mM) and it hydrolyzed the presumed natural substrates, Man(beta 1-4)GlcNAc and Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc. The nonlysosomal beta-mannosidase B had an apparent molecular weight of 43,000 +/- 2,000 and an isoelectric point of pH 5.5. beta-Mannosidase B was activated by Triton X-100 (0.1%) and was inhibited by cysteine (20 mM). Hydrolysis of Man(beta 1-4)GlcNAc, but not of Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, followed incubation with beta-mannosidase B. 1,5-Dideoxy-1,5-imino-D-mannitol did not inhibit the A enzyme and only feebly (Ki = 0.3 mM) inhibited the B enzyme; beta-D-mannopyranosylmethyl p-nitrophenyl triazene did not inactivate either enzyme but 1,2-anhydro-1,2,3,5,6/4-cyclohexane hexol inactivated the B enzyme only. The radical mechanistic differences between the two enzymes argue against their having the same genetic origin.