A metalloprotease (MprIII) involved in the chitinolytic system of a marine bacterium,Alteromonas sp. strain O-7

Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50 degrees C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.     

Identification and characterization of the gene cluster involved in chitin degradation in a marine bacterium, Alteromonas sp. strain O-7.

Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when alpha-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc)(2) from chitin. The optimum temperature and pH of ChiD were 50 degrees C and 7.0, respectively.     

A Metalloprotease (MprIII) Involved in the Chitinolytic System of a Marine Bacterium, Alteromonas sp. Strain O-7

Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50°C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.     

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-I^WT), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (M^WT) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-I^WT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-I^WT released soluble peptides from SSP faster than did M^WT. Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass.     

Characterization of chitinase genes from an alkaliphilic actinomycete,Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Characterization of Chitinase Genes from an Alkaliphilic Actinomycete, Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiBΔ, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiBΔ was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBΔ were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBΔ, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.     

Enzymatic deacetylation of chitin by extracellular chitin deacetylase from a newly screened Mortierella sp. DY-52

Among more than a hundred colonies of fungi isolated from soil samples, DY-52 has been screened as an extracellular chitin deacetylase (CDA) producer. The isolate was further identified as Mortierella sp., based on the morphological properties and the nucleotide sequence of its 18S rRNA gene. The fungus exhibited maximal growth in yeast peptone glucose (YPD) liquid medium containing 2% of glucose at pH 5.0 and 28 degrees C with 150 rpm. The CDA activity of DY-52 was maximal (20 U/mg) on the 3rd day of culture in the same medium. The CDA was inducible by addition of glucose and chitin. The enzyme contained two isoforms of molecular mass 50 kDa and 59 kDa. This enzyme showed a maximal activity at pH 5.5 and 60 degrees C. In addition, it had a pH stability range of 4.5-8.0 and a temperature stability range of 4-40 degrees C. The enzyme was enhanced in the presence of Co2+ and Ca2+. Among various substrates tested, WSCT-50 (water-soluble chitin, degree of deacetylation 50%), glycol chitin, and crab chitosan (DD 71-88%) were deacetylated. Moreover, the CDA can handle N-acetylglucosamine oligomers (GlcNAc)2-7.     

Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.     

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7.

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 degrees C, respectively. Chi-A was stable in the range of pH 5-10 up to 40 degrees C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulans WL-12.     

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.     

Analysis of the chitin recognition mechanism of cuticle proteins from the soft cuticle of the silkworm, Bombyx mori

Insect cuticle is composed mainly of chitin, a polymer of N-acetylglucosamine, and chitin-binding cuticle proteins. Four major cuticle proteins, BMCP30, 22, 18, and 17, have been previously identified and purified from the larval cuticle of silkworm, B. mori. We analyzed the chitin-binding activity of BMCP30 by use of chitin-affinity chromatography. The pH optimum for the binding of BMCP30 to chitin is 6.4, which corresponds to hemolymph pH. Competition experiments using chitooligosaccharides suggested that BMCP30 recognizes 4-6 mer of N-acetylglucosamine in chitin fiber as a unit for binding. The comparison of the binding properties of BMCP30 with those of BMCP18 showed that their binding activities to chitin are similar in a standard buffer but that BMCP30 binds to chitin more stably than BMCP18 in the presence of urea. BMCPs possess the RR-1 form of the R&R consensus, about 70 amino acids region conserved widely among cuticle proteins mainly from the soft cuticle of many insect and arthropod species. Analysis of the binding activity using deletion mutants of BMCPs revealed that this type of conserved region also functions as the chitin-binding domain, similarly to the RR-2 region previously shown to confer chitin binding. Thus, the extended R&R consensus is the general chitin-binding domain of cuticle proteins in Arthropoda.     

Molecular cloning of the gene encoding a novel beta-N-acetylhexosaminidase from a marine bacterium, Alteromonas sp. strain O-7, and characterization of the cloned enzyme

We have reported that the chitinolytic system of Alteromonas sp. strain O-7 consists of chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), beta-N-acetylglucosaminidases (GlcNAcasesA, GlcNAcaseB, and GlcNAcaseC), and a novel transglycosylative enzyme (Hex99). The gene encoding a beta-hexosaminidase with an unusual substrate specificity (hex86), located upstream of the hex99 gene, was cloned and sequenced. The gene encoded a protein of 761 amino acids with a calculated molecular mass of 86,758 Da. The deduced amino acid sequence of Hex86 showed sequence similarity with beta-hexosaminidases belonging to family 20. The hex86 gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme rapidly cleaved p-nitrophenyl-beta-N-acetyl-D-glucosaminide and slowly cleaved p-nitrophenyl-beta-N-acetyl-D-galactosaminide. Unexpectedly, the enzyme did not hydrolyzed chitin oligosaccharides under the assay conditions for synthetic glycosides. However, after prolonged incubation with excessive quantities of the enzyme, Hex86 hydrolyzed chitin oligosaccharides. These results indicate that Hex86 is a novel enzyme that prefers p-nitrophenyl-beta-N-acetyl-D-glucosaminide to chitin oligosaccharides as a substrate.     

Characterization and cloning of chitin deacetylases from Rhizopus circinans

Chitin deacetylase catalyzes hydrolysis of the acetamido groups of N-acetylglucosamine of chitin in fungal cell walls. Here a chitin deacetylase secreted by Rhizopus circinans was purified to homogeneity and partially characterized. The enzyme exhibits an apparent molecular weight of approximately 75kDa. At 37 degrees C it shows optimal activity at pH 5.5-6. Its pH stability and thermal stability are good. Mn(2+) and Mg(2+) slightly enhance the activity of the enzyme and Cu(2+) strongly inhibits it. An R. circinans cDNA library was constructed and screened with a homologous probe synthesized by RT-PCR or with synthetic primers derived from the N-terminal amino-acid sequence of the native purified chitin deacetylase. Three chitin deacetylase cDNAs (RC, D2, and I3/2) were isolated from the cDNA library and sequenced. These cDNAs exhibit features characteristic of chitin deacetylase sequences: the presence of a polysaccharide deacetylase domain, a metal-binding triad, the conserved catalytic residues, and high homology with various chitin deacetylase genes. The cDNAs were cloned in a Pichia pastoris expression system and produced as polyhistidine-tagged proteins. Only one recombinant enzyme (called RC) was active under the tested conditions. It was purified to homogeneity in a single step and further characterized. The protein showed an apparent molecular mass of approximately 75kDa and, like the native enzyme, showed optimal activity at pH 5.5-6 at 37 degrees C. It was strongly inhibited by Cu(2+). The isolation of several chitin deacetylase cDNAs from the same microorganism is discussed.     

Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium <Emphasis Type="Italic">Moritella marina</Emphasis> and biochemical characterization of the recombinant enzyme

The marine psychrophilic bacterium Moritella marina, isolated from a sample raised from a depth of 1,200 m in the northern Pacific Ocean, secretes several chitinases in response to chitin induction. A gene coding for an extracellular chitinolytic enzyme was cloned and its nucleotide sequence was determined. The chitinase gene consists of an open reading frame of 1,650 nucleotides and encodes a protein of 550 amino acids with a calculated molecular weight of 60.788 kDa, named MmChi60. MmChi60 has a modular structure consisting of a glycosyl-hydrolase family 18 N-terminal catalytic region as well as a C-terminal chitin-binding domain (ChBD). The new chitinase was purified to homogeneity from the intracellular fraction of Escherichia coli. The optimum pH and temperature of the recombinant MmChi60 were 5.0 and 28 degrees C, respectively. The mode of action of the new enzyme on N-acetylchitooligomers, chitin polymers, and other substrates was examined, and MmChi60 was classified as an endochitinase. Thermal unfolding of MmChi60 was studied using differential scanning microcalorimetry and revealed that the protein unfolds reversibly at 65 degrees C. On the basis of the crystal structure of the chitinase C of Streptomyces griseus, a homology-based 3-D model of the ChBD of the MmChi60 was calculated.     

Structural and functional definition of the human chitinase chitin-binding domain

Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.     

Isolation and characterisation of chitosanase from Bacillus sp. 739 strain

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 has been determined. Maximum enzyme activity was observed in a medium containing the biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity using chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme, assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. Temperature and pH optima of the purified chitosanase were in the ranges 45-55 degrees C and 6.0-6.5, respectively. Time to half-maximum inactivation of the enzyme at 50 degrees C was equal to 1 h. With colloidal chitosan as the substrate, the value of K(M) of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

Isolation and characterization of the genes encoding two metalloproteases (MprI and MprII) from a marine bacterium, Alteromonas sp. strain O-7

An extracellular alkaline metalloprotease (MprI) from Alteromonas sp. strain O-7 was purified and characterized. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE. The optimum pH and temperature were pH 10.0 and 60 degrees C, respectively. The gene (mprI) encoding MprI was cloned and its nucleotide sequence was analyzed. The deduced amino acid sequence of MprI showed significant similarity to metalloproteases classified into the thermolysin family. Furthermore, sequence analysis showed that another metalloprotease (MprII)-encoding gene was located downstream from mprI. The deduced amino acid sequence of MprII showed high similarity to metalloproteases of the aminopeptidase family. Similar repeated C-terminal extensions were found in both MprI and MprII.