Production of Chitinases and beta-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

The in vitro production of chitinases and beta-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO(3). The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for beta-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and beta-1,3-glucanases were detected. S. elegans culture filtrates, possessing beta-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.     

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.     

Molecular cloning,characterization, and expression of a cDNA encoding an endochitinase gene from the mycoparasite Stachybotrys elegans

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogenic fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall degrading enzymes such as chitinases. This report details the cloning by RACE-PCR and characterization of a full-length cDNA clone, sechi44, that appears to encode an extracellular endochitinase. An analysis of the sechi44 sequence indicates that this gene contains a 1269-bp ORF and encodes a 423-aa polypeptide. The SECHI44 protein has a calculated molecular weight of 44.1kDa and pI of 5.53. Since the SECHI44 protein also appears to encode a signal peptide, an extracellular location for the corresponding protein is predicted. Comparison of SECHI44 sequence with known sequences of fungal endochitinases revealed that SECHI44 is grouped with endochitinases from other mycoparasites. Real-time quantitative RT-PCR analysis showed an elevated level of expression of sechi44 (21-fold) in chitin-rich (induced) as compared to no-carbon (non-induced) culture conditions. In dual culture, the temporal expression of sechi44 increased after 2 days of contact with R. solani, reaching a 10-fold increase after 9 days, followed by a decrease to basic expression level at 12 days. Interestingly, inhibition of sechi44 expression was observed when S. elegans hyphae were in close proximity with R. solani hyphae.     

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism.

The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.     

Chitinases of Streptomyces olivaceoviridis and significance of processing for multiplicity.

Five extracellular chitinases of 20.5, 30, 47, 70, and 92 kDa purified from the culture filtrate of Streptomyces olivaceoviridis ATCC 11238 differed in their sequences at the amino termini of the protein chains. In the native state, the chitinases were found to be resistant to proteolysis by trypsin, papain, and Staphylococcus aureus V8 protease. The latter produced several fragments of identical molecular mass from chitinases denaturated with sodium dodecyl sulfate. Five proteases were detected in the protein concentrate from the culture filtrate, and two of them showing ability to cleave chitinases in the native state were purified. One, a protease of 42 kDa, released a 30-kDa protein from the 70-kDa chitinase that reacts with anti-30 kDa chitinase antibodies; the other, a protease of 29 kDa, split the 30-kDa chitinase into 20.5-, 18-, and 16-kDa fragments. From these results, it was deduced that the 70-kDa chitinase is the precursor protein of the 30- and 20.5-kDa chitinases.     

Subunit identity of the dimeric 17 beta-hydroxysteroid dehydrogenase from human placenta.

Human placental 17 beta-hydroxysteroid dehydrogenase has been purified with a new rapid procedure based on fast protein liquid chromatography, yielding quantitatively a homogeneous preparation with high specific activity catalyzing the oxidation of 7.2 mumol of estradiol/min/mg of enzyme protein at 23 degrees C, pH 9.2. This preparation was shown to have a subunit mass of 34.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while having a molecular mass of 68 kDa by both Superose-12 gel-filtration and native pore gradient gel electrophoresis. When 17 beta-hydroxysteroid dehydrogenase was expressed in HeLa cells or overproduced in insect cells using the baculovirus expression system, both from its cDNA encoding a protein of 34 kDa, the enzyme had the same migration in native and sodium dodecyl sulfate-gel electrophoresis as the purified one from human placenta and eluted from the Superose-12 column at the same elution volume. Moreover, all the above forms of this enzyme have similar specific activity. These results clearly demonstrate the identity of the three enzyme forms. The enzyme produced from the cDNA is expressed as a dimer, and its two subunits are identical. 17 beta-Hydroxysteroid dehydrogenase subunit identity is thus proved. The NH2-terminal analysis revealed a unique sequence of Ala-Arg-Thr-Val-Val-Leu-Ile for the purified enzyme from placenta, further confirming the above conclusion.     

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.     

Isolation and characterization of a lectin from peanut roots.

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.     

Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step

The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.     

Isolation and characterization of three chitinases from Trichoderma harzianum.

Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with chitin as the sole carbon source. Purification was carried out after protein precipitation with ammonium sulphate, adsorption to colloidal chitin and digestion, and, finally, chromatofocusing. By this procedure, two chitinases of 42 kDa (CHIT42) and 37 kDa (CHIT37) were purified to homogeneity, as judged by SDS/PAGE and gel filtration, whereas a third, of 33 kDa (CHIT33), was highly purified. The isoelectric points for CHIT42, CHIT37 and CHIT33 were 6.2, 4.6 and 7.8, respectively. The three enzymes displayed endochitinase activities and showed different kinetic properties. CHIT33 was able to hydrolyze chitin oligomers of a polymerization degree higher than n = 4, its Km for colloidal chitin being 0.3 mg/ml. CHIT42 and CHIT37 were able to hydrolyze chitin oligomers with a minimal polymerization degree of n = 3, their Km values for colloidal chitin being 1.0 mg/ml and 0.5 mg/ml respectively. With regard to their lytic activity with purified cell walls of the phytopathogenic fungus Botrytis cinerea, a hydrolytic action was observed only when CHIT42 was present. Antibodies against CHIT42 and CHIT37 specifically recognized the proteins and did not display cross-reaction, suggesting that each protein is encoded by a different gene.     

Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme

We report the identification and purification of a novel enzyme from soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reaction is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase. The enzyme was purified from crude extracts of soybean root nodules approximately 100-fold to apparent homogeneity with a final specific activity of 10 micromol/min/mg. The enzyme exhibited a native molecular mass of approximately 68 kDa by gel filtration chromatography and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 +/- 2 kDa. The purified enzyme obeyed normal Michaelis-Menten kinetics, and the K(m) for 5-hydroxyisourate was determined to be 15 microM. The amino-terminal end of the purified protein was sequenced, and the resulting sequence was not found in any available data bases, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin.     

Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M. flavoviride, and Beauveria bassiana during Fungal Invasion of Host (Manduca sexta) Cuticle

Extracellular chitinases have been suggested to be virulence factors in fungal entomopathogenicity. We employed isoelectric focusing and a set of three fluorescent substrates to investigate the numbers and types of chitinolytic enzymes produced by the entomopathogenic fungi Metarhizium anisopliae, Metarhizium flavoviride, and Beauveria bassiana. Each species produced a variety of N-acetyl-(beta)-d-glucosaminidases and endochitinases during growth in media containing insect cuticle. M. flavoviride also produced 1,4-(beta)-chitobiosidases. The endochitinases could be divided according to whether they had basic or acidic isoelectric points. In contrast to those of the other two species, the predominant endochitinases of M. anisopliae were acidic, with isoelectric points of about 4.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the acidic chitinases of M. anisopliae into two major bands (43.5 and 45 kDa) with identical N-terminal sequences (AGGYVNAVYFY TNGLYLSNYQPA) similar to an endochitinase from the mycoparasite Trichoderma harzianum. Use of polyclonal antibodies to the 45-kDa isoform and ultrastructural immunocytochemistry enabled us to visualize chitinase production during penetration of the host (Manduca sexta) cuticle. Chitinase was produced at very low levels by infection structures on the cuticle surface and during the initial penetration of the cuticle, but much greater levels of chitinase accumulated in zones of proteolytic degradation, which suggests that the release of the chitinase is dependent on the accessibility of its substrate.     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein.All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.     

Involvement of cell surface sugars in recognition, attachment, and appressorium formation by a mycoparasite

Fluorescein isothiocyanate labeled lectin binding techniques have revealed differences in the distribution pattern of glycosyl residues at the cell wall level between fungi that are hosts and those that are nonhosts of the mycoparasite Piptocephalis virginiana, and at the protoplast level between compatible and incompatible hosts. The cell wall of the compatible hosts (Choanephora cucurbitarum and Mortierella pusilla) and an incompatible host (Phascolomyces articulosus), as well as that of the mycoparasite itself, contains glucose and N-acetylglucosamine. However, the cell wall of a nonhost (Mortierella candelabrum) tested positive with lectins specific for various sugars, including not only glucose and N-acetylglucosamine, but also fucose, N-acetylgalactosamine, and galactose. These latter sugars could also be exposed at the surfaces of hosts and of the mycoparasite, but only after mild treatment with proteinase or when grown in a liquid culture. Pretreatment of the mycoparasite with glucose and N-acetylglucosamine inhibited its attachment to the host cell surface, but had no obvious effect on appressorium formation. On the other hand, appressorium formation was inhibited by heat treatment of host cell wall fragments which still permitted attachment, thus indicating that the factors responsible for attachment and for appressorium formation are different. The protoplast surfaces of compatible hosts contained all the sugars listed above and these protoplasts could attach to the germ tube of the mycoparasite. Only lectins specific for N-acetylglucosamine and for glucose were bound at the protoplast surface of the incompatible host; these protoplasts did not attach to the mycoparasite germ tube. Key words: mycoparasite, appressorium formation, lectins, host cell surface, attachment, protoplast surface.     

Purification of an ultraviolet-inducible, damage-specific DNA-binding protein from primate cells.

A UV-inducible, damage-specific DNA-binding (DDB) protein with high affinity for double-stranded UV-irradiated DNA has been identified recently in monkey kidney (CV-1) cells (Hirschfeld, S., Levine, A. S., Ozato, K., and Proti?, M. (1990) Mol. Cell. Biol. 10, 2041-2048). We have now purified the DDB protein from extracts of CV-1 cells using hydroxylapatite, phosphocellulose, Mono S, and DNA-affinity column chromatography. The DDB activity, either from mock-treated or UV-induced cells, is heterodisperse in column chromatography, and separation of three forms of the protein was obtained on a phosphocellulose column. Analysis of purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that greater than 90% of all three forms is a protein of approximately 126 kDa. The size of the native DDB protein was deduced from gel filtration and native polyacrylamide gel electrophoresis to be approximately 210 kDa, which suggests that the native DDB protein in solution is a homodimer. Preparations of partially purified DDB protein from UV-treated cells have enhanced levels of DDB activity and the protein when compared with similar preparations from mock-treated cells. This damage-recognition protein, alone or in conjunction with other subunits, may be of general importance for the initial recognition of DNA damage in mammals.     

Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut

Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest k(cat)/K(M) value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.     

Expression, purification, and biochemical characterization of a recombinant Iectin of Sarcocystis muris (Apicomplexa) cyst merozoites

The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia coli as a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.