Three Extracellular Chitinases in Suspension-cultured Rice Cells Elicited by N-Acetylchitooligosaccharides

In rice suspension culture, a large part (about 90% of total activity in the culture) of the chitinase activity was found in the medium. Two extracellular chitinases (which we named RCH-A and -B) were separated from the cell suspension by DEAE-cellulofìne column chromatography. When cells were treated with N-acetylchitooligosaccharides (chitin oligosaccharides) for 3 days, extracellular chitinase activity increased about 3-fold over the control culture. After the treatment, another extracellular chitinase (named RCH-C) appeared in addition to increases in the levels of RCH-A and -B. Partial amino acid sequences of these enzymes indicated that RCH-A (33.5 kDa) and -B (34kDa) were class Ib chitinases but RCH-C (27kDa) was a class III chitinase. RCH-A and -B were capable of actively degrading water-insoluble chitin with high affinities, while RCH-C had less affinity for the substrate. However, when a water-soluble chitin derivative, 6–O-hydroxyethylchitin (glycolchitin) was used, RCH-C as well as RCH-A and -B degraded actively with a high affinity. A synergistic effect was observed when these three chitinases acted simultaneously in the hydrolysis of chitin.     

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1

The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.     

Purification and characterization of extracellular chitinase from Aeromonas schubertii

A locally isolated stain Aeromonas schubertii was cultured and induced by powdered chitin for the production of chitinases. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts, and then preparative isoelectric focusing (IEF) to yield several chitinases. The purified enzymes were analyzed by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with and without glycol chitin and were found to be SDS-resistant. The chitinase present in the highest abundance was the one with an estimated molecular weight of 75 kDa. The Michaelis constant and turnover number were determined to be 0.29 mM and 1 s⁻¹, respectively, for this enzyme using colloidal chitin azure as the substrate. However, the ethanol treatment of this enzyme could significantly increase its chitinolytic activity. Other chitinases obtained in the same IEF fraction were determined to have molecular weights of ca. 30, 38, and 110 kDa. Since the proteins with highest chitinase activity were collected from IEF fraction tube with pH value of 4.8, those chitinase were believed to be acidic. An activity assay method using colloidal chitin azure as the substrate was recommended since it possessed a broader range of linearity in comparison with conventional reducing sugar equivalent method.     

Synthesis of Extracellular Chitinase by Wild-Type B-10 and Mutant M-1 Strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, whereas M-1 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused oversynthesis of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the synthesis of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Myco-chitinases as versatile biocatalysts for translation of coastal residual resources to eco-competent chito-bioactives

Chitinases (EC 3.2.1.14) are the glycoside hydrolases (GH) that catalyse the cleavage of β-(1,4) glycosidic linkages of chitin, which is a key element of fungal cell wall and insect's exoskeletons. Fungi have been considered as an excellent source for the production of extracellular chitinases, which could further be employed for chitin degradation to generate a range of bioactive chito-derivatives, i.e., oligosaccharides and glucosamine. Moreover, chitinases have diverse roles in various physiological functions, i.e., autolysis, cell wall remodeling, mycoparasitism and biocontrol. The advent of technology led to the sequencing of several fungal genomes and enabled the manipulation of novel effective chitinase genes to investigate their mechanistic and structural insights to decode the variabilities in chitin degradation. Further, the comprehensible understanding of attributes including substrate-binding sites and catalytic domains could give an insight into chitin catabolism for value-added products development. The review summarized various aspects of fungal chitinases viz. structure, mechanism, classification, properties, functions and application in the present precis. The study has also underlined the recent research related to the framework of substrate-binding clefts in fungal chitinases and its correlation with the hydrolytic and transglycosylation (TG) activity for the production of oligosaccharides with variable degrees of polymerization.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.     

Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Invertase in cultured Daucus carota cells

Invertase activity of cultured carrot cells was distributed between cell wall and supernatant fractions of the cell homogenate. The enzyme associated with the cell wall fraction was solubilized by alkaline NaCl solution and the proportions found in the cell wall and soluble fractions depended on the concentration of NaCl. Formation of protoplasts by the action of cellulase and pectinase was accompanied by release of 50–60% of the invertase activity from the cells.     

Chitinase system of Aeromonas salmonicida,and characterization of enzymes involved in chitin degradation

ABSTRACT

The genes encoding chitin-degrading enzymes in Aeromonas salmonicida SWSY-1.411 were identified and cloned in Escherichia coli. The strain contained two glycoside hydrolase (GH) families 18 chitinases: AsChiA and AsChiB, two GH19 chitinases: AsChiC and AsChiD, and an auxiliary activities family 10 protein, lytic polysaccharide monooxygenase: AsLPMO10A. These enzymes were successfully expressed in E. coli and purified. AsChiB had the highest hydrolytic activity against insoluble chitin. AsChiD had the highest activity against water-soluble chitin. The peroxygenase activity of AsLPMO10A was lower compared to SmLPMO10A from Serratia marcescens. Synergism on powdered chitin degradation was observed when AsChiA and AsLPMO10A were combined with other chitinases of this strain. More than twice the increase of the synergistic effect was observed when powdered chitin was treated by a combination of AsLPMO10A with all chitinases. GH19 chitinases suppressed the hyphal growth of Trichoderma reesei.     

A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function.

Enzymological evidence has been sought for the purported involvement of chitinolysis in vegetative growth of filamentous fungi. A procedure has been developed for the production of fast growing and morphologically homogeneous exponential phase mycelium of the non-septate dimorphic zygomycete Mucor rouxii. A partially purified extract of this material has been subjected to gel-permeation chromatography and the chitinolytic activity of eluate fractions has been assessed using colloidal and nascent chitin and 3,4-dinitrophenyl tetra-N-acetylchitotetraoside [3,4-DNP-(GlcNAc)4] as substrates. Exponentially growing (td = 1.1 h) mycelium consisting of single short-branched hyphae contains at least seven chitinases. The two particulate ones have not been studied in detail. The soluble chitinases hydrolyse (pseudo)chito-oligomers by random cleavage of internal beta-1,4-bonds (and not by processing) and have a minimum chain-length requirement of n = 4. They are clearly distinct from beta-N-acetylglucosaminidase (beta-GlcNAc'ase) with respect to their chromatographic behaviour, substrate chain-length specificity, inhibition by chitobionolactone oxime (Ki = 175 microM), and non-inhibition by the specific beta-GlcNAc'ase inhibitor N-acetylglucosaminono-1,5-lactone oxime. Their pH optima are similar (6.5-7.0), and all can hydrolyse 3,4-DNP-(GlcNAc)4 as well as nascent chitin. With respect to their charge, response to protease treatment, behaviour upon gel-permeation chromatography and ability to use colloidal chitin as a substrate, the soluble chitinases do, however, represent two distinct groups. Type A chitinases are acidic, display partial latency, show an unusual affinity to dextran gel and act weakly on colloidal chitin. Type B chitinases are basic (or neutral) and non-zymogenic, do not behave anomalously upon gel filtration and can degrade performed chitin. An hypothesis is presented for the function of the complex chitinolytic system of the fungal hypha in branching and, possibly, also in apical growth.     

Inhibitory effect of chitinases isolated from Semillon grapes (Vitis vinifera) on growth of grapevine pathogens

Characteristics and antifungal activity of chitinases in Semillon grapes were investigated. Chitinases were isolated from the juice of Semillon grapes by chitin affinity chromatography. Native and SDS-PAGE analyses of the fraction showing chitin affinity (active fraction) demonstrated only the presence of protein bands of chitinases. Three types of class IV chitinases (chi-1a, chi-1b and chi-2) were purified from the active fraction. These chitinases actively hydrolyzed chitin under acidic conditions (pH 4.0–4.5). The isoelectric points and the molecular weights of chi-1a, chi-1b and chi-2 were 4.73, 4.60, and 7.87, and 32.1 kDa, 31.6 kDa, and 29.0 kDa, respectively. The active fraction was found to inhibit Botrytis cinerea mycelial growth and the inhibitory effect was due to the activity of chitinases. The active fraction inhibited twenty strains of B. cinerea collected from the experimental vineyard. The effect of chitinases was enhanced in media containing more than 20% sugar. When the active fraction was tested on Glomerella cingulata, the growth inhibitory effect observed was markedly less than that seen on B. cinerea.     

A Comparative Study of Multiple: Acid Phosphatases in Cellular Fractions of Bean Hypocotyl

Six kinds of acid phosphatases were solubilized with TritonX-100 from the cell wall (W-I, W-II), mitochondrial (M-I, M-II,M-III) and microsomal (Ms) fractions of bean hypocotyl, andthey were partially purified by using Sephadex gel filtrationand DEAE-cellulose column chromatography. Acid phosphatasesfrom the soluble fraction were also fractionated into 12 isozymesby electrophoresis, and the properties of the isozymes werecompared. The soluble isozymes showed pH optima at 5·0,5·3 and 5·6; the isozymes possessed high affinityto p-nitrophenyl phosphate and ADP, and their mol. wts rangedfrom 30000 to 45000. Among the solubilized phosphatases, W-I,M-I and M-III showed maximum activity at pH 5·0 and theirmol. wts were between 50000 and 110000. W-I proved to have highaffinity to ATP and bis-p-nitrophenyl phosphate as M-I did top-nitrophenyl phosphate and M-III to phenyl phosphate. The characteristicsof these solubilized isozymes were different from those of thesoluble isozymes. On the contrary, W-II, M-II and Ms were quitesimilar to those of the solubles in pH optima, substrate specificity,K_m value, affinity to DEAE-cellulose and gel electrophoreticpatterns. These results suggest that W-II, M-II and Ms werederived from the soluble isozymes. Phaseolus vulgaris L., bean, hypocotyl, acid phosphatases, Michaelis constant     

An Extracellular Insoluble Protein in Cultures and Seeds of Carrot

An 18-kDa extracellular insoluble protein (EIP18) was foundin the culture medium of calli and in seeds of carrot (Daucuscarota L.). EIP18 was found in amorphous particles suspendedin the culture medium of callus and was not solubilized by treatmentof these particles with EDTA, with Triton X-100 plus NaCl orwith LiCl, but it was partially solubilized by treatment withNaSCN and was entirely solubilized by treatment with urea. EIP18seems not to belong to any known family of cell wall proteinsbecause of the complete absence of glycan moieties and its lowlevels of hydroxyproline, proline and glycine. The protein inthe medium seemed to have become insoluble as a result of formationof a homopolymer. In the plant, the protein was found only inthe seed, being located both in the embryo and at the inneredge of the endosperm that faced the interspace between theembryo and the endosperm. EIP18 might be an extracellular matrixprotein specific to seeds. (Received August 19, 1994; Accepted December 20, 1994)     

Chitinolytic enzymes produced by ovine rumen bacteria

Two strains of clostridia, isolated from the rumen fluid of sheep as potential antagonists toward anaerobic fungi showed a complete array of chitinolytic enzymes. Enzyme tests in cultures demonstrated endochitinase, exochitinase,N-acetylglucosaminidase, chitosanase and chitin deacetylase activities mainly in the extracellular fractions. In all samples, the highest was the activity of exochitinase (600–1100 nmol mL⁻¹ h⁻¹); the activity of endochitinase (280–500 nmol mL⁻¹ h⁻¹) was also significant. Chitinases were stimulated in the presence of reducing compounds and no dependence on cations was observed. In both strains different isoforms of chitinases of molar mass 36–96 kDa were detected. The chitinases from our isolates lyzed cell walls of anaerobic fungiin vitro and inhibited the activity of fungal β-1,4-endoglucanases. Of the two bacteria examined, one was more effective in both antifungal effects.     

Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan(HA), a large polysaccharide abundant in the extracellular matrixof vertebrate tissues. Six genes have been predicted to encodehyaluronidases in humans, but the protein products of only SPAM1,HYAL1, and HYAL2 have been characterized. We have now expressedthe mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in BabyHamster Kidney (BHK) cells and demonstrated the presence ofmultiple forms of Hyal3 ranging from 45 to 56 kDa in expressionlysates. Complete and partial digestions of the expressed proteinwith PNGase F showed three N-linked oligosaccharides accountedfor all forms of Hyal3 detected in expression lysates. Mostof these oligosaccharides were Endo H sensitive, indicatingthat they were high mannose or hybrid N-linked oligosaccharides.Subcellular fractionation of Hyal3-expressing BHK cells by densitygradient centrifugation revealed most Hyal3 in a low-densityvesicular population. Low levels of Hyal3 were detected in higherdensity vesicles, but no colocalization with the late endosomal/lysosomalmarker Lamp1 was found by immunofluorescence microscopy. BHKcells stably expressing Hyal3 had increased acid-active hyaluronidaseactivity, but no such activity was detected when Hyal3 was transfectedinto Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpressionof Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels,suggesting that the increased hyaluronidase activity in thesecells was due to Hyal1 rather than Hyal3. The results indicatethat Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidaseactivity and that Hyal3 may contribute to HA metabolism by augmentingthe activity of Hyal1.     

Substrate specificities of tobacco chitinases

Ten tobacco chitinases (1,4-N-acetyl-β-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a β-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endo-type enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)_4–6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.     

Tryptophan Oxidizing Enzyme and Basic Peroxidase Isoenzymes in Arabidopsis thaliana (L.) Heynh.: Are They Identical?

The in vitro conversion of [³H]tryptophan by a plasma membraneenriched fraction from Arabidopsis thaliana (L.) Heynh. seedlings,grown in liquid culture, revealed indole-3-acetaldoxime (IAOX)as the only detectable reaction product. The pH optimum of thereaction was at pH 8, the K_m value for tryptophan 12 µM.The formation of IAOX was stimulated about 10-fold by H₂O₂ Incubationexperiments with solubilized proteins and membrane vesiclesshowed that the investigated enzyme(s) were bound covalent tothe plasma membrane. Tryptophan oxidizing enzyme (TrpOxE) andperoxidase activity were not only found in the plasma membrane,but also in the culture medium. Specific IAOX forming activitywas 74-fold and 6-fold higher compared to the crude extractand the plasma membrane fraction, respectively. After isoelectricfocusing of solubilized plasma membrane and precipitated mediumproteins, TrpOxE activity co-migrated with two prominent highpI peroxidase bands stained with benzidine-guaiacol. The zonesof the IEF gel with peroxidase and TrpOxE activity were analyzedby SDS PAGE and revealed in all fractions a main protein bandof ca. 55 kDa. TrpOxE activity and peroxidase activity wereboth inhibited by antisera directed against tobacco and horseradishperoxidase. TrpOxE activity and peroxidase activity were determinedduring plant development. TrpOxE activity peaked after 8 and42 days, whereas peroxidase activity was consistently presentduring the whole life cycle. The inhibitory effects of indolederivatives, especially indole-3-glyoxylic acid, on (i) seedlingdevelopment and (ii) on TrpOxE and peroxidase activity werealso compared. (Received November 1, 1991; Accepted September 2, 1992)     

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.     

球孢白僵菌两种胞外几丁质酶的诱导和纯化

球孢白僵菌突变株CH-1316在完全培养基中培养至对数前期后转入以胶体几丁质为唯一碳、氮源的液体诱导培养基中继续培养20～25h,几丁质酶被诱导产生;在对数生长期胞外几丁质酶活力最高。发酵液经(NH_4)_2SO_4沉淀、DEAE-纤维素层析和凝胶过滤分离出二种几丁质酶组分,在聚丙烯酰胺凝胶电泳图上显示出两条均一的带,并且每条带都具有几丁质酶活力。几丁质酶1既是外切酶又是内切酶,而几丁质酶2只表现内切酶活力。分子排阻法测得这两种酶的分子量分别为52000和39000。