A Method for the Quantitative Assessment of Wound-induced Chitinase Activity in Potato Tubers

A method for the quantitative assessment of chitinase activity was developed. Dilution series of crude potato tuber chitinase extracts were assayed with a colorimetric microtitre plate assay. using CM-chitin-RBV as enzyme substrate. Linearity between absorbance values mea-sured (540 nm) and enzyme concentration was found to be limited to the low concentration range. where depletion of the substrate was no longer limiting. As as absorbance of 0.1 always fell within the concentration range for which absorbance-concentration linearity was valid. one unit of enzyme activity was defined as the amount of enzyme needed to yield and A of 0.1. A more reliable method for the assessment of chitinase activity was established by basing the difinition of enzyme, activity on a concentration rather than on as absorbance value. as was done previously. Using this method, differences in the rate of chitinase induction upon wounding were demonstrated for six commercial potato cultivars.     

Detection of bacterial chitinase activity in transformed plant tumour cells using a specific exochitinase substrate

Methods for the detection of bacterial chitinase activity were compared. The soluble substrate p-nitrophenyl-ß-D-N,N diacetyl chitobiose (NDC) was more sensitive in detecting purified chitinase of Serratia marcescens than assays measuring degradation of a solid chitin substrate by either radiochemical or colorimetric means. A chimaeric gene containing a S. marcescens chitinase gene under control of a Cauliflower Mosaic Virus 35S promoter and nopaline synthase terminator sequences was constructed and transferred to tobacco tumour cells using Agrobacterium tumefaciens as a vector. The rate of hydrolysis of the NDC substrate was three fold greater with cell extracts of both pooled and individual tumours carrying the chimaeric chitinase gene than in control tumours. It was calculated from the enzyme activity data that the foreign bacterial chitinase contributed 0.1% of the total soluble protein in transformed plant cells. This level of expression of this gene was not detectable using the less sensitive assays employing solid chitin substrate. These results indicate that NDC is a preferable substrate for assaying bacterial chitinase in transformed plant cells.     

Characterization of chitinases and β-1,3-glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit ( Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β-1,3-glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS-PAGE were probed with antibodies raised against purified citrus chitinase and β-1,3-glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β-1,3-glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β-1,3 linkages for activity, were active against both soluble and insoluble β-1,3 glucans and generated similar products.     

A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide.

A total of 101 strains of bacteria from environmental and clinical sources, most of which were gram negative, were tested for chitobiase activity by using a filter paper spot test with 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide as the substrate. The results were compared with those obtained by a conventional plate method for chitinase activity by using colloidal chitin as the substrate. There was excellent agreement in the results for both methods. The filter paper spot test with 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide has the advantages of being rapid, simple to perform, and inexpensive. This method should be adaptable to a wider range of microorganisms, particularly those with unusual growth requirements.     

A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide

A total of 101 strains of bacteria from environmental and clinical sources, most of which were gram negative, were tested for chitobiase activity by using a filter paper spot test with 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide as the substrate. The results were compared with those obtained by a conventional plate method for chitinase activity by using colloidal chitin as the substrate. There was excellent agreement in the results for both methods. The filter paper spot test with 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide has the advantages of being rapid, simple to perform, and inexpensive. This method should be adaptable to a wider range of microorganisms, particularly those with unusual growth requirements.     

Characterization of gastrointestinal chitinase in the lizard Sceloporus undulatus garmani (Reptilia: Phrynosomatidae)

Most studies on chitinase activity in lizards have been concerned with Palaearctic (European) and Laurasian (Middle Eastern and Asian) taxa. Several genera of Old World lizards, Anguis, Uromastix, Chamaeleo and Lacerta, have been shown to possess chitinolytic activity. To date, only one New World lizard, Anolis carolinensis, has been reported to exhibit chitinolytic activity. In the present study, chitinase activity was characterized in a second New World taxon, Sceloporus undulatus garmani, a New World, phrynosomatid lizard. Chitinolytic activity was measured by incubating tissue extracts with a radioactive chitin substrate, acetyl-[H³]chitin and determining acid soluble radioactivity as an estimate for chitin hydrolysis. Chitinolytic activity was present in stomach, small intestine and pancreas extracts, with the stomach and pancreas having the highest specific activities. Chitinolytic activity was higher at pH 4.5 than at pH 7.5. The stomach chitinase is immunologically similar to the gastric chitinase previously described for rainbow trout. Western blot analysis showed anti-chitinase cross-reactivity in the extracts of the stomach, but no cross-reactivity in the pancreatic or intestinal extracts, suggesting different isoforms of chitinase. There was no detected lysozyme activity (less than 0.01 mg/ml lysozyme) present in the extracts of the stomach, small intestine and pancreas. The localization of chitinolytic activity in S. u. garmani is in agreement with earlier reptilian reports on the distribution of chitinase.     

Expression of mammalian Rab Escort protein-1 and -2 in yeast Saccharomyces cerevisiae

A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from mold (Aspergillus giganteus MDH 18894) has been developed. alpha-Sarcin and AFP were purified simultaneously by chitin affinity column chromatography and gel filtration. By this method, 4.5 mg of pure alpha-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Compared with other purification methods such as ion-exchange column chromatography, this procedure was very simple and specific. The purified alpha-sarcin and AFP were homogeneous as characterized by SDS-polyacrylamide gel electrophoresis. Both alpha-sarcin and AFP exhibited the binding activity to generated chitin. Soluble glycochitin decreased the intensity of fluorescence of alpha-sarcin and made the lambda(em)m shift from 340 to 347 nm. Titration of alpha-sarcin with N-bromosuccinimide under native conditions revealed that two tryptophans (Trps) were all located in the core part of alpha-sarcin molecule. This indicated that Trps were not involved in the binding of alpha-sarcin to chitin. Glycochitin in the culture medium increased the expression of alpha-sarcin, while it had no effect on the expression of AFP. Unlike other ligands such as Cibacron blue for the affinity purification of alpha-sarcin and AFP, glycochitin increased the nuclease activity of alpha-sarcin.     

Quantitative fluorometric analysis of plant and microbial chitosanases.

A quantitative fluorometric assay for chitosanase activity in bacterial and plant tissues was developed. The assay can be conducted with either finely milled preparations of chitosan in suspension or dissolved chitosan; activity is based on measurements of glucosamine (GlcN) or oligomers of GlcN. GlcN is detected fluorometrically after reaction with fluorescamine with detection in the nanomole range. Fluorescence measurements of chitosanase activity and radioassay of chitinase in commercial preparations of chitinase from Streptomyces griseus revealed that both activities were present. Specific activities for the S. griseus chitosanase using suspended and soluble chitosans were respectively 1.24 and 6.4 mumol GlcN.min-1.mg protein-1. Specific activity of the S. griseus chitinase was 0.98 mumol GlcN.min-1.mg protein-1. Sweet orange callus tissue was tested for chitosanase and chitinase activity. It was necessary to remove small amine-containing molecules from the callus preparations before chitosanase activity could be assayed. The specific activity for chitinase and chitosanase in desalted extracts of nonembryogenic Valencia sweet orange callus tissue was determined to be 18.6 and 89.4 nmol GlcN.min-1.mg protein-1, respectively.     

A method for the study of fungal growth inhibition by plant proteins

A bioassay is described for the study of inhibitory activity of plant proteins on fungal growth. Fungal spores were germinated in liquid growth medium and pipetted into wells of a microtitre plate. Fungal growth was followed spectrophotometrically. The bioassay was tested using crude protein extracts from plant tissues known to have high activities of chitinase and beta-1,3-glucanase, and with purified enzymes. Crude protein preparations and combinations of the purified enzymes produced a temporary reduction of growth but no permanent growth inhibition.     

A Microtiter Plate Assay for Screening Antioxidant Activity in Extracts of Marine Organisms

A novel microtiter plate assay was developed to determine the total peroxyl radical-trapping activity of antioxidants extracted from marine organisms by measuring the inhibition rate of dye-substrate oxidation. We compared use of dihydrorhodamine-123, dihydrofluorescein, and dichlorodihydrofluorescein as reduced substrates for oxidation by peroxyl radicals generated from 2,2-azobis(2-amidinopropane) dihydrochloride. The oxidation products of these highly reactive substrates are intensely colored dyes that absorb maximally in the wavelength region, _max = 489 to 512 nm, and their concentrations were determined photometrically using a 96-well, microtiter plate reader. The microtiter plate method provides for concurrent multisample analysis with automated data storage, regression analyses, and calculation of oxidation inhibition rates. Dihydrorhodamine was selected as the preferred substrate for screening crude extracts, and typical assay results are presented. Novel lead antioxidants are selected from active extracts by chromatographic analysis with electrochemical detection.     

Biochemistry of insect differentiation: A system for studying the mechanism of chitinase activity in vitro

Results obtained with an in vitro system for the study of chitinase are described. The system involves soluble enzyme protein(s) and an insoluble substrate preparation. With insect molting fluid chitinase, it shows properties that parallel those observed during in vivo breakdown of cuticle during the molt. For example, molting fluid chitinase activity not previously exposed to chitin is stronly and specifically adsorbed to the substrate, in contrast to other enzymatic activities including hexosaminidase (chitobiase) present in molting fluid. This leads to partial purification of molting fluid chitinase activity reflected in increased specific activity of chitinase associated with the insoluble chitin substrate; we have previously reported increase of specific chitinase activity of (deproteinized) cuticle resulting from its incubation with molting fluid (M. L. Bade and A. Stinson, 1978, Biochem. Biophys. Res. Commun.84, 381–388). Soluble end product is generated rapidly and linearly with time by the in vitro system; the end product is assumed to be N-acetylglucosamine since the specific radioactivity of this compound is unchanged during the 10 min required for assay. Molting fluid chitinase activity may involve a number of polypeptides ranging in molecular weight from 145,000 to less than 20,000 daltons. The system described gives results consistent with a processive mechanism for molting fluid chitinase, i.e., data are given demonstrating that molting fluid chitinase continues to act on the same chitin particle(s) with which it initially associates rather than diffusing freely from substrate particle to substrate particle, and the product of its action appears to be a monosaccharide rather than a mixture of oligosaccharides. Processive behavior for chitinase would be predicted from the known structure, and the in vivo measured rate of breakdown, of cuticle chitin during the molt; the preliminary nature of this conclusion, based on what is so far known about the structure of the substrate used in the in vitro system, is briefly discussed.     

Comparison of chitinase isozymes from yam tuber--enzymatic factor controlling the lytic activity of chitinases

To evaluate the anti-pathogen activity of chitinases, we developed a new method for measuring the lytic activity, and investigated the correlation of the lytic activity with the enzymatic properties by using four chitinase isozymes, Chitinases E, F, H1 and G, which had been purified from yam tubers by column chromatography. Chitinases E, F and H1 had high lytic activity against the plant pathogen, Fusarium oxysporum, but Chitinase G did not. Chitinase E, which is the family 19 chitinase, was similar to Chitinases F and G in its antigenecity, but not to Chitinase H1 or H2. Chitinases H1 and H2 were recognized by the anti-Bombyx mori chitinase antibody, suggesting that Chitinases H1 and H2 are family 18 chitinases like B. mori chitinases. Chitinases E, F and H1 had two optimum pH ranges of 3-4 and 7.5-9 toward glycolchitin, but Chitinase G had only one optimum pH value of 5. Chitinases E, F and H1 had higher affinity to the polymer substrate, glycolchitin, than Chitinase G. These results suggest that the lytic activity of plant chitinases may be related to the chitin affinity and probably to the characteristic optimum pH value, or two values, but not related to its classification. The correlation of the lytic activity of a chitinase isozyme with its elicitor specificity is also discussed.     

A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes

We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the K_m of ATP binding to Cdk1 to be 3.5 μM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity.     

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.     

Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption

Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. To establish this new assay method, a chitin-active LPMO from Bacillus atrophaeus (BatLPMO10) was cloned as a model enzyme. An expression system using B. subtilis as the host cell yielded a simple purification process without complicated periplasmic fractionation, as well as improved productivity by 3.7-fold higher than that of Escherichia coli BL21(DE3). At the optimum pH determined using a newly developed assay, BatLPMO10 showed the highest activity in terms of promoting chitin degradation by a chitinase. In addition, the assay method indicated that BatLPMO10 was inhibited by sodium ions, and BatLPMO10 and a chitinase mutually enhanced each other’s activities upon degrading chitin as the substrate. In conclusion, this hydrolase-free ascorbate assay allows quantitative analysis of BatLPMO10 without a coupled enzyme.     

Purification and characterization of chitinase produced by Streptomyces erythraeus

A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.     

Identification of secreted proteins of Aspergillus oryzae associated with growth on solid cereal substrates

Filamentous growth of Aspergillus oryzae on solid cereal substrates involves secretion of substrate converting enzymes and a solid substrate specific polarised hyphal growth phenotype. To identify proteins produced under these specific conditions, the extracts of A. oryzae grown on wheat-based media were analysed using N-terminal sequence analysis. In a submerged wheat-based growth medium of A. oryzae, besides alpha-amylase, also an arabinosidase and xylanase were abundantly produced. In the extracts of A. oryzae grown on wheat-based solid substrate besides alpha-amylase and chitinase, two new proteins of 16 and 27 kDa were identified. These hypothetical proteins showed only close homologies to filamentous fungal proteins.     

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.     

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.     

Determination of peptidylglycine alpha-amidating monooxygenase activity in human serum by thin-layer chromatography

We developed a simple assay system for the quantitative evaluation of peptidylglycine alpha-amidating monooxygenase activity using as substrate a 125I-labeled synthetic tripeptide, 125I-D-Tyr-Val-Gly, thin-layer chromatography, and a radiochromatoscanner. The basic principle of this method is that thin-layer chromatography separates the reaction product, 125I-D-Tyr-Val-NH2, from the substrate in an assay mixture. The 125I activities of both substrate and product separated from each other on a thin-layer chromatography plate were quantified with a radiochromatoscanner and the rate of conversion of the substrate to the product was calculated from their counts. Human serum was used as an enzyme source and the values of alpha-amidation activity obtained by our method under optimal conditions were almost identical to those of the published method using ion-exchange chromatography (sulphopropyl-Sephadex C-50 column) and a gamma-counter. Our method makes it possible to estimate the 10-pmol level of the product using 10 microliters of human serum and to assay a large number of samples rapidly and easily. It is therefore thought to be very useful for screening various tissues for alpha-amidation activity.