The Enzymatic Approach of Zygomycosis - Causing Mucorales
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Hee Sun Ko et al: The Enzymatic Approach of Zygomycosis - Causing Mucorales Kor J Med Mycol 12(1), 2007 Original Article The Enzymatic Approach of Zygomycosis - Causing Mucorales Hee Sun Ko1, Hideaki Taguchi2, Kayoko Takizawa2, Kazutaka Fukusima2 and Hyun Soo Kim1,3 Center for Traditional Microorganism Resources, Keimyung University, Dalseo-gu, Daegu 704-701, Korea1, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba 260-8673, Japan2 and Department of Microbiology, Collage of Natural Science, Keimyung University, Dalseo-gu, Daegu 704-701, Korea3 Various oxidases and hydrolytic enzymes were analyzed to investigate the relationship between these enzymes and the skin pathogenicity of 18 Mucorales strains. Each strain was cultured in a nutrient medium containing starch as a carbon source. The cells grew quickly and were at a good state of growth after incubation for three days. Oxidase activity was not detected in any strain, whereas Mucor spp. including Mucor racemosus IFM47053 typically had high alcohol dehydrogenase (ADH) activity and all the strains had catalase activity. The culture filtrate and the cell free extract of each strain were applied to APIZYM test system, which revealed that all the strains examined produced many hydrolytic enzymes both inside and outside their mycelia. In the case of Absidia corymbifera strains, lipase activity was comparatively high, and polysaccharide hydrolytic enzymes such as α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase were produced. [Kor J Med Mycol 2007; 12(1): 9-17] Key Words: Zygomycosis, Alcohol dehydrogenase (ADH), Catalase, Hydrolases coming common as this disease can be caused by INTRODUCTION other members of the Zygomycete class of fungi in addition to the Mucor species. Most species of Mucor are filamentous fungi of the class Zygo- Mucor are unable to grow at 37℃ but the strains mycetes that are found in soil, plants, decaying isolated from human infections are usually ther- fruits and vegetables. These saprophytic fungi grow motolerant. A characteristic feature of human zygo- fast and appear early in the process of material mycosis is that it causes a fence-sitting infectious decomposition. Organisms of the Mucor species disease by weakening the immunity of a host. are distributed worldwide. Not only ubiquitous in The role of oxidation-reduction enzymes in the nature but also a common laboratory contaminant, skin pathogenicity of Zygomycetes has seldom some Mucorales such as genus Rhizopus, Mucor, been investigated. In order to study of the relation- Absidia, Mortierella may cause the disease mucor- ship between enzymes and pathogenicity, we mea- mycosis1,2 or zygomycosis. The latter term is be- sured various oxidizing and hydrolysing enzymes † Corresponding author: Hyun Soo Kim, Department of by applying the APIZYM 20 test system to the 18 Microbiology, Collage of Natural Science, Keimyung University, pathogenic strains of Zygomycetes. Dalseo-gu, Daegu 704-701, Korea. Tel: +82-(0)53-580-5284, Fax: +82-(0)53-580-5509 e-mail: [email protected] - 9 - Kor J Med Mycol 12(1), 2007 Table 1. Strains belonged to Mucorales used in the MATERIAL AND METHODS present experiments Strain 1. Organism, medium and culture conditions Mucor circinelloides IFM47052 Eightteen strains of Mucorales were obtained Mucor ramosissiumus IFM46006 from Chiba University Research Center for Patho- Mucor circinelloides IFM46508 genic Fungi and Microbial Toxicoses, Chiba. The Mucor racemosus IFM40781 strains (Table 1) were maintained on potato dex- Mucor racemosus IFM47053 trose agar (PDA; Becton Dickinson, Sparks, MD, Mucor hiemalis IFM41559 USA). The medium (liquid medium) for the pro- Cunninghamella bertholletiae IFM46110 duction of the oxidoreductases contained the follo- Cunninghamella bertholletiae IFM46111 wing compounds (g per liter): starch, 20; NaNO3, Cunninghamella elegans IFM46109 5; K2HPO4, 5; MgSO4 · 7H2O, 1; yeast extract, 0.2; trace salt solution, 2 ml; vitamin solution, 0.2 ml. Cunninghamella elegans IFM47050 The trace salt solution had the following compo- Rhizopus oryzae IFM40515 sition (g per 100 ml): FeC6H5O7 · nH2O, 0.6; CaCl2 Absidia corymbifera IFM41345 · 2H2O, 0.4; ZnSO4 · 7H2O, 0.2; MnCl2 · 4H2O, Absidia corymbifera IFM47048 · 0.01; KI, 0.01; 6(NH4) Mo7O24 4H2O, 0.01; CoCl2 Absidia corymbifera IFM5335 · 6H O, 0.01; H BO , 0.01; NaCl, 0.5. The vitamin 2 3 4 Absidia corymbifera IFM40776 solution had the following composition (g per 100 Absidia cylindrospora IFM40501 ml): biotin, 0.002; Ca-pantothenate, 0.4; pyridoxin- Absidia cylindrospora IFM40777 HCl, 0.4; p-aminobenzoate, 0.2. The medium (150 ml) in a 500-ml Erlenmeyer flask was sterilized Absidia glauca IFM41346 by autoclaving at 121℃ for 20 min. The flask was inoculated and incubated at 30℃ on a rotary consumed 1 μmol of oxygen per min. shaker (180 rpm). After incubation for four days, 2) Catalase acitivity cells were collected by centrifugating the culture. Catalase activity was measured in cell free extract The resultant cells were washed twice with 50 mM and culture filtrate by the method of Aebi4. The potassium phosphate buffer (pH 7.2) and stored at decomposition of the substrate H2O2 was moni- -20℃ until use. tored spectrophotometrically at 240 nm. Specific activity was defined as the micromole substrate 2. Enzyme assays decomposed per minute per milligram of protein 1) Alcohol oxidase activity (i.e. U/mg protein). Alcohol oxidase activity was assayed by mea- 3) Dehydrogenase activity suring oxygen consumption using an oxygen elec- Enzyme assays were carried out in a 1 ml volume trode (Oxygraph 9; Central Science, Tokyo, Japan)3. with 50 μM β-NAD+ cofactor, 1 M substrate The incubation cell (1 ml) contained 100 μmol of (alcohol, lactate, malate, mannitol, alanine etc.) and alcohol, 100 μmol of potassium phosphate buffer they were monitored for reduction of the cofactor -1 -1 5 (pH 7.2) and 100 μl of enzyme preparation, and it at 340 nm, e340 = 6220 M cm . The spectro- was incubated at 30℃. One unit (U) of alcohol photometer used was a Shimadzu UV-2500 (Kyoto, oxidase was defined as the amount of enzyme that Japan). For the enzyme kinetic characterization, - 10 - Hee Sun Ko et al: The Enzymatic Approach of Zygomycosis - Causing Mucorales 50 mM potassium phosphate buffer was used at pH 7.4 at a temperature of 30℃. RESULTS 4) Laccase activity 1. The growth of Zygomycetes Laccase activity was determined by measuring the oxidation of 2, 2'-azinobis (3-ethylbenzthiazo- Each Zygomycete was grown in the medium line-6-sulfonic acid) (ABTS) at 420 nm (ε = 3.6× composition and under the culture conditions de- 104 cm-1 M-1)6. The reaction mixture contained scribed above. Genus Cunninghamella and Mucor 0.03% ABTS, 0.1 M sodium acetate buffer, pH 5.0 circinelloides demonstrated fast growth, approa- and a suitable amount of enzyme solution. One ching the maximum mycelia production after in- laccase activity unit was defined, as the amount of cubation for three days. The mycelia production of enzyme that oxidized 1 μmol ABTS per min. genus Absidia was quite good, but the level achie- 5) Hydrolase activity APIZYM application ved after incubation for two days was maintained Cell free extract and culture filtrate of each strain without further increasing. Mucor ramosissimus were inoculated into the APIZYM test system, grew very slowly and their number of mycelia also incubated for 4 h at 30℃ and processed according increased slowly. Rhizopus oryzae showed poor to the manufacturer's directions7 in order to deter- mycelia production with a spool-like form. mine the strain's enzyme profile. The APIZYM 2. Oxido-reductase production system (BioMerieux Vitek) consists of 19 hydrolase substrates, alkaline phosphatase, esterase, esterase 1) Oxidase activity lipase, lipase, leucin arylamidase, valine arylami- Various cell free extracts from 18 strains of dase, cysteine arylamidase, trypsin, chymotrypsin, Zygomycetes were screened using Oxigraph, to acid phosphatase, naphtol-AS-BI-phosphohydro- detect oxidase activity against each substrate (glu- lase, alpha-galactosidase, beta-galactosidase, alpha- cose, methanol, ethanol, mannose, mannitol, guaia- glucosidase, beta-glucosidase, N-acetyl-beta glu- col, glycin, maleic acid, nicotinamide, lactose, tyro- cosaminidase, alpha-mannosidase, and alpha- sine, inositol, lysine, xylose, fructose, glucosamin fucosidase. APIZYM allows the rapid detection of and arabinose). No oxidase activity was demon- constitutive enzymes based on a colorimetric assay. strated for any of the 18 strains (data not shown). The intensity of color development following the Moreover, no trace of laccase was detected in the addition of reagents, determines the presence and cell free extract. Therefore we conclude that the level of enzyme activity. Zygomycetes do not produce oxidases as their main enzymes. 3. Protein assay 2) Catalase activity Protein was measured by the method of Brad- Catalase activity was assessed in the cell free ford8 with bovine serum albumin as the standard extract of the Zygomycetes. All the Zygomycetes protein. had some degree of catalase activity. Genus Cun- ninghamella showed the most abundant catalase 4. Chemicals production (Table 2). Mucor spp. also had some The other reagents which are not referred to catalase production in their culture filtrates (data specifically are from Wako Pure Chemical (Osaka). not shown). 3) Dehydrogenase activity Various dehydrogenase activities were screened - 11 - Kor J Med Mycol 12(1), 2007 Table 2. Catalase and alcohol dehydrogenase activity by various strains of Mucorales Catalase ADH Strain (U/mg) (U/mg) M. circinelloides IFM47052 148 0.53 M. ramosissiumus IFM46006 133 0 M. circinelloides IFM46508 139 0.64 M. racemosus IFM40781 157 0.034 M. racemosus IFM47053 143 1.022 M. hiemalis IFM41559 235 0.50 Fig. 1. Alcohol dehydrogenase activity of Mucor spp. C. bertholletiae IFM46110 94 0 ○, Mucor circinelloides IFM47052; ●, Mucor circine- C. bertholletiae IFM46111 57 0 lloides IFM46508; □, Mucor racemosus IFM47053; ▲, Mucor hiemalis IFM41559. C. elegans IFM46109 562 0 C. elegans IFM47050 214 0 order to examine the production of hydrolases by R.