X~olia Mierobiol. 26, 142--146 (1981)

Production of a-Amylase by Microscopic Fungi J. AUOUSTIN a, g. ZEMEK a, O. FASSATIOV-~ b and E. KUNIAK a

alnstitute of Chemistry, Slovak Academy of Sciences, 809 33 Bratislava, Czechoslovakia, and bDepartment of Botany of Lower Plants, .Faculty of Science, Charles University, Prague

Received November 15, 1979

ABSTIIAET. The amylolytic activity and especially the production of ~-amylase (EC 3.2.1.1) and ~-gluco- sidasc (EC 3.2.1.20) was screened in imperfect fungi, mucoral fungi and some ascomycetes. The character of the polysaccharide system, which is responsible for the utilization of ~ (1 --~ 4) glucan, was specified with a concominant screening of growth on soluble starch. The amylolytic activity was found in 29 strainm ou$ of ~he 49 tested.

At present a-amylase is obtained by isolation from media of the production strains of Bacillus subtilis or Streptomyces aureofaciens, a-Amylases are known to be produced by some representives of imperfect fungi especially from the genera Aspergillus, Rhizopus, Penici lI ium, Paecilomyces, Alternaria etc. (Dunn et al. 1959; Dvatsa tova 1958; Kamachl and Tanaka 1957; Tsujisaka et al. 1958; Sakamoto and Shuzui 1957; Sakamoto 1958; Kuvikova 1961). Hayashida (1957) isolated erytalline a-amylase from Aspergil lus awamori and Ozaki (1957) from Aspergillus usami. Kalashnikov and Trainina (1961), and Dass-Gupta and Verma (1962) studied the amylolytic act ivi ty in various strains of nonspecified genera of Aspergillus, Rhizopus, M u t e r and Alternaria. Similar studies were performed b y Kanie (1956), Loginova (1960), Oda and Iguohi (1963), Misaki et al. (1961), Sawada et al. (1966), Herada (1963) and Galich et al. (1974). The present s tudy is concerned with the a(1 -> 4) glucan utilizing ability of some imperfect fungi, mueorM fungi and ascomycetes, and with the specifi- cation of the type of the hydrolase system which is responsible for the degradation process.

MATER/ALS AND M E T H O D S

The strains studied (Table I) were from the Collection of Fungal Cultures of t l~ Facul ty of Science (Depar tment of Bo tany of Lower Plants, Charles University, Prague). Selection of strains was performed according to their ability to grow in the presence of soluble starch or maltose as the sole carbon sources in the cultivation medium.

Growth in the presence of soluble starch was tested on a liquid cultivation medium with Difco Yeast Nitrogen Base (YI~B), containing 2 ~ soluble starch. The pur i ty of the starch, i.e. its content of saeeharide components, maltose or glucose, was tested chromatographically. The a-glueosidase act iv i ty was tested according to Tal- mage and Albersheim (1969) using 4,nitrophenyl a-D-glucopyranoside. The amylase- producing ability was screened according to the technique of Zemek and Kuniak

1981

TABLE I. List of tested strains

PRODUCTION OF a-AMYLASE BY FUNGI |45

Genus and species Production of ~-Amylaso

Strain Coll. a-amylase activity No. (liquefaction

of gel, d) U/L

Avremonium kilience G]ai3Tz 571 Alternaria tenuissima (FRIES) WILSHIRE 1493 Aspergillus oryzae (AHLBURG) COHN 1036 A. oryzae 1550 A. parasiticus SPEARE 1131 Aureobasidium pullulans (DE BARY) BERKI~. 1138 Beauveria bassiana (BALs.) VUILL. 264 Botrytis einerea PERSOOI~ 917 Byssochlamys fulva OLLIVEX et G. S~ITH 1487 B. nivea WESTLINO 1558 Chrysosporium pannorum (LINK) HUdHES 271 Cladosporium cladoaporioides (FREs.) VRIES 1431 C. resinae (LI~DAU) VINES 1127 Cylindrocarpon radicicola WOLLENWEBER 950 Cunninghamella elegans LENDN. 1050 Diaporthe impulsa (CKE. et PEcx) SACC. 1470 Diplocladium maius BONORD. 276 Doratomyces stemonitis (PERs.) COI~T)~- 922 ~usarium culmorum (W.G. SM.) SAcc. 1117 ~. oxysporum var. dianthi (P~ILL. et DEL) RAIL~O 1358 F. oxysporum SCHLECHT. GilmanieUa humicola BARROW 528 Gliocladium catenulatum GILMAN et ABBOTT 1494 G. roseum (LK.) BAIN 1495 Humicola brevis (GILZtAN et ABBOTT) GILMAN 1054 H. fuscoatra TP.AAEN 1341 H. grisea TRAAEN 1429 Hypoxylon .fuscus PARS. ex Fm 1463 Metarrhizium anisopliae (ME(~N.) SOR. 966 Mucor globosus FISCHER 476 Myrothecium striatisporum PaESTON 1409 Paecilomycesfarinosus (DICKS. ex FRO BROWN et S~ITg 343 P. varioti BAINIE~ 341 Penicillium albidum SoPP. 1481 P. cyelopium WESTLING 1154 P. expansus LI~K 1443 l~hizopus arrhizus FIS0~.R 100 Seopulariopsis brevicaulis BAINIER 1347 Sordaria fumieola Stachybotrys chartarum (EHREI~B. ex LINK) HU(mES 506 Stemphylium botryosum WALL~. 585 Thamnidium elegans LINK 468 Thielavia basicola (B. et BR.) ZO~'F. 1376 Torula herbarum (PE~s.) LINK 1370 Triehoderma boningii OVD. 1455 T. viride PERS. eX S . F . GRAY 1403 Triehothecium roseum LINK. 395 Verticill iumfalcatum (PETC~.) W. GAMS 1390 Zygorhynehus heterogamus (VuiIm.) VUIL~-. 1490

+ (3)

+ (I) 421

+ (4) 96

+ (5) 74

+ (4) lOr

+ (5) 65 + (1~) 25

+ (7) 39

+ (3) 148 + (2) 262 + (3) 169 + (12) 19 + (5) 82 + (6) 51 + (4) 9o + (4) 98 + (4) 81 + (9~ 32

+ (3) 127 + (4) 91 + (6) 65

+ ( ~ 52

+ (5) 85 + (5) 71 + (9) 45 + (12) 28

+ (2) 293 + (3) 148 + (7) 46

|44 J. AUGUST:~N et a l . Vol. 26

(1978) based on cultivating the microorganism on the surface of a gel prepared from amylose crosslinked with 2-chloromethyloxirane (Fig. 1). The amylose gel (8 % dry weight) contained diffusion-distributed components of the YNB (6 g/L). Triangular blocks (side length 4.5 mm) were cut from the gel, placed into Petri dishes, immersed in the YNB solution reaching to half of the gel thickness and sterilized at 100 ~ in streaming steam for 1 h. No degradation of polysaccharide macromolecules occur- red during the sterilization of the crosslinked amylose or of the dissolved starch. Sterile gels were inoculated with the tested microorganisms. Strains that produce ~-amylase during growth liquefy the gel since ~-amylase degrades the tridimensional lattice of the crosslinked po]ysaccharide. Enzymes attacking ~(1 --> 4) glucau from the nonreducing termini (~-glucosidase, ~-amylase, amyloglueosidase) do not liquefy the gel of the crosslinked amylose, splitting only a portion of the external chains up to the crosslink. Chromatographic analysis of the cultivation medium with liquefied gel descending paper chromatography, Whatman 1 paper, ethylacetate-pyridine- water, 8 : 2 : 1, 18 h) revealed the accumulation of maltose-type oligosaccharides (especially maltose, maltotriose and maltotetraose) and glucose indicating the amylolytic splitting of ~(1 -+ 4) glucan.

The biological purity was checked by microscopic and cultivation tests. Activity of ~-amylase released into the liquid fraction was tested after liquefaing the gel by a chromolytic method according to Kuniak and Zemek (1979). The analysis was performed in 0.5 mL of medium (pH set at 5.0 by 0.5 mL of 50 mM acetate buffer with 50 mM NcC1). The obtained absorbance values were transformed into ~-amylase activity units on the grounds of an effect of isolated ~-amylase from Aspergillus oryzae and of a reference radiochemical method using U714C-dtarch according to Zemek et al. (1978).

The ~-amylase activity is expressed in U/L units which correspond to micromoles of the maltose-type oligosaccharides released in 1 min from starch at 30 ~ in 1 L of the cultivation medium.

RESULTS AND DISCUSSION

Most of the tested imperfect and mucoral fungal species and some ascomycetes were found to be capable to utilizing ~(1-~ 4) glucan with concomitant gel lique- faction. Of the total of 49 tested strains six strains (Acremonium kiliense, Bys- sochlamys nivea, Cunninghamella elegans, Hypoxylon fuscus, Stemphylium botryosum and Fusarium oxysporum SCHLEC~T.) were capable of degrading ~(1-~ 4) glucan without concomitant gel liquefaction indicating their ability to utilize the external parts of ~(l -~ 4) glucan, splitting only a portion of the external chains up to the first crosslink. This was reflected in growth of microorganisms on the surface of the gel. Twenty strains were not capable of utilizing ~(1 --> 4) glucan by an endo-mechanism despite their ability to grow on maltose and dissolved starch. The ability of the tested microorganisms to produce ~-amylase, as described by Dvatsatova (1958), Sak~moto (1958), Dass-Gupta and Verma (1962), Sawada et al. (1966), etc., was confirmed. The ~-amylase production was found in 29 strains out of the 36 tested fungal genera.

The method used, with a test period up to 28 d, made it possible to detect minute ~-amylase activities due to the cumulative hydrolase effect. The high number of ~-amylase producers suggests that the production of the enzyme does not coincide ~ with the general ability of imperfect fungi, mucorM fungi and some ascomycetes to split the ~(1 -> 4) glucan chains since these may be equally split by other enzyme systems (~-glucosidase, glucoamylase).

1981

61_...>46 ~

PRODUCTION OF c~-AMYLASE BY FUNGI ~4~

/, I l ; -G ~ G --.-.-.~

1' CH~HOH-CH z

I' i HiCHOH-CH

6 FIG. 1. Schemat ic i l lustration of eross l inked amylose wi th d i s t inguished bonds fez;mad in cross-

0 .' G , ~ l inking; (3 ~ -~ -g lucopyranosy l unit.

A c c o r d i n g to t h e r e su l t s of a s e m i q u a n t i t a t i v e a n a l y s i s of e - a m y l a s e a c t i v i t y - - t h e r a t e of gel l i q u e f a c t i o n - - t he t e s t e d s t r a i n s m a y be d i v i d e d i n t o t h r e e g r o u p s : t h e h i g h - p r o d u c t i o n s t r a i n s ( l i q u e f a c t i o n in 4 d: 14 s t r a i n s of Aspergillus oryzae 1036, Fusar ium oxysporum var . dianthi 1358 a n d Trichoderma koningii 1455 e x h i b i t e d t h e h ighes t ra tes ) , t h e m e d i u m - p r o d u c t i o n s t r a i n s ( l i q u e f a c t i o n in 4 - - 8 d; 8 s t r a ins ) a n d t h e l o w - p r o d u o t i o n s t r a i n s ( > 8 d; 5 s t r a ins ) . These r e su l t s are i n a n a g r e e m e n t w i t h t h e v a l u e of e - a m y l a s e a c t i v i t y d e t e r m i n e d i n a l ique f i ed m e d i u m b y a c h r o n o l y t i e m e t h o d . The e - a m y l a s e a c t i v i t y of t h e m o s t r a p i d l y p r o d u c i n g s t r a i n (Aspergillus oryzae 1036) was c o n s i d e r a b l y h i g h e r t h a n t h a t of o t he r s t r a i n s (Tab le I). I t shou ld , howeve r , be n o t e d t h a t t he e - a m y l a s e a c t i v i t y of Bacillus subtilis CCM 2722 in t h e s a m e m e d i u m r e a c h e d t h e leve l of 2873 U / L o n l y a f t e r 42 h.

R E F E R E N C E S

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a-amylase activity in biological materials (In Czech). Czech.Pat. 192 210 (1979). KUVIKOVA A.F.: Technology of enzyme separation. Proizv.Novylch.Vidov Produkt.Spirt.Prom.Sb. 109

(1961). LOGINOVA L.G.: Activity of hydrolytic enzymes in the Aspergillus nidula~s variant formed by ultraviolet

irradiation. Mikrobiologiya 29, 683 (1960). MISAKI T., YASW It., SAW.~DA J., TANAKA K.: Acid stable hydrolytic enzyme. Identification protease and

amylase of Aspergillus oryzae var. mierosporus TPR-18. Nippon Nogeikagaku Kaiashi 3,~, 1258 (1961). ODA K., IouCHI ~q.: Genetic and biochemical studies uu the formation of protease in A~pergiUus soyae.

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|46 J . AUGUSTJ[N et al. Vol. 26

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