Mycelial glucoamylases produced by the thermophilic fungus Scytalidium thermophilum strains 15.1 and 15.8: purification and biochemical characterization.

Two strains (15.1 and 15.8) of the thermophilic fungus Scytalidium thermophilum produced high levels of intracellular glucoamylases, with potential for industrial applications. The isoform I of the glucoamylase produced by 15.1 strain was sequentially submitted to DEAE-Cellulose and CM-Cellulose chromatography, and purified 141-fold, with 5.45% recovery. The glucoamylase of strain 15.8 was purified 71-fold by CM- Cellulose and Concanavalin A-Sepharose chromatography, with 7.38% recovery. Temperature and pH optima were in the range of 50-60°C and 5.0-6.0, respectively, using starch and maltose as substrates. The glucoamylase of S. thermophilum 15.8 was more stable (t50 > 60 min) than that of S. thermophilum 15.1 (t50= 11-15 min), at 60°C. The glucoamylase activities were enhanced by several ions (e.g. Mn(2+) and Ca(2+)) and inhibited by β- mercaptoethanol. The glucoamylase from 15.1 strain showed a Km of 0.094 mg/ml and 0.029 mg/ml and Vmax of 202 U/mg prot and 109 U/mg prot, for starch and maltose, respectively. The hydrolysis products of starch and maltose, analyzed by TLC, demonstrated glucose as end product and confirming the character of the enzyme as glucoamylase. Differences were observed in relation to the products formed with maltose as substrate between the two strains studied. S. thermophilum 15.8 formed maltotriose in contrast with S. thermophilum 15.1.

INTRODUCTION

Glucoamylases (GAs) (EC 3.2.1.3) are exo-amylases which hydrolyze a-1,4 glycosidic linkages in raw or soluble starches and related oligosacharides, producing β-glucose by inversion of the anomeric configuration (26). In addition, they also hydrolyze, but at a lower rate, a-1,6 glycosidic linkages of starch (20).

The conversion of starch into sugar, syrups and dextrins by action of amylases represents the larger part of the starch processing industry (27). The hydrolytes are used as carbon source in fermentation, as well as sources of sweetness in a range of manufactured food products and beverages. Amylases are used in the liquefaction of insoluble starch, manufacture of oligosaccharides mixtures (substitutes sucrose preventing crystallization in foods and keeping a certain level of hardness of the texture during storage), maltotetraose syrup (to control the freezing points of frozen foods) and of high molecular weight branched dextrins (used as extender and glozing agent for production of powdery foods and rice cakes). Manufacturing starch to maltose is important since that this disaccharide is widely used as sweeter and also as intravenous sugar supplement. In food industries is used due of low tendency to be crystallized and relative nonhygroscopic. To manufacture of high fructose containing syrups, the starch is first converted to glucose by enzymes liquefaction and saccharification.

Several fungal species produce glucoamylases, for instance, Aspergillus awamori, A. terreus and Rhizopus oryzae (21).Glucoamylases produced by fungi, generally, occur in multiple forms. They have two domains, a catalytic domain and a starch binding domain. These domains are connected by an O- glycosylated polypeptide linker located at the N-terminus (20). Total or partial proteolytic excision of the starch binding domains leads to the formation of glucoamylase capable of hydrolyzing only soluble starch (8).

The fungus Scytalidium thermophilum belongs to the thermophilic Torula-Humicola complex which has more than thirty isolates with species very variable in macroscopical and microscopical features (24). In our laboratory we previously studied Scytalidium thermophilum 15.1 and 15.8, which were excellent producers of trehalase (14) and of conidial and mycelial alkaline phosphatases (13). Regarding amylase activity, substantial differences were detected between S. thermophilum 15.1 and 15.8 isolates. The aim of the present study was to compare some biochemical properties of the glucoamylase produced by the mycelial extracts of fungi S. thermophilum 15.1 and 15.8. The high amylolytic activity produced by the two strains suggested potential for applications in the saccharification starch process.

MATERIALS AND METHODS

Microorganism and culture

The S. isolates 15.1 (= CBS 619.91) and 15.8 (= CBS 671.88 = ATCC 669.38) were maintained on slants of solid 4% oatmeal baby food (Quaker) medium, at 45°C. Liquid cultures were prepared in 50 ml of M-5 liquid medium (22), inoculated with of 2.0 ml of a spore suspension (105 spores/ml), in 250 ml Erlenmeyer flasks. The culture conditions were standardized according to the exigency of each fungus for maximal enzymatic production, i.g., incubated at 45°C, without agitation, and for 5 or 7 days, respectively, for 15.1 and 15.8 isolates.

Preparation of crude mycelial extract

The cultures were harvested by vacuum filtration on Whatman #1 and the mycelial pads were ground in a porcelain mortar with twice their weight of acid- washed sea sand, at 4°C, extracted in distillated water and centrifuged at 23,700xg for 15 min. The supernatant was the source of crude mycelial extract.

Determination of glucoamylase activity and protein

The glucoamylase activity was determined using 1% starch as substrate in 0.1M sodium acetate buffer, pH 5.5. The reducing sugars formed were quantified according to Miller (18) using dinitrosalicylic acid (DNS). The glucoamylase activity was also determined on maltose, starch, amylopectin and glycogen, as substrates by glucose oxidase method according to Bergmeyer & Bernt (3). One unit of glucoamylase activity was defined as the amount of enzyme that releases one mmol of glucose per minute. The protein was quantified according to Lowry (16) using bovine serum albumin as standard. Specific activity corresponds to μmol/min/mg prot.

Purification and biochemical characterization

Mycelial glucoamylase of S. thermophilum 15.1 was purified using a DEAE-Celullose column (21 x 60 mm) equilibrated in 0.01 M sodium acetate buffer, pH 5.5 and two forms (I e II) were separated. The form I did not interact with the resin, and the fractions with amylolytic activity were pooled and applied to a CM-Celullose chromatographic column (21 x 60 mm) equilibrated in the same buffer. The enzyme was eluted as single form, using a linear gradient of NaCl (0-0.5 M), in the same buffer. The fractions with amylase activity were pooled, dialyzed overnight at 4°C, and used for biochemical characterization. The form II was eluted using a linear gradient of NaCl (0-1.0 M) in the same buffer but it has not representative amylolytic levels.

Glucoamylase of S. thermophilum 15.8 was purified also by two chromatographic steps. The crude extract was applied to a CM-Celullose column (21 x 60 mm) and eluted as a single form under the same condition mentioned above. The fractions with amylase activity were pooled, dialyzed overnight at 4°C and applied to a Concanavalin A-Sepharose column (10 x 50 mm), equilibrated with 0.020 M Tris-HCl buffer, pH 7.5 added of 0.5 M NaCl, 0.5 mM Mn++ and Ca++. The enzyme was eluted as a single form using a linear gradient of methyl α-D-mannopyranoside (0- 0.5 M), pooled, dialyzed overnight at 4°C, and used for biochemical characterization.

Electrophoresis

The samples of purified enzymes were run under nondenaturing electrophoresis in polyacrylamide 7% gels PAGE, Davis (9) and stained for protein with AgNO3 according to Blum (4).

Molecular mass and carbohydrate content determination

The molecular mass of glucoamylase produced by S. thermophilum was estimated by gel filtration in a Sephadex G- 100 column (20 x 860 mm), equilibrated in 0.05 M Tris-HCl buffer, pH 7.5 added of 0.1 M KCl, using as molecular mass markers alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa) and cytochrome-c (14 kDa). The protein carbohydrate content was determined according to Dubois (10).

Kinetic parameters

The kinetic parameters (Km and Vmax) were determined using starch, maltose, amylopectin and glycogen as substrate by the Lineweaver-Burk graphic representation (15).

Thin layer chromatography

Chromatographic analysis of the reaction end products of glucoamylase activity produced by S. thermophilum 15.1 e 15.8 was carried out using TLC. The reaction mixture was concentrated and applied (10 μl) on silica gel, and two ascendent chromatographic steps were applied, using butanol/ethanol/ water (5:3:2) as solvent system. Spots were developed by spraying the air-dried plate with H2SO4 and methanol (1:9) containing 0.2% orcinol and heating at 100°C (12).

RESULTS AND DISCUSSION

Enzyme purification

Crude mycelial extract of S. thermophilum 15.1 was submitted to chromatography in DEAE-Cellulose (Fig. 1A). Two isoforms (I and II) with amylolytic activities were separated. The form I contained approximately 86% of total amylolytic activity and it did not interact with the resin. Form II, with 14% of total amylolytic activity was eluted as single form with 0.58 M NaCl, using a linear gradient (0-1.0M). The fractions of form I were pooled, dialyzed against buffer and applied to a CM-Cellulose chromatographic column (Figure 1B). The amylolytic activity eluted as single form with 0.16-0.28 M NaCl, with a linear gradient (0-0.5M).

Figure 1

Chromatographic profiles of the glucoamylases produced by fungi S. thermophilum 15.1 (A and B) and 15.8 (C and D), on DEAE-Cellulose (A), CM-Cellulose (B and C) and Concanavalin A-Sepharose (D). Symbols: (●) glucoamylase activity; (○) absorbance 280 nm.

S. thermophilum 15.8 crude mycelial extract was applied to a CM-Cellulose chromatographic column (Fig. 1C), and eluted with a linear NaCl gradient (0-0.5M). The fractions with glucoamylase activity eluted as a single form with 0.18 M NaCl. The activity fraction were pooled, dialyzed, applied to a Concanavalin A-Sepharose column (Fig. 1D) and eluted as single form with 0.05-0.1 M methyl a-D-mannopyranoside using a linear gradient (0-0.5M).

The enzymes of the strains 15.1 and 15.8 were purified 141-fold and 71.23-fold, with recovery of 5.45% and 7.38%, respectively (Table 1). Only a single band on 7% PAGE was observed for the intracellular glucoamylase of strain 15.1, stained for protein by silver and stained for glucoamylase activity (Fig. 2).

Table 1

Purification of glucoamylases produced by S. thermophilum 15.1 and 15.8.

S. thermophilum 15.1						S. thermophilum 15.8
Step	Activity (Total U)	Protein (Total mg)	Specific Activity (U/mg prot)	Yield (%)	Purification fold (X)	Activity (Total U)	Protein  (Total mg)	Specific Activity (U/mg prot)	Yield (%)	Purification fold (X)
Crude extract	72.27	235.06	0.31	100	1	40.92	305.80	0.13	100	1
DEAE-Celullose	7.39	1.98	3.73	10.22	12.03	-	-	-	-	-
CM-Celullose	3.94	0.09	43.77	5.45	141.2	4.37	1.32	3.31	10.68	25.46
Con A- Sepharose	-	-	-	-	-	3.02	0.32	9.44	7.38	71.23

Figure 2

7% PAGE stained for glucoamylase activity using starch (A) and maltose (B), and stained for protein by silver (C).

Molecular mass determination

Molecular masses of intracellular glucoamylases of S. thermophilum 15.1 and 15.8 determined by Sephadex G-100 gel filtration were, respectively, 54 kDa and 98.7 kDa. These values are in agreement with the literature that reports that the molecular masses of fungal glucoamylases are in the range 48-90 kDa (20), as those of T. lanuginosus (26), Chaetomium thermophilum (7) and Sclerotinia sclerotiorum (17). However, a higher value of 125 kDa was reported for Aspergillus niger glucoamylase (25).

Usually, the carbohydrate content of fungal glucoamylase is in the range of 10-20%, as observed for Rhizopus niveus (29). But, lower values were observed such as that of the glucoamylase of Neurospora crassa (5.1%) (23). Intracellular glucoamylases of S. thermophilum 15.1 and 15.8 had carbohydrate contents of 20% and 51%, respectively.

Temperature and pH optima and thermal stability

The values of temperature optima for glucoamylases of the strains 15.1 and 15.8 were compatible with the literature, which reports values among 50°C to 70°C (20). Intracellular glucoamylase of S. thermophilum 15.1 exhibited optimum temperature of 50°C and 55°C, with maltose and starch as substrates (Fig. 3A), and 60°C with either substrates for the intracellular glucoamylase of S. thermophilum 15.8 (Fig. 3B). A temperature optimum of 50°C is reported for S. sclerotiorum glucoamylase (17). Higher activity temperatures were verified also for glucoamylase of Thermomyces lanuginosus (26), Chaetomium thermophilum (7) and Trichoderma reesei (11). Mycelial enzymes of S. thermophilum have t50 of 11 min and 15 min (strain 15.1) (Fig. 3C), and more than 60 min for strain 15.8 (Fig. 3D), at 60°C, when starch and maltose, respectively, were used as substrates. Glucoamylase of Chaetomium thermophilum retained 50% active after 60 min, at 65°C (7). The high percentage of carbohydrate content of the glucoamylase of S. thermophilum 15.8, could be responsible by the higher thermal stability compared to S. thermophilum 15.1 glucoamylase.

Figure 3

Optima temperature (A and B) and thermal stability on 60°C (C and D) of the glucoamylases produced by S. thermophilum (A and C) and 15.8 (B and D), using starch (●) and maltose (○) as substrate.

Intracellular glucoamylase of S. thermophilum 15.1 had a pH optimum of 5.0 when starch or maltose used as substrates (Fig. 4A), and the mycelial glucoamylase of S. thermophilum 15.8 has pH optimum in the range of 5.5-6.0, either with starch or maltose (Fig. 4B). Similar results were obtained for extracellular glucoamylase of S. thermophilum 15.1 (5), Thermomyces lanuginosus (26), Arthrobotrys amerospora (20) and Neurospora crassa (23). The values obtained were higher than those reported for S. sclerotiorum (3.5-4.0) (17). Generally, glucoamylases obtained from fungi are active at acidic pH values. However, several forms have variable values of pH optimum.

Figure 4

Optimum pH of activity for the glucoamylases produced by S. thermophilum 15.1 (A) and 15.8 (B), using starch (●) and maltose (○) as substrate.

Influence of ions on the glucoamylase activity

The influence of different ionic compounds on the glucoamylase activities is shown in Table 2. The intracellular glucoamylase activity of S. thermophilum 15.1 was enhanced 23-36% by addition of Mn2+, but strongly inhibited by Cu2+, Hg2+ and β-mercaptoethanol. These last two metallic ions drastically reduced the 15.8 glucoamylase activity, but in contrast, Cu2+ enhanced 23 or 74% the glucoamylase activity, depending on the substrate, starch or maltose, respectively.

Table 2

Effect of ions and other compounds on the glucoamylases produced by S. thermophilum 15.1 and 15.8.

Ions/Compounds	S. thermophilum 15.1		S. thermophilum 15.8
	Starch	Maltose	Starch	Maltose
	Relative activity (%)
None	100	100	100	100
β-mercaptoethanol	40	24	0	0
BaCl2	70	67	121	98
CaCl2	78	65	109	139
CuCl2	48	38	123	174
HgCl2	7	26	0	0
MgCl2	101	80	140	108
MgSO4	94	86	134	86
MnCl2	123	136	59	203
NaCl	95	73	116	76
NH4Cl	104	76	131	88
ZnCl2	93	12	83	61

The inhibition promoted by β-mercaptoethanol indicates the existence of disulfide bonds in the enzyme molecule. S. thermophilum 15.8 glucoamylase was also enhanced by 5mM Ba2+, Mg2+, Na+, NH4+ and Ca2+. For both species of S. thermophilum were verified considerable differences on glucoamylase activity determined with the substrates starch or maltose in the presence of Mn2+ and Zn2+. Glucoamylase of C. thermophilum was more active in the presence of Ca2+, Mg2+, Na+ and K+, and inhibited by Fe2+, Ag+ and Hg2+ (7). Mn2+ and Zn2+ enhanced glucoamylase activity from Humicola grisea var. thermoidea (28) and lower concentrations of Mn2+ and Ca2+ enhanced the activity of glucoamylase of S. sclerotiorum (17).

Kinetic properties

The kinetic parameters obtained for the glucoamylase of S. thermophilum 15.1 are shown in Table 3. The enzyme possesses greater affinity to maltose (Km 0.029 mg/ml) than for starch (Km 0.094 mg/ml), which was confirmed by the values of catalytic efficiency. But, probably, the hydrolysis of starch and maltose occurred at the same catalytic site, considering that the rate of hydrolysis of a mixture of starch and maltose (35 U/ml) was intermediary when compared with that of the individual substrates (42 U/ml for starch and 12.5 U/ml for maltose), and lower than the addition of both (54.5 U/ml approximately). The enzyme showed reduced affinity by glycogen.

Table 3

Determination of kinetic parameters of the purified glucoamylase produced by S. thermophilum 15.1.

Substrate	Km (mg/ml)	Vmax (μmol/min/mg prot)	Vmax/ Km
Starch	0.094	0.202	2.15
Maltose	0.029	0.109	3.76
Starch + Maltose	0.003	0.070	2.33
Glycogen	0.158	0.185	1.17
Amylopectin	0.045	0.016	0.35

Both enzymes, GAs 15.1 and 15.8, hydrolyzed starch and maltose as substrate, and glucose was always the main hydrolysis product (Fig. 5), but S. thermophilum 15.8 formed maltotriose when maltose was the substrate, in contrast with S. thermophilum 15.1. In according to Thorsen (26), di-, tri- and tetrasacharide can be formed reversibly from high glucose concentrations.

Figure 5

Thin layer chromatography of the hydrolysis products of the glucoamylases of Scytalidium thermophilum 15.1 (A,B) and Scytalidium thermophilum 15.8 (C,D) using starch (A,C) and maltose (B,D) as substrates. G = glucose; m = maltose. Time is represented in hours, except 15 (minutes).

We previously reported the purification of the extracellular amylases produced by S. thermophilum 15.1 and 15.8 and significant differences were detected between the enzymes of the two isolates. The first one (15.1) produced an extracellular glucoamylase (1) and an a-amylase (2), which were isolated and purified by chromatographic methods. But, in contrast, under the same conditions, it was no detected a-amylase activity in S. thermophilum 15.8, but two isoforms of glucoamylase were separated and characterized (5;6). Here, mycelial extracts from two species were purified by two different sequential chromatographic steps. Optimal temperature and pH, for starch and maltose as substrates, were close between the two species (50-60°C and pH 5.0-6.0) but differences were observed as for effect of metallic ions, thermostability, kinetic constants, molecular masses 15.1 and 15.8 and products of hydrolysis. In relation at molecular mass, for example, for S. thermophilum 15.1, extra (1) and intracellular glucoamylase had close values (60 kDa and 54 kDa, respectively), but in contrast, differences were observed to extracellular GAI (68.5 kDa) (6), GAII (83 kDa) (5) and to the intracellular form (98.7 kDa) from S. thermophilum 15.8. These results reflect differences structural or in the carbohydrate content since that extracellular glucoamylases of S. thermophilum 15.1 and 15.8 had carbohydrate contents of 9.8% and 10-25% (1,5,6), but intracellular glucoamylases of S. thermophilum 15.1 and 15.8 had carbohydrate contents of 20% and 51%, respectively. Then, S. thermophilum is a potential thermophilic fungus with highest amylolytic levels either extra or intracellular with great possibility industrial application.