Extracellular isoamylase produced by Bacillus circulans MIR-137

Journal of Applied Bacteriology 1992, 73, 520-523

Extrace1lular isoamy lase produced by Bacillus circulans G.R. Castro'*', G.F. Garcia' and F. Siiieriz' 'Laboratorio de Microbiologia,Departamento de Ouimica Biolbgica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, and 'CBtedra de Microbiologia Superior, Facultad de Bioquimica , Quimica y Farmacia, Universidad Nacional de Tucumdn-PROIMI-MIRCEN,TucumAn, Argentina 4139/02/92: accepted 30 May 1992

A strain designated MIR-137 was selected after a screening programme for amylolytic enzymes and was characterized as Bacillus circulans. Batch cultures showed the highest amylolytic activities in the stationary phase. Extracellular isoamylase activity exhibited an optical temperature of 60°C at pH 5.0. These G.R. CASTRO, G.F. GARCIA AND F. S I N E R l Z . 1992.

properties would allow its use in normal saccharification processes in the starch industries.

INTRODUCTION

The genus Racillus is important in bacterial fermentations for the production of extracellular enzymes and certain metabolites. One of the most important extracellular enzymes is a-amylase, used in the starch industries for the production of sweeteners. This process requires high temperatures and low pH values for optimal yields. In the past decade several strains have been isolated and used to produce thermostable a-amylases (Priest 1989). However, the a-amylases are unable to hydrolyse the a-l,6 glucoside linkages of amylopectin, the major component of the industrial starches. For this purpose, debranching enzymes could be used in combination with other enzymes (e.g. /Lamylase) to raise the saccharification rates and yields (Fogarty & Kelly 1980). It has already been reported that various enzymes related to starch degradation are present in strains of Bacillus circulans (Siggens 1987; Sata et al. 1989; Hoffman et al. 1989). To our knowledge, however, the presence of isoamylase and a-amylase activities in Bacillus circulans have not so far been described. On the other hand, the isoamylase activities were reported in several bacteria and yeasts (Gunja-Smith et al. 1961, 1970; Ueda & Nanri 1967; Jeanningros et al. 1975; Harada 1968; Spencer-Martins 1982). However, the isoamylase activities described were not able to operate under the conditions employed in the starch industries (temperatures above 60°C and pH 4-5 to 5.5). The object of this work was to study the production in batch culture of extracellular isoamylase and a-amylase Correspondenceto: Dr G.R. Castro, PROIMI, Av. Belgrano y fie, Caseror, 4000 Tucuman, Argcntina.

synthesized by a thermoresistant strain characterized as Bacillus circulans MIR-137. The pH and temperature profiles of isoamylase and a-amylase were studied.

MATERIALS AND METHODS

Strains Several strains of the genus Bacillus were isolated in a screening programme (Castro et al. 1992a). One was selected according to the methods previously described (Sato 8i Park 1980) and characterized according to international criteria (Claus & Berkeley 1986). The statistical treatment was developed by the Jacquard coeflicient (S,)(Colwell & Austin 1981). The matrix frequencies were derived from the data of previous workers (Claus & Berkeley 1986; Priest et al. 1988).

Medlum Batch cultures were performed in a peptone starch (PS) medium containing (g/l): (NH4)2S04, 1.0; KH2P04, 3.0; KZHPO,, 6.0; MgS04 . 7H20, 0.01 ; GCI2 . 2H20, 0.05; MnSO, . 7H20, 0.01; FeSO, . 7H20, 0-001; ZnSO, . 7H20, 0.001; trisodium citrate, 1.0; soluble starch, 5.0; and peptone, 0.2. These reagents were obtained from Merck (Darmstadt, Germany).

Bacterlal cultures In PS medium The selected strain was cultured for activity prior to batch culture. It was grown in Erlenmeyer flasks (500 ml) on a shaker (200 rev/min) with 125 ml of medium at 45°C.

AMYLOLYTIC ENZYMES FROM B . CIRCULANS 521

Growth was measured as a function of optical density at 560 nm, in a spectrophotometer, diluting the sample with 145 mmol/l NaCl when necessary.

1 e

.-

2

Analytical procedures

The proteins were determined by the Coomassie Blue G-250 method, the starch was assayed by the blue index method, and glucose was measured with glucose oxidaseperoxidase system according to Castro et al. (1992b). Reducing sugars as maltose were determined by the method of Somogyi (1954). All reagents were analytical grade from Sigma. Alpha amylase was measured by the dextrinizing activity with I, as described by Castro et al. (1992b). Isoamylase activity was determined with oyster glycogen described by Gunja-Smith et al. (1961). Extracellular activities were determined by centrifuging the cultures for 20 min at 10000g at 2-4"C.

40

Jz t

6 + J

-a L 0

0.1

20

5 0

c

O I 0.l01 0l

6

3

12

9

15

18

21

Time ( h )

Flg. 1 Kinetics of growth

(m) and extracellular proteins ( 0 ) -I 0 . 4

71-

Optimal conditions for enzyme activities

The effect of temperature and of pH on the activities of the different enzymes was assayed under optimum conditions for the other parameters. The determinations were performed with the crude enzyme preparations. The effect of pH on the a-amylase activity was measured at 45°C in 50 mmol/l phosphate (pH 5.8-8.0) and 40 mmol/l veronal (pH 6.8-8.0). The effect of pH on isoamylase was assayed at 55°C during 30 min in 100 mmol/l citrate buffer (pH 3-0-6.0). The effect of temperature on the enzymatic activities was determined in 100 mmol/l citrate buffer (pH 5.0) for the isoamylase activity, and in 50 mmol/l phosphate buffer (pH 7.0) for the a-amylase activity.

0

Y

0

3

6

9

12

15

18

21

Time ( h

Fig. 2 Kinetics of production of reducing sugars as maltose (0)

and degradation of starch (V)

9 0 -25

5 r

RESULTS AND DISCUSSION

The strain MIR-137 was characterized by 80 morphological and biochemical tests (results not shown) but the results of these tests did not correspond to any of the recognized species of the genus Bacillus. This is not uncommon for environmental isolates belonging to the genus Bacillus (Priest et al. 1988). The use of the statistical treatment with the Jacquard coeficient gave a tentative identification of the strain MIR-137 in the B. circulans cluster and B. circulans species (S, = 0.810). The results with the batch culture of Bacillus circulans MIR-137 are shown in Figs 1 to 4. The batch culture can be divided into three stages: the first stage involving protein synthesis and exponential cell growth (0-6 h); the

0

3

6

9

12

15

18

21

Time ( h

Flg. 3 Kinetics of production of glucose (A) and extracellular

amylase activity ( 0 ) second, in which the maximum expression of the extracellular enzymatic activities was achieved (6 to approximately 12 h); and the third, where it appeared that extracellular enzyme production was depleted, possibly by the presence of small oligosaccharides (such as maltose and glucose) in the culture (12-21 h).

522 G . R . CASTRO ET A L .

\

n

60

3

I

\

20 30

0

3

6

9

i\

'0°[ 80

12

Time ( h

15

18

1

I

35

40

21

1

Fig. 4 Kinetics of production of isoamylase. Extracellular ( 0 )

/x\x

50

45

60

55

65

Temperature (OC)

Fig. 5 Effect of temperature on isoamylase ( x ) and a-amylase (+) activity

and total ( 0 )activity

The low level of extracellular a-amylase activity in the exponential phase of growth could be attributed to the regulation pattern, which was partially constitutive. The basal activity levels resulted in the catalytic breakdown of the starch, thus increasing the branched oligosaccharide concentration. Furthermore, the activity of a-amylase was increased by additional synthesis possibly induced by these oligosaccharides. The a-amylase activity of B . circulans MIR-137 reached a maximum of 4.2 kU/ml after culture for 10 h; thereafter the enzyme activity was reduced. This effect could possibly be attributed to the presence of proteases (results not shown) and the uptake of extracellular proteins by the cells characteristic of the sporulation mechanism in the genus Bacillus (Fig. 1). Bacillus circulans MIR-137 grew in starch medium producing isoamylase (Fig. 4). The extracellular and total activities of the medium have very similar profiles, and the expression of this enzyme is temporary, as it is rapidly diminished. The maximum activity was reached in the middle of the stationary phase of the cell-growth, and afterwards the activity was reduced in the same way as the aamylase activity. Alpha glucosidase activity was expressed in extracellular and total samples at low levels; it was detected in the second stage of the batch culture in accordance with previous enzyme profiles (results not shown). Other enzymes previously reported in different strains of B . circulans species, such as fl-amylase (Siggens 1987), pullulanase (Sata et al. 1989) and cyclodexmn-glycosyltransferases (Hoffman et al. 1989) could not be detected in B . circulans MIR-137 under our experimental conditions. This may be explained by the heterogeneous nature of the circulans cluster (Nakamura & Swezey 1983). The pH and temperature profiles for a-amylase activity showed an optimum pH at 7.5 and temperature at 37°C (Figs 5 and 6, respectively).

The effect of pH on B . circulans MIR-137 isoamylase activity at 55°C is shown in Fig. 5. The pH profile of this activity is similar to that reported for the enzyme from Lysobacter sp. (Gunja-Smith et al. 1970), Streptomyces sp. (Ueda et al. 1978) and Lipomyces kononekoe (SpencerMartins 1982), where optimal activity occurred between pH 4.5 to 5.5 (normal industrial saccharification conditions). The pH range of the maximum activity of isoamylase in Escherichia coli Ueanningros et al. 1975) and in Pseudomonas sp. (Harada et al. 1968) are higher and lower respectively than the conditions for the industrial operation. The optimum temperature for isoamylase activity at pH 5.0 was obtained at 60°C (Fig. 6), although at 65°C for 30 min it retained 80% of the maximum activity. The majority of the isoamylases previously reported in Saccharomyces cerevisiae (Gunja-Smith et al. 1961), Escherichia intermedia (Ueda et al. 1978), Lysobacter (Gunja-Smith et al. 1970), Escherichia coli Ueanningros et al. 1975), Pseudomonas sp. (Harada et al. 1968) and Lipomyces kononekoe (Spencer-Martins 1982) are thermolabile enzymes, which have an optimum temperature below 53°C and they are not

I00