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Optimum production and characterization of thermostable amylolytic enzymes from B. stearothermophilus GRE1.

Optimum production of extracellular, thermostable amylolytic enzymes ([alpha] and [beta]-amylase) by a newly isolated bacterium, Bacillus stearothermophilus, was investigated in a batch bioreactor. Starch and lactose at 1.0% and 3.0% (w/v) respectively were found to be optimum for maximum enzyme production. Optimization of cultural conditions (pH 7.0 and temperature 45[degrees]C) resulted in high bacterial specific growth rate (0.64 [h.sup.-1]), yielding 2.20 g[L.sup.-1] biomass, 11.43 Um[L.sup.-1] [alpha]-amylase and 10.04 Um[L.sup.-1] of [beta]-amylase. Hydrolysis of native starches from wheat, cassava, corn and potato at 60[degrees]C using the crude enzyme showed 60-80% saccharification with potato starch showing the least and wheat starch showing the greatest hydrolysis. The [K.sub.m] and [V.sub.max] values of the crude [alpha]-amylase for starch were 4.78 mg starch/mL and 6.67 mg/mL.min respectively.

On a etudie dans un bioreacteur discontinu la production optimale d'enzymes extracellulaires thermostables ([alfa] et amylase-[beta]) par une bacterie nouvellement isolee, Bacillus stearothermophilus. On a trouve que l'amidon et le lactose a 1,0% et 3,0% (w/v), respectivement, etaient optimums pour la production maximale d'enzymes. L'optimisation des conditions de culture (pH de 7,0 et temperature de 45[grados]C) entraine une vitesse de croissance bacterienne specifique elevee (0,64 [h.sup.-1]), produisant 2,20 g[L.sup.-1] de biomasse, 11,43 Um[L.sup.-1] d'amylase-[alfa] et 10,04 Um[L.sup.-1] d'amylase-[beta]. L'hydrolyse d'amidons indigenes a partir de ble, de manioc, de mais et de pomme de terre a 60[grados]C a l'aide de l'enzyme brute montre une saccharification de 60-80%, l'amidon de pomme de terre montrant le taux d'hydrolyse la plus faible et l'amidon de ble le taux le plus eleve. Les valeurs de [K.sub.m] et de [V.sub.max] de l'amylase-[alfa] brute pour l'amidon sont de 4,78 mg d'amidon/ml et de 6,67 mg/mL.min, respectivement.

Keywords: B. stearothermophilus, thermostable amylolytic enzymes, Ethiopian hyperthermal spring

INTRODUCTION

Starch can readily be hydrolyzed by hydrochloric acid completely. However, low glucose yield, the formation of large amount of salt and the need to use corrosion resistant equipments are among some of the disadvantages of using acid hydrolysis. Enzymatic hydrolysis of starch on the other hand has several advantages, such as the specificity of enzymes allows the production of sugar syrups with well-defined physical and chemical properties, and the milder enzymatic reaction conditions result in few side reactions and less browning (Poonam and Dalel, 1995; Haki and Rakshit, 2003a).

Glucose, maltose and maltooligosaccharides find important applications in food, beverage and pharmaceutical industries. They had earlier been produced by hydrolysis of starch using amylases from higher plants such as barley, sweet potato, soybean, cassava and wheat and also from certain mesophilic bacteria. However, those enzymes are relatively expensive and above all thermally unstable (Hyun and Zeikus, 1985a, b). A high value is thus, placed on thermostable and thermoactive amylolytic enzymes in the bioprocessing of starch (Norman, 1979).

The main industrial thermostable enzymes used in starch degradation ([alpha]- and [beta]-amylases, glucoamylases and pullulanases) are either of fungal or bacterial origin. Enzymes exhibiting an optimal temperature for activity, ranging from 70-100[degrees]C were reported from Bacilli alone. These include Bacillus amyloliquefaciens (Underkofler, 1976), Bacillus licheniformis (Viara et al., 1993), and Bacillus stearothermophilus (Vihinen and Mantsala, 1990; Jeayoung et al., 1989; Wind et al., 1994; Amartey et al., 1991; Campus et al., 1992; Kim et al., 1989; Lee et al., 1991; Suvd et al., 2001). Most of the reports dealt with the purification and characterization of the enzymes.

Based on earlier shake flask studies, starch and lactose were identified as the best nutrient sources, enhancing amylolytic enzyme production by the newly isolated Bacillus stearothermophilus GRE1 (Haki and Rakshit, 2003b). The present work describes the optimization of the concentration of carbon sources (starch and lactose) and culture conditions (temperature, initial pH of the medium, controlled and uncontrolled pH) for the production of amylase in batch bioreactor. In addition, the kinetic properties of the crude amylase and native starch hydrolysis efficiency of the enzyme were studied.

MATERIALS AND METHODS

Organism and Growth Conditions

The aerobic bacterial strain B. stearothermophilus GRE1, newly isolated from a hyperthermal spring in Ethiopia (Haki and Rakshit, 2003b, 2004), was used in this study. The medium for inoculum preparation contained (g/L): starch, 5.0; beef extract, 3.0; peptone 5.0 and sodium chloride, 8.0. The pH of the medium (100 mL in 500 mL Erlenmeyer flask) was adjusted to 6.5 prior to sterilization at 115[degrees]C for 15 min. The cultures were grown on a rotary shaker at 150 rpm. An exponentially overnight-grown culture was used as inoculum for amylase production by B. stearothermophilus in a bioreactor.

The fermentation medium for enzyme production comprised (g/L): starch, 10.0; bacteriological peptone, 5.0; lactose, 5.0; NaCl, 1.0; K[H.sub.2]P[O.sub.4], 2.0; Ca[Cl.sub.2], 0.1; MgS[O.sub.4], 0.1 and yeast extract, 1.0. The pH was adjusted to around 7 prior to sterilization.

Bioreactor Cultivation

The optimization experiments were carried out in 2x1 L double-jacketed bioreactor (Biostat-Q, B. Braun, and Germany). 450 mL of the fermentation medium supplemented with proper carbon sources was added to reactor vessel before autoclaving at 115[degrees]C for 15 min. The medium was inoculated with 50 mL (10% v/v) overnight culture. Air was passed at 0.2 L/min through a perforated aluminum cylinder fitted inside and the dissolved oxygen was monitored by using dissolved oxygen (DO) probe. The culture was stirred at 200 rpm and the pH was measured with the pH probe and maintained at the desired value by automatic addition of 2N acid or base. A temperature of 45[degrees]C was maintained by circulating hot water through the jacket of the bioreactor.

Quantification of Biomass

Samples (5 mL each) were withdrawn at regular intervals under sterile conditions. Optical densities (OD) were measured after appropriate dilution at 600 nm using a UV-spectrophotometer (UNICAM). The biomass (g/L) was determined based on a Biomass-OD standard curve. Biomass values (g/L) were obtained after two times centrifugation of an appropriate amount of sample (taken during stationary phase) at 6000 rpm for 20 min each, with intermediate washing using saline (6% NaCl in water) and subsequent drying at 105[degrees]C for 24 h.

Enzyme Assay

Bacterial culture broth was centrifuged at 6000 rpm for 25 min and the clear supernatant was used in enzyme assays. Soluble starch (1% w/v, in phosphate buffer, 0.02 mol/L and pH 6.9) was used as a substrate for assaying the activity of [alpha]-amylase at pH 6.9 and [alpha]-amylase at pH 4.8, according to the DNS method (Bernfeld, 1955). Reducing sugars released by the action of amylases were determined as maltose equivalent by the DNS method, in which, one amylase unit is defined as that amount of enzyme, which liberates 1[micro]mol maltose per minute under specified conditions. Enzyme assay was carried out in duplicate and the average values are given.

Optimization of Carbon Sources

Varying concentrations of starch (0, 0.2, 0.5, 0.8, 1.0, 1.5 and 2.0% (w/v)) and lactose (0, 0.5, 1.0, 2.0, 3.0 and 4.0% (w/v)) were added in the fermentation medium. The specific growth rate and enzyme activity were then monitored.

Optimization of Environmental Factors of Fermentation

The optimum temperature for B. stearothermophilus cultivation was determined by performing fermentation at various temperatures (40, 45 and 50[degrees]C). Starch and lactose were maintained at 1% and 3% (w/v) level (optimum for enzyme production) respectively. The effect of initial pH on the growth and enzyme production of the strain was investigated by adjusting the pH at a desired level (pH 5.0, 6.0, 7.0 and 9.0), which was then allowed to float with the fermentation course. For pH-controlled experiments, the pH was maintained at 6.0 or 6.9 throughout the fermentation process by automatic addition of HCl and NaOH (both 1 mol/L). A bioreactor experiment, allowed to run without any pH control, was used as a control.

Digestion of Raw Starch

Hydrolysis experiments were carried out in a 15 mL centrifuge tubes containing 25 mg starch, crude [alpha]-amylase solution (10 U/mL) and 1 mL phosphate buffer. Toluene (100 [micro]L) was added to each tube to prevent microbial growth. The tubes were incubated at different temperatures (40, 50 and 60[degrees]C) in a shaking water bath at 150 rpm. Samples were collected at regular time intervals (6, 12, 18, 24 and 36 h) and the production of reducing sugars was estimated by the method of DNS.

The raw starch digestion rate ([r.sub.d]) was defined by the following equation:

[r.sub.d] (%) = ([A.sub.1]/[A.sub.0]) x 100 (Iefuji et al., 1996).

Where [A.sub.1] = weight of reducing sugar obtained from calibration curve for maltose and,

[A.sub.0] = weight of whole raw starch before the reaction.

Morphological changes in starch granules after digestion were monitored by a Scanning Electron Microscope (SEM, Philips, XL 30 CP) at Chulalangkorn University, Bangkok, Thailand. Starch granules both native and digested were washed with ethanol, dried and attached to an SEM stub with a silver plate. The mounted samples were coated with gold/platinum by sputter coater, polaron, SC 7610 before observation.

Kinetic Study

Enzyme kinetics was studied using soluble starch as a substrate. A volume of [alpha]-amylase solution (10 U/mL) was incubated with various concentrations (1-3 mg/mL) of soluble starch (5 mL each) and 1 mL of phosphate buffer. After different time intervals in the range of 0 to 1 minute, 20 [micro]L acidic iodine reagent ([I.sub.2]KI) were added (keeping tubes in ice cold water) to make inactive the enzyme and terminate the reaction (Yoo et al., 1987). The absorption was measured at 580 nm after which reducing sugar was estimated. [K.sub.m] and [V.sub.max] were then calculated using the Lineweaver-Burk plot.

RESULTS AND DISCUSSION

Optimization of Starch Concentration

Soluble starch was reported to be the best carbon source for thermostable amylolytic enzymes production (Swamy and Seenayya, 1996a, b; Reddy et al., 1998, 1999). In the present study, enzyme production by B. stearothermophilus GRE1 was dependent on starch concentration (Figure 1). After 12 h of fermentation, the medium containing 1% (w/v) of starch gave the highest enzyme level, in which [alpha]- and [beta]- amylase activities reached 5.69 and 4.81 U/mL, respectively. The maximum biomass production was 0.22 g/L.

[FIGURE 1 OMITTED]

Figure 2 depicted that the specific growth rate increased with the starch concentration. The maximum specific growth rate was obtained at a starch concentration of 0.5 to 2% (w/v) and reached 0.6 [h.sup.-1]. The specific growth rate when no starch was added was as high as 0.45 [h.sup.-1]. Thus, 1% (w/v) of starch in the basal medium is optimum for the production of amylases by B. stearothermophilus GRE1. Enzyme production however, was suppressed at higher concentrations of starch. This might be attributed to the production of organic acids (lactate, acetate and propionate) at higher concentrations of starch, changes in the viscosity of the medium, repression by starch hydrolysis products such as glucose and a difference in [O.sub.2] availability.

[FIGURE 2 OMITTED]

Optimization of Lactose Concentration

Preliminary shake flask studies in our laboratory showed that lactose induced enzyme production. The time course effect on amylolytic enzyme productivity using different lactose concentration (0-4% (w/v)), in a bioreactor, is shown in Figure 3. The results indicated that 3% lactose gave the highest enzyme activity with 11.43 U/mL of [alpha]-amylase and 10.07 U/mL of [beta]-amylase (5-6 fold increase compared to shake flask studies). The specific growth rate ([[micro].sub.max]) was 0.63 [h.sup.-1], while the biomass ([X.sub.max]) was 2.08 g/L. In a similar study, conducted using Bacillus sp. IMD 435, an amylase activity of 26 U/mL was obtained using 4% lactose in the enzyme production medium (Hamilton et al., 1999).

[FIGURE 3 OMITTED]

Optimization of Fermentation Temperature

There was no obvious difference in the values of the maximum specific growth rates at 40[degrees]C ([[micro].sub.max] = 0.61 [h.sup.-1]) and 45[degrees]C ([[micro].sub.max] = 0.63 [h.sup.-1]). The maximum biomass was 2.08 g/L at 45[degrees]C, and 1.98 g/L at 40[degrees]C. At 50[degrees]C, [[micro].sub.max] and [x.sub.max] respectively, were 0.57 h-1 and 1.5 g/L.

Figure 4 shows the effects of cultivation temperature on the amylolytic enzyme production. Optimum cultivation temperature for bacterial growth and enzyme production (11.43 U/mL for [alpha]-amylase and 10.07 U/mL for [beta]-amylase) was found to be 45[degrees]C. Similar values were reported for amylase production by other B. stearothermophilus strain (Wind et al., 1994). The optimum temperature for the [alpha]-amylase activity (partially purified) produced by B. stearothermophilus GRE 1 is 60[degrees]C (Haki and Rakshit, 2003b).

[FIGURE 4 OMITTED]

Optimization of Initial pH of the Medium

Figure 5 depicted that pH played a sensitive role in enzyme production and growth of B. stearothermophilus GRE1. High and an almost similar growth rate ([[micro].sub.max] [[approximately equal to] 0.63 [h.sup.-1]), biomass ([X.sub.max] [approximately equal to] 2.11 g/L) and enzyme production ([alpha]-amylase [approximately equal to] 11.30 U/mL and [beta]-amylase [approximately equal to] 10.00 U/mL) were observed (after 12 h of fermentation), when the initial pH of the medium was adjusted to 6.0 and 7.0. Beyond a pH of 7.0, both biomass and enzymes productions were retarded. According to Vihinen and Mantsala (1987), the pH optima for [alpha]-amylase production by B. stearothermophilus vary, but generally are in the range of 4.5-8.0.

[FIGURE 5 OMITTED]

Effect of Controlled pH on Enzyme Production

In a number of fermentation processes, uncontrolled pH change enhanced enzyme production (Rakshit and Sahal, 1991). In our experiments, pH was controlled to be constant throughout the fermentation process at 6.0 and 6.9 in separate fermentation tanks. At a controlled pH value of 6.9, production of amylolytic enzymes (Figure 6) and biomass were high. The activity of [alpha]-amylase reached 12.33 U/mL, while that of [beta]-amylase was 10.40 U/mL. In both controlled and uncontrolled conditions, the specific growth rates are similar (0.63-0.64 [h.sup.-1]), while the maximum biomass production ranged between 2.08 to 2.20 g/L.

[FIGURE 6 OMITTED]

Hydrolysis of Starch Granules by the Crude Enzyme

The rate of degradation of four types of starch (wheat, corn, cassava and potato raw starch) by the [alpha]-amyalse solution was investigated. Wheat starch was the most susceptible to hydrolysis and at 50 to 60[degrees]C, about 80% conversion to sugar was obtained. Corn and cassava starches were also hydrolyzed efficiently (Figure 7). At 40[degrees]C potato starch hydrolyzed slowly, and after 36 h of incubation 30% conversion was achieved. At 60[degrees]C, the corresponding value was 60%, which was doubled compared to that at 40[degrees]C.

[FIGURE 7 OMITTED]

Miyoshi et al. (1986) reported that degradation efficiency of raw starch granules by amylase was increased by raising the reaction temperature from 40 to 60[degrees]C. In addition, structure and size of starch granules affect enzyme attack (Kimura and Robty, 1995). Our observations indicate that, at 60[degrees]C all the starch granules showed the highest degradation rate, which might be attributed to their gelatinization temperature that ranges between 58 to 72[degrees]C. On the other hand, the highest granular size of potato starch (28 [micro]m) has made it more resistant to the enzyme attack. Wheat (6 [micro]m) was well hydrolyzed, followed by cassava (16 [micro]m) and corn (15 [micro]m). Figure 8 shows the morphology of cassava starch granules (native and enzyme treated) observed by scanning electron microscopy. The surfaces of the untreated granules were smooth, while the treated granules were rough.

[FIGURE 8 OMITTED]

Determination of Kinetic Constant

The [K.sub.m] and [V.sub.max] values of the [alpha]-amylase were calculated from the Lineweaver-Burk plot (Figure 9). The values respectively are 4.78 mg starch/mL and 6.67 mg/mL.min.

[FIGURE 9 OMITTED]

CONCLUSIONS

Enzyme production by B. stearothermophilus GRE1 is growth associated. Soluble starch and lactose were found to be the best carbon sources for optimum enzyme production. The [alpha]-amylase obtained could be used in the hydrolysis of starch in the granular form.
NOMENCLATURE

DNS dinitosalicylic acid colour reagent
w weight (g)
v volume (mL)
rpm rotational speed per minute


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Manuscript received July 12, 2005; revised manuscript received January 19, 2006; accepted for publication January 23, 2006.

S. M. Z. Hossain (1 *), G. D. Haki (2) and S. K. Rakshit (3)

(1.) Department of Chemical and Petroleum Engineering, University of Calgary, 2500 University Drive, Calgary, AB, Canada T2N 1N4

(2.) Debub University, P.O. Box 05, Awassa, Ethiopia

(3.) Food Engineering and Bioprocess Technology Program, School of Environment, Resources and Development, Asian Institute of Technology, P.O. Box 4, Klong Luang, Pathumthani 12120, Thailand

* Author to whom correspondence may be addressed.

E-mail address: zhossain@ucalgary.ca
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