EXTRACTION OF THERMO-STABLE ALPHA AMYLASE FROM FERMENTED WHEAT BRAN

BIOLOGIA 2001, 47 (1&2), PP 47 52 ISSN 0006 3096 EXTRACTION OF THERMO-STABLE ALPHA AMYLASE FROM FERMENTED WHEAT BRAN *HAMAD ASHRAF, IKRAM UL HAQ, AND JAVED IQBAL Biotechnology Research Laboratory, Department of Botany, Govt. College, Lahore, Pakistan. ABSTRACT: The present study is concerned with the optimization of conditions for the extraction of alpha amylase from fermented wheat bran. Phosphate buffer (ph 7.5) was used as an extractant, bran to extractant ratio of 1:10 was found to be optimum for extraction of maximum enzyme. There was a correlation between agitation intensity, time of agitation and temperature for the extraction of extracellular & thermostable alpha amylase. Time duration of agitation (for extraction of enzyme) was decreased as the agitation intensity was increased from 100 rpm. The maximum enzyme was extracted (3600 IU/g/min) when the agitation intensity was maintained at 200 rpm for one hour. The extraction of enzyme was increased after every 5 o C rise in temperature and reached optimum (3850 IU/g/min) at 65 o C. Keywords: Alpha amylase, Bacillus, Fermentation, Phosphate buffer, Solid state, INTRODUCTION The enzyme, alpha amylase, which converts starches into oligosaccharides is the most important product obtained for human needs through microbial sources. A large number of industrial processes in the area of textile, food biotechnology, pharmaceutical and detergent utilize the thermostable alpha amylase to great extent (Nigam & Sing, 1995). The enzymes from Bacillus subtilis were found to be more thermostable; therefore, it can be preferred for industrial production of alpha amylase (Ludikhuyze et al., 1996). Solid-state fermentation holds tremendous potential for the production of alpha amylase. It can be of special interest in those processes where crude fermented product may be used directly as enzyme source. Different agricultural byproducts such as wheat bran, rice bran, cottonseed meal, soybean meal etc., can be used for their exploitation as substrates by solid-state fermentation (Ramesh & Lonsane, 1990; Ramesh & Lonsane, 1991; Babu & Satyanarayana, 1995). The enzyme produced by solid substrate cultivation extracellularly and remains attached with the fermentation medium (Ramadas et al., 1995). For the commercial production of enzyme as well as for its maximum activity, it is necessary to separate the maximum enzyme from fermented wheat bran (Ramesh & Lonsane, 1989). The extraction from dried bran by buffers not only yielded higher enzyme titre but also show its maximum activity. Phosphate buffer (ph 7.5) was found to be most suitable for the extraction of alpha amylase from the fermented wheat bran (Haq et al., 1998). The present study is concerned with the optimisation of conditions for the extraction of extra-cellular alpha amylase from the fermented wheat bran.

BIOLOGIA HAMAD ASHRAF, ET AL 48 MATERIALS AND METHODS Organism The mutant strain of Bacillus licheniformis GCUCM-30 (a thermophilic strain, enzyme active at 60-70 o C) isolated and tested for alpha amylase production in this laboratory was used for present study. The organism was maintained at nutrient broth starch agar slants. The strains were stored in paraffin oil containing nutrient starch agar medium. Inoculum Preparation Vegetative inoculum was used in present studies. Fifty ml of inoculum medium containing (%w/v) nutrient broth 1.0, soluble starch 1.0, NaCl 0.5, CaCl 2 0.2 in 100 ml of phosphate buffer was transferred to each of 250 ml cotton plugged Erlenmeyer flask and sterilized at 15lb pressure (121 C) for 15 minutes. After cooling at room temperature, a loopful of bacteria was aseptically transferred to each flask. The flasks were then rotated in the rotary shaking incubator (200 rpm) at 40 C for 24 hours. Fermentation Technique Solid-state fermentation technique was employed. Ten gram solid substrate (wheat bran 7.0g & cotton seed meal 3.0 g) was transferred to 250 ml Erlenmeyer flask and moistened with 10 ml phosphate buffer. The flasks were sterilized in the autoclave and cooled at room temperature. The flasks were inoculated with one ml of vegetative inoculum and incubated at 40 C for 48h. After 48h 100ml of phosphate buffer was transferred to each flask and then rotated in the rotary incubator shaker at 200 rpm for one hour. After one hour the fermented bran was filtered and filtrate was used for the estimation of alpha amylase. All the experiments were run parallel in triplicates. Enzyme Assay Alpha amylase estimation was carried out according to the method of Rick & Stegbauer (1974). The enzyme solution at ph 7.5 was incubated at 60 o C using 1% soluble starch solution. The reducing sugars were measured by adding 3,5-dinitro salicylic acid reagent, boiling for 5 min, cooling and measuring the O.D at 540 nm in the spectrophotometer (Model CECIL CE7200) against maltose as standard. One unit of activity is equivalent to that amount of enzymes, which in 10 minutes librates reducing group from 1% Lintner s soluble starch corresponding to 1 mg maltose hydrate. Statistical Analysis Treatment effects were compared by the method of Snedecor & Cochrane (1980). RESULTS AND DISCUSSION The separation of alpha amylase from fermented wheat bran is very essential for its commercial application. Phosphate buffer (ph 7.5) was used as an extractant because previously (Haq et al., 1998) it was found to be the best extractant and stabilizer of alpha amylase. The ratio of extractant to fermented bran is a critical factor for maximum separation of enzyme. Different ratios (1:2.5-1:20) of bran to extractant were evaluated and the maximum extraction of alpha amylase (3500 IU/g/min) was obtained when bran to extractant ratio was 1:10. As the amount of extractant was increased, there was gradual reduction in the extraction of enzyme. It might be due to the fact that high ratios of bran to extractant acted as a hindrance for proper agitation and separation of

Vol. 47(1&2) ALPHA AMYLASE EXTRACTION FROM FERMENTED BRAN 49 enzyme (Ramadas et al., 1995). At low ratios of bran to extractant the extraction of alpha amylase was insignificant. It might be due to the fact that at low ratio, the amount of extractant was insufficient to extract the attached enzyme from fermented wheat bran as the extractant becomes more concentrated. Therefore, 1:10 ratio of bran to extractant was selected for subsequent experiments. The agitation intensity and its time duration play a very critical role in the extraction of attached enzyme from fermented wheat bran. For this purpose the relationship between agitation intensity and time of agitation was investigated (Fig., 2). It was observed that with increase in the agitation intensity, the time duration for the extraction of enzyme was decreased. The extraction of enzyme was found to be optimum (3600 IU/g/min) after one hour when the enzyme was extracted at 200 rpm. Further increase in the agitation intensity was found to be insignificant. Below 200 rpm the insignificance of the results might be due to the fact that agitation was not proper, thus causing insufficient separation of enzyme from fermented bran. Padmanabhan et al. (1992) have reported that the separation of enzyme from fermented bran was increased as the time of agitation was increased from 60 to 120 min. However, in this study the maximum separation of the enzyme (3850 IU/g/min) was achieved only after 60 min. Thus, this finding is more encouraging than the work reported by Padmanabhan et al. (1992) because it takes lesser time for the extraction of enzyme; it would lead towards not only safe energy but also economy of the country. Thus, agitation intensity of 200 rpm for one hour was selected for extraction of enzyme from fermented wheat bran. The temperature also plays a very critical role in the extraction of thermostable enzyme because the stability and activity of alpha amylase is very sensitive to temperature (Babu & Satyanarayana, 1995). It was observed that there was a correlation between temperature and separation of enzyme from fermented wheat bran (Fig. 3). The extraction of enzyme was increased with increase in the temperature after every 5 o C rise in temperature and reached maximum at 65 o C. It might be due to the fact that at high temperature, the permeability of the mash was increased and hence the leaching of the enzyme from fermented bran was also increased (Prescott & Dunn, 1987). Further increase in the temperature caused decrease in the extraction of the enzyme. It might be due to the fact that denaturing of the enzyme started at high temperature (Ludikhuyze et al., 1996) that resulted decrease in the activity of alpha amylase. Therefore, agitation intensity of 200 rpm at 65 o C for one hour was optimized for the preparation of crude alpha amylase at industrial level. CONCLUSION From the present study it was concluded that there was a correlation between agitation intensity, time of agitation and temperature for the extraction of extracellular and thermostable alpha amylase. By using the optimum conditions for extraction of alpha amylase from fermented bacterial bran, energy and economy of the country can be saved which makes the present study more encouraging.

BIOLOGIA HAMAD ASHRAF, ET AL 50 REFERENCES Babu, K.P. & Satyanarayana, T., 1995. Alpha-amylase production by thermophilic Bacillus coagulans in solid state fermentation. Process Biochem. 30(4): 305-309. Haq, I., Ashraf, H., Zahara, R. & Qadeer, M. A., 1998. Biosynthesis of alpha amylase by Bacillus subtilis GCB-12 using agricultural by products as substrates. Biologia, 44(1&2): 154-163. Ludikhuyze, L., De-Cordt, S., Weemaes, C., Hendrickx, M. & Tobback, P., 1996. Kinetics for heat and pressuretemperature inactivation of Bacillus subtilis alpha amylase. Food Biotechnol. 10(2): 105-109. Nigam, P. & Sing, D., 1995. Enzyme and microbial system involved in starch processing. Enzyme Microb. Technol., 17: 770-778. Padmanabhan, S., Ramakrishna, M., Lonsane, B.K. & Kishnaiah, M.M., 1992. Enhanced leaching of product at elevated temperature: Alpha amylase produced by Bacillus licheniformis M 27 in solidstate fermentation system. Lett. Appl. Microbiol., 15 (6): 235-238. Prescott, S. & Dunn, A., 1987. Industrial microbiology. 4 th Ed. CBS Publishers and Distributors, New Delhi, India. pp: 550-565. Ramadas, M., Holst, O. & Mattiasson, B.,1995. Extraction and purification of alpha amylase produced by solid state fermentation with Aspergillus niger. Biotechnol. Tech., 9 (12): 901-906. Ramesh, M.V. & Lonsane, B.K., 1989. Purification of thermostable alpha amylase produced by Bacillus licheniformis M27 under solid state fermentation. Process Biochem., 24 (5): 176-178. Ramesh, M.V. & Lonsane, B.K., 1990. Critical importance of moisture contents of the medium in alpha amylase production by Bacillus licheniformis M27 in a solid state fermentation system. Appl. Microbiol. Biotechnol., 33 (5): 501-505. Ramesh, M.V. & Lonsane, B.K., 1991. Regulation of alpha amylase production in Bacillus licheniformis M 27 by enzyme end products in submerged fermentation and its overcoming in solid state fermentation system. Biotechnol. Lett., 13 (5): 355-360. Rick, W. & Stegbauer, H. P., 1974. α- Amylase measurement of reducing groups. In H.V. Bergmeyer (Ed.), Methods of Enzymatic Analysis, 2 nd Ed., Vol. 2, Academic Publisher. Snedecor, G. W. & Cochrane, W. G., 1980. Statistical Methods, 7 th Edition. Ames, Iowa: Iowa State University Press. ISBN 0-81381560-6.

Enzyme activity (IU/g/min) Vol. 47(1&2) ALPHA AMYLASE EXTRACTION FROM FERMENTED BRAN 51 4000 3500 3000 2500 2000 1500 1000 500 0 25 50 75 100 125 150 175 200 Amount of extractant (ml) IU/g/min Figure 1: Selection of bran to extractant ratio for the extraction of alpha amylase from fermented bran Each value is an average of three replicates. Y bars indicated the standard error. Agitation intensity (rpm) Figure 2: Selection of time and agitation intensity for the extraction of alpha amylase from fermented bran Each value is an average of three replicates. Y bars indicated the error.

Enzyme activity (IU/g/min) BIOLOGIA HAMAD ASHRAF, ET AL 52 4500 4000 3500 3000 2500 2000 1500 1000 500 0 30 35 40 45 50 55 60 65 70 75 80 Temperature (C) Figure 3: Effect of temperature on the extraction of alpha amylase from fermented bacterial bran Each value is an average of three replicates. Y bars indicated the error.