The present study is concerned with the selection of a suitable Bacillus strain and optimization of cultural conditions for the biosynthesis of alpha amylase. Two hundred and seventy Bacillus species were isolated from the soil and tested for alpha amylase production. Of all the species tested, the Bacillus licheniformis GCB-36 gave maximum production of alpha amylase (200 U/g/min, 65 U/ml/min) in both solid state and submerged fermentation media. This strain was selected as parental strain for the optimization of cultural conditions.
Different agricultural by-products such as wheat bran, rice bran, oat bran, nee husk. sunflower meal, cotton seed meal, guar meal, rape-seed meal and soybean meal were tested for the production of alpha amylase using solid state fermentation conditions. Of all the substrates tested, wheat bran along with cotton seed meal-at the ratio of 7.5: 2.5 was selected as basal medium. The production of alpha amylase following growth of the organism was found to be optimum (260 U/g/min) as the basal medium was moistened with phosphate buffer (0.02 M) and incubated at 40°C for 48 h. The production of enzyme was increased (300 U/g/min) with the addition of starch (2%) and ammonium sulphate (1.0% nitrogen) to the fermentation medium.
Eight culture media were tested for the production of alpha amylase in 250 ml shake flask. The culture medium M-3 containing (g/l) nutrient broth 10.0, soluble starch 10.0, (NH4)2SO4 2.0, NaCI 1.0, CaCI2 2.0, MgSO4 2.0 in phosphate buffer (0.02M) was found to be the best for the production of alpha amylase. The production of alpha amylase following growth of the organism was found to be optimum (76 U/ml/min) as the fennentation medium (pH 7.5) was incubated at 40oC for 48 h. The productivity of the enzyme was increased (88 U/ml/min) with the addition of ammonium chloride (0.5%) in the fermentation medium. Different metallic salts were supplemented in the fermentation medium and CaCI2 was found to be best for the productivity of alpha amylase.
For the improvement of Bacillus licheniformis strain, the parental strain, after optimization of the cultural conditions, was subjected to UV irradiations for 5-35 minutes. Among 257 mutants isolated, the mutant Bacillus licheniformis GCUM-15, isolated after 15 min of UV irradiations, gave maximum production of alpha amylase (389 U/ml/min and 670 U/g/min). This mutant was further subjected to NTG treatment for 5-60 minutes. The amount of NTG 200 μg/ml for 30 min was found to be best for the production of stable and viable mutants. The mutant Bacillus licheniformis GCCM-23, better producer of enzyme was again treated alternatively with UV/NTG and the best alpha amylase secreting mutant Bacillus licheniformis GCUCM-30 was selected. This mutant gave 8 times more enzyme (788 U/ml/min and 2800 U/g/min) than the original parental strain. When this mutant was again treated with UV or chemically, complete death of the bacteria was observed. The kinetic parametric study indicated that the yield of the enzyme by cell mass formation (Yp/x) was ten times high by the mutant than the - parental strain. The volumetric rates of ,biomass formation (Qx) and enzyme formation (Qp) were also high by Bacillus licheniformis GCUCM-30 and varied significantly (p<0.05) than the parental and other mutant derivatives.
To obtain the maximum production of alpha amylase, the fermentation was again carried out by both solid state and submerged fermentation conditions. Different carbon sources were added to the bran medium and tested for alpha amylase production. The production of alpha amylase by mutant strain was found optimum (3050 U/g/min) in the presence of starch (1.5%) and lactose (1.0 %) in the fermentation medium. The kinetic values such as Yp/x, Qp and Qx also gave promising results in the presence of above carbon sources. Both organic and inorganic nitrogen sources were tested for production of alpha amylase by parental and mutant strain of Bacillus licheniformis. The volumetric production of enzyme was found to best in the fermentation medium containing nutrient broth (1 %) and ammonium sulphate (1.0 % nitrogen). The production of. alpha amylase was found to be optimum (3475 U/g/min) as the fermentation medium (pH 7.5) was inoculated with 10% inoculum for 48 h.
Optimization of conditions for the extraction of alpha amylase from fermented bacterial bran was carried out. Phosphate buffer (0.02M) was used as extractant and bran to extractant ratio of 1:10 was found to be optimum for the extraction of maximum enzyme. Time duration of agitation was decreased as the agitation intensity was increased from 100 rpm. The maximum enzyme was extracted (3600 U/g/min) when the agitation intensity was maintained at 200 rpm for 1.0 h. The- extraction of enzyme was increased after every 5°C rise in temperature and reached optimum (3850 U/g/min) at 65°C.
The optimization of cultural conditions was also carried out by parental and mutant strain of Bacillus licheniformis in 250 ml shake flask. Different carbon and nitrogen sources were tested for the productivity of alpha amylase. The starch (l %) and lactose (0.5%) as carbon source while, nutrient broth (1.0%) and amnionium sulphate (0.25%) as nitrogen source was optimized for the maximum production of alpha amylase. The production of alpha amylase by mutant strain was reached optimum (860 U/ml/min) at 44 h as the fermentation medium (pH 7.5) was inoculated with 2% inoculum.
The effect of different alcohols such as sorbitol, manitol, glycerol, propylene diol or ethylene glycol was investigated on the production of alpha amylase by parental and mutant strain of Bacillus lichel1iformis. The production of alpha amylase was significantly improved by mutant strain as the sorbitol (0.3 M) was added in the fermentation medium. However, the productivity of the enzyme by parental strain was greatly inhibited in the presence of alcohols.
Economical medium is preferable than the costly medium. In. this connection different agricultural by-products were. supplemented in tire above optimized liquid medium. The production of enzyme following growth of the organism was significantly improved as the wheat bran at 1.25% level was supplemented in the fermentation medium. Different starchy substrates were added to the fermentation medium to replace the costly soluble starch. The production of enzyme by Bacillus licheniformis GCUCM-30 gave promising results (960 U/ml/min) as the soluble starch was replaced with Pearl millet starch. With the addition of above medium, the supplementation of nutrient broth was decreased from 1.0% to only 0.5%.
The selected media (solid, liquid and semi-synthetic) were compared for the production of alpha amylase. The production of alpha amylase was increased with increase in the incubation period and 48, 44 and 40 h were found optimum by solid, liquid and semi-liquid medium respectively. The kinetic study revealed that the yield of the enzyme by cell mass formation and substrate addition was highly significant by semi-synthetic medium and varied significantly (p<0.05) than the solid and liquid medium.
After optimizing the cultural conditions in shake flask, the scale up studies were carried out in 7.5L stirrer fermentor. Selection of suitable fermentation medium is very essential for optimum production of alpha amylase in biofermentor. Two culture media were tested for amylase synthesis and medium M-2 containing g/1 Wheat bran 15 ,Pearl millet starch 10, lactose 5.0, nutrient broth 5.0, (NH4)2SO4 5.0, CaCl2 2.0, NaCI 1.0, MgSO4 2.0 in 1000 ml of phosphate buffer (0.02M) was found to be best for the production of alpha amylase. The production of enzyme following growth of the organism was found optimum when volume of the medium at 60%, agitation intensity at 250 rpm. and air supply at 1.0 1 1-1 m-1 was maintained in the fermentation medium. The production of the enzyme was reached optimum (1520 U/ml/min) at 36 h as the fermentation medium (pH 7.5) was inoculated with 4% inoculum. The production , of alpha amylase was stimulated as the sorbitol was added to the femidntation medium 24 h after inoculation.
After optimizing the cultural conditions the alpha amylase was partially purified by acetone and ammonium sulphate precipitation. The specific activity of alpha amylase was found to be 8 fold increased after partially purifying the enzyme. The thennostability of Ca+2 dependent and the activity of alpha amylase was found optimum at pH 6.5, at 65°- 85°C.
The desizing of fabric by crude and partially purified alpha amylase was carried out. The maximum desizing (100.%) of fabric was obtained after 30 min of incubation when the desizing took place at pH 6.5 and at 80°C.