This action might not be possible to undo. Are you sure you want to continue?
Rica Mae A. Palongpalong1, Percival G. Garcia2
Student, School of Chemical Engineering and Chemistry, Mapua Institute of Technology Adviser, School of Chemical Engineering and Chemistry, Mapua Institute of Technology
Abstract Amylases have great significance in biotechnology and are commercially important enzymes in various starch processing industries. The microbial amylases meet industrial demands and have almost completely replaced chemical hydrolysis of starch in starch processing industry. This study explored the use of jackfruit seed as carbon source in the production of alpha-amylase. A production medium composed of ground jackfruit seed and yeast extract powder was mixed with 10% inoculum of Bacillus licheniformis 1331 cultured in nutrient broth at 30°C for 18 hours. The effects of temperature and carbon to nitrogen ratio were determined. Triplicate analysis was done at 10%, 15%, 20% carbon source with 20% nitrogen source set at pH 7 and temperature of 30°C and 37°C. It was found out that alpha-amylase production was optimal at 10% carbon, 20% nitrogen, pH 7, and 37°C. Amylase production was conducted in 1000 ml medium and incubated with shaking. Samples were taken at regular intervals at 0, 1, 2, 4, 6, 8, 18, 24, 27, and 30 hours of fermentation. Enzyme activity and protein concentration were determined using Alpha-amylase assay and Bradford assay, respectively. Biomass was determined by dry cell weight (DCW) analysis. Alphaamylase production was highest during exponential growth at 4-8 hours of fermentation. Keywords: alpha-amylase, Bacillus licheniformis, jackfruit seed, yeast extract
1. Introduction In the environment abound possible sources of essential materials or substances necessary for the development or production of certain products like enzymes, which will be useful to humans, plants, and other animals. These substances can be produced in significantly large amounts with the advent of biotechnology, but primarily the materials and the factors that influence its production source must be identified. Papain was prepared from high quality latex of Carica papaya (Baines, B. S. and Brocklehurst, K., 1978). Some wastes from fruits and vegetables can also be a good substrate for enzyme production. One study was done on banana fruit stalk, which was
used as a substrate for alpha-amylase production by Bacillus subtilis under solid-state fermentation (Krishna and Chandrasekaran, M., 1996). A similar study was also done using potato peelings (Shukla, J. and Kar, R., 2000). Trypsin/ chymotrypsin inhibitor was isolated from jackfruit by ammonium sulfate fractionation and chromatography (Sai Annapurna, S. and Siva Prasad, D., 1990). These studies showed that some enzymes could be produced using components of fruits and vegetables as substrate. This particular study aims to produce an enzyme by a Bacillus sp. using the seeds of a locally grown fruit as substrate. This particular fruit is jackfruit scientifically known as Artocarpus heterophyllus or locally
known as “nangka” or “langka”, which is a favorite dessert of Filipinos and is a widely grown fruit crop in the Philippines. It contains carbohydrates, protein, calcium, iron, sodium, potassium, B-complex, ascorbic acid, and small amounts of fats, ash, and iron. Analysis of jackfruit food composition per 100 gram edible portion showed that the carbohydrate content of the seed is 34.90 g (Agriculture and Fisheries Information Science, Department of Agriculture). Based on the above composition, the jackfruit seed having a high content of carbohydrate can be used as a substrate for the production of the enzyme amylase. The production of amylase is of great significance in biotechnology. It is commercially important in some processing industries such as beverages, food, textile, detergents, and paper industries (Pandey et al., 2000). This enzyme can be produced using a specific microorganism or fungi under controlled conditions. The amylases can be derived from several sources ranging from bacteria to plants to humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extracellular digestion. When they have broken down the insoluble starch, the soluble end products such as glucose or maltose are absorbed into their cells. This particular study will use Bacillus licheniformis for amylase production. This is an exploratory study to determine alpha-amylase production by Bacillus licheniformis using jackfruit seed as substrate. This study is limited to optimization of cultural and environmental conditions such as temperature and carbon/nitrogen ratio.
No enzyme purification and characterization will be done. The significance of this study is that another substrate will be added to the existing list of carbon sources for amylase production by microorganisms. Many industrial processes use waste materials from plants or animals. Part of solving the environmental problem of waste management can be addressed by the utilization of waste products. Jackfruit seeds as waste from fruit can be a potential material for enzyme production. Being a substrate it will become a source of income for fruit vendors and farmers. This study becomes important to the biotechnology industry and likewise contributes to environmental management and economic stability. 2. Methods 2.1 Preparation of jackfruit seed One hundred (100) g of jackfruit seeds was autoclaved in 1L beaker at 121°C (15 lbs psi) for 15 minutes. The outer seed coating was removed. Seeds were grinded by using a coffee grinder. Grinded seeds were placed in zip locked plastic bag and stored in the freezer. 2.2 Bacterial strain The bacteria, Bacillus licheniformis 1331, was obtained from the Philippine National Collection of Microorganisms (PNCM), BIOTECH, University of the Philippines, Los Baños, Laguna. 2.3 Medium Preparation HIMEDIA M001 was used for maintenance of organism. The
composition of nutrient agar is shown in Appendix A. Twenty-eight (28) g of nutrient agar was mixed with 1L-distilled water in 1L cotton plugged Erlenmeyer flask. The medium was sterilized by autoclaving at 121°C (15 lbs psi) for 15 minutes. From the pure culture, cells were subcultured by streaking them on nutrient agar plate and incubated at 30°C for 30 h. Several growing colonies were transferred to nutrient agar slants and incubated at 30°C for 18 h. Nutrient agar slants were stored in refrigerator and subcultured every three (3) weeks. 2.4 Preparation of inoculum Nutrient broth was used for preparation of inoculum. The medium contains (in g/L): HIMEDIA RM 027 yeast extract, 3; peptone, 5; and NaCl, 8. The medium was sterilized at 121°C (15 lbs psi) for 15 minutes.
and HIMEDIA RM 027 yeast extract powder as nitrogen source at 20%. The pH of the production medium was 7. The inoculum size was 10% (v/v) of the production medium. The culture was incubated at 30°C for 24 h. 2.6 Optimization of cultural conditions Factors such as temperature and source of carbon and nitrogen affecting production of amylase were optimized by varying parameter one ats a time. The experiment was conducted in 250 ml cotton plugged Erlenmeyer flask containing 100 ml of production medium. The pH of 7, temperature at 30°C and 37°C, carbon source at 10%, 15%, and 20%, and nitrogen source at 20% were used. The optimum C/N ratio was determined by varying carbon and nitrogen source of production medium. A factorial design shown below was conducted. Table 1. Factorial design at pH 7 and 30°C
Three (3) loopfuls of B. licheniformis 1331 from nutrient agar slant was inoculated in 100 ml nutrient broth in 250 ml cotton plugged Carbon source (w/v) Erlenmeyer flask. It was incubated at 10% 15% 20% 30°C for 18 h.
20% (10%, 20%) (15%, 20%) (20%, 20%)
Nitrogen source (w/v)
2.5 Production medium The production medium consists of grinded jackfruit seeds as carbon source and HIMEDIA RM 027 yeast extract powder as nitrogen source. One hundred (100) ml of production medium in 250 ml Erlenmeyer flask contains jackfruit seed as carbon source at 10%, 15%, and 20%
Table 2. Factorial design at pH 7 and 37°C Carbon source (w/v)
10% 15% 20% (10%, 20%) (15%, 20%) (20%, 20%)
The experiment was done in triplicate. After determining the optimum temperature and C/N ratio, 500 ml of production medium in 1L cotton plugged Erlenmeyer flask was made. Samples were taken at regular intervals (0, 1, 2, 4, 6, 8, 18, 24, 27 and 30h) and analyzed for biomass, protein concentration, and alpha-amylase activity. 2.7 Preparation of crude enzyme extract Five (5) ml culture broth in micro test tube was centrifuged for 20 minutes at 5000 rpm. The supernatant was used as crude enzyme solution to determine protein concentration (mg/ml) and alphaamylase concentration (units/ml). 2.8 Alpha (α)-amylase assay 2.8.1 Iodine reagent The stock solution was a mixture of two solutions in separate 100 ml volumetric flask. The two solutions were 0.5 g iodine in 100 ml water and 5 g Potassium iodide in 100 ml water. The freshly prepared working solution was a mixture of 1 ml stock solution, 5 ml HCl (5N), and 500 ml deionized water in 500 ml volumetric flask. 2.8.2 Buffer solution
The KH2PO4 solution was a mixture of 0.6803 g of KH2PO4 (0.05M) and distilled water in 100 ml volumetric flask. The NaOH solution was a mixture of 0.1999 g NaOH and distilled water in 100 ml volumetric flask. One-hundred (100) ml KH2PO4 solution was placed in 250 ml beaker and 10-11 ml of NaOH solution was added to have 0.05M KH2PO4-NaOH buffer at pH 6.0. 2.8.3 Substrate: Starch solution 0.2 g soluble starch was dissolved in boiling 0.05M KH2PO4NaOH buffer at pH 6.0 and was cooled to 40°C. 2.8.4 Analytical procedure For the assay, 1.0 ml of the diluted enzyme solution (0.5 ml crude enzyme and 0.5 ml buffer) was placed in a test tube and warmed to 40°C in a water bath for 10 min. Two (2.0 ml) of starch solution was added and incubated for 10 minutes. Then 0.2 ml was removed from the test tubes and placed in another tube containing 5.0 ml of iodine reagent. The absorbance at 620 nm was measured against a blank (0.2 ml buffer + 5 ml of iodine reagent). The substrate control contains 1.0 ml buffer + 2 ml substrate in place of diluted enzyme solution. α-Amylase activity was calculated from the absorbances by using the equation: α-Amylase activity (U/ml)= Ac − At × 40 D , where: Ac is the Ac absorbance of substrate control, At is the absorbance of test sample, 40 represents 4.0 mg starch present in the reaction tube
times 10, and D is the enzyme dilution factor. One unit of alpha-amylase is defined as the amount of enzyme that will hydrolyze 0.1 mg of starch in 10 min at 40°C when 4.0 mg of starch is present. Activities which resulted in absorbances of less than 0.125 after 10 min required dilution to give linear reactions over the 10-min period (Smith and Roe, 1949). 2.9 Protein Bradford assay Curve determination by
calculating the actual protein concentration of the enzyme solution. 2.10 Biomass determination Using a pipette, 5 ml of culture broth was placed in pre-weighed micro test tubes that have been dried overnight at 105°C and cooled in a desiccator. The sample was centrifuged for 5 minutes at 5000 rpm. The supernatant was discarded. The pellet was resuspended in 5 ml distilled water using a vortex mixer. It was again centrifuged for 5 minutes and the supernatant was discarded. The micro test tubes with pellets were dried at 105°C overnight and cooled in a desiccator. The sample was weighed and the dry cell weight (DCW) of biomass was calculated. 3. Results and discussion The starch content of jackfruit seeds was verified at the Bureau of Plant Industry (BPI) before the actual experiment. The starch content was 37.68% by using Luffshoorl method. The average of the results of the alpha-amylase activity for three trials at pH 7 and 30°C is shown in Table 5. It can be seen that the highest alphaamylase activity was at 10% carbon and 20% nitrogen. Table 3. Alpha-amylase activity at pH 7 and 30ûC
2.9.1 Preparation of Standard Twenty (20) µl of each standard solution of bovine serum albumin (BSA) was prepared at 0 (blank), 125, 250, 500, 1000 and 1500 µg/ml. Two (2) ml of Bradford dye reagent was added to each solution. The solution was equilibrated for two minutes to one hour, after which the absorbance of each solution was measured at 595 nm, using distilled water as a blank. 2.9.2 Determination unknown protein content of
Two (2) ml of Bradford dye reagent was added to 400 µl of the crude enzyme solution. The solution was equilibrated for two minutes to one hour, after which the absorbance of each solution was measured at 595 nm, using distilled water as a blank.
Alpha-amylase activity (U/ml) 10% Carbon, 20% Nitrogen 0.1592 If the enzyme solution has 15% Carbon, 20% Nitrogen 0.059 20% Carbon, 20% Nitrogen 0.1061 absorbance reading outside the range
established by the standard curve, the sample enzyme solution was diluted with water and absorbance measurement at 595 nm was redone. Dilution factors were taken into account when
0.18 Alpha-amylase activty (U/ml) 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 1 2 % Carbon, % Nitrogen 3
of the fermentation medium that resulted in the decreased agitation and poor mixing of air, which were essential for the growth of organism and production of enzyme (Haq et. al., 1998). Also, alpha-amylase production was possibly affected by the presence of a catabolite repressing carbon source in the growth medium. Table 5. Protein concentration at pH 7 and 30°C
Figure 1. Alpha-amylase activity at pH 7 and 30°C
Protein concentration (mg/ml) 3.1338 The average of the results of the 10% Carbon, 20% Nitrogen 15% Carbon, 20% Nitrogen 3.3168 alpha-amylase activity for three trials at 20% Carbon, 20% Nitrogen 3.1464
pH 7 and 37°C is shown in Table 4. The highest alpha-amylase activity was also at 10% carbon and 20% nitrogen. Table 4. Alpha-amylase assay at pH 7 and 37ûC
10% Carbon, 20% Nitrogen 15% Carbon, 20% Nitrogen 20% Carbon, 20% Nitrogen
3.35 3.3 3.25 3.2 3.15 3.1 3.05 3 1 2 % C a r bo n , % N i t r o g e n 3
Alpha-amylase activity (U/ml) 0.6374 0.609 0.1658
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 % Ca r bon, % N i t r og e n 3
Figure 3. Protein concentration at pH 7 and 30°C Table 6. Protein concentration at pH 7 and 37°C
10% Carbon, 20% Nitrogen 15% Carbon, 20% Nitrogen 20% Carbon, 20% Nitrogen Protein concentration (mg/ml) 2.8833 2.7915 3.0993
Figure 2. Alpha-amylase activity at pH 7 and 37°C Figure 1 and Figure 2 shows that the higher concentration of carbon source has an adverse effect on the production of alpha-amylase particularly at 37°C. It may be due to the thickness
3.2 3.1 3 2.9 2.8 2.7 2.6 1 2 % C a r bo n, % N i t r o ge n 3
Figure 4. Protein concentration at pH 7 and 37°C The total protein concentration in the sample was determined by the Bradford assay. As shown in Table 5 and Table 6, the highest protein concentration was obtained at 30°C, 15% carbon and 20% nitrogen. Table 7. Alpha-amylase activity, Protein concentration, and Biomass concentration of samples taken at regular intervals
and C/N ratio of 10% carbon and 20% nitrogen. Samples were taken at regular intervals (0, 1, 2, 4, 6, 8, 18, 24, 27, and 30h) for analysis of alpha-amylase activity, protein concentration and biomass (DCW), as shown in Table 7. Figure 5 shows the alphaamylase activity in the course of fermentation as shown in Alpha-amylase activity was highest during exponential growth at 4-8 hours and protein concentration was highest at 6 hours of fermentation.
The values of protein concentration in mg/ml were higher than Time (h) Alpha-amylase activity (U/ml) Protein concentration (mg/ml) Biomass (mg/ml) the values of alpha-amylase in U/ml. It 0 0.2879 2.6988 0.2 may be due to the fact that protein 1 0.3165 2.9745 0.26 concentration in jackfruit seeds was 2 0.3275 3.0455 0.2 high.
4 6 8 18 24 27 30 0.4045 0.5868 0.6484 0.6593 0.6593 0.6659 0.6725
3.2888 0.36 Ajayi (2008) reported that the 3.3288 0.38 moisture, ash, protein, crude0.3 crude oil, 3.224 fiber, and carbohydrate contents of 3.2825 0.22 Artocarpus heterophyllus seeds to be 3.2975 0.32 3.2318 0.12 2.78%, 6.72%, 20.19%, 11.39%, 7.10%, 3.2688 0.38 and 51.82%, respectively. Bobbio et al.,
3.5 3 Concentrations (mg/ml) 2.5 2 1.5 1 0.5 0 0 10 20 time (h) 30 40 Alpha-amylase activity (U/ml) Protein concentration (mg/ml) Biomass concentration (mg/ml)
Figure 5. Plot of Alpha-amylase activity, Protein Concentration, and Table 8. Yields and Productivity Biomass Concentration during course of fermentation Time (h) Yamylase/protein Yp/x Productivity 0 0.1067 1.4395 N/A The production of alpha-amylase 1 0.1064 1.2173 0.3165 was conducted in 500 ml of medium at 2 0.1075 1.6375 0.1637 pH 7 and optimum temperature of 37°C
(1978) reported protein, crude lipids, and carbohydrate contents of jackfruit seeds as 31.9%, 1.3%, and 66.2%, respectively. Kumar et al. (1988) also reported composition of seeds from two varieties of jackfruit. Protein, crude lipids, and carbohydrate content were 17.8-18.3%, 2.1-2.5%, and 76.1%, respectively. The protein content of Artocarpus heterophyllus reported by Bobbio et al. (1978) was very high; however the seeds were reported to have been collected from fruits of various varieties of jackfruit.
4 6 8 18 24 27 30
0.123 0.1763 0.2011 0.2009 0.2 0.2061 0.2057
1.1236 1.5442 2.1613 2.9968 2.0603 5.5492 1.7697
0.1011 0.0978 0.081 0.0366 0.0274 0.0247 0.0224
Arunava, B., Pal, S.C. and S.K. Sen (1993). Alpha-amylase production in lactose medium by Bacillus circulanse. J. Microbiologia., Volume 9 (2),142148. Awal, H. M. A. and S. Gheyasuddin (1991). Biochemical parameters of jackfruit seed-meal. Bangladesh Journal of Agricultural Research, Volume 16 (1), 17-22. Baines, B. S. and K. Brocklehurst (1979). A Necessary Modification to the Preparation of Papain from Any HighQuality Latex of Carica papaya and Evidence for the Structural Integrity of the Enzyme Produced by Traditional Methods. Biochem J., Volume 177 (2), 541-548. Bhat, A. V. and T. N. Pattabiraman (1989). Protease inhibitor from jackfruit seed (Artocarpus integrifolia). Journal of Biosciences, Volume 14 (4), 351-365. Bernfeld, P. (1955). Amylases, α, β, Methods in Enzymology. Volume 1, 149-155. Bobbio, F. O., El-Dash, A. A., Bobbio, P. A., and L. R. Rodrigues (1978). Isolation and characterization of the physico-chemical properties of the starch of jackfruit seed (Artocarpus heterophyllus). Cereal Chem, Volume 55, 501-511. Borchet T.V.F., Lassen, S. E., Svendsen, A. and H.B. Frantzen (1995). Oxidation stable amylase for detergent. J. Biotechnol., Volume 10,175-179. Burhan A., Nisa U., Gokhan C., Omer C., Ashabil A., Osman G. (2003). Enzymatic properties of a novel
Table 8 shows the yields and productivity in the course of fermentation. It can be seen that alphaamylase production was highest during exponential growth and was almost complete during the stationary phase. 4. Conclusion Alpha-amylase was successfully produced using jackfruit seed as solid substrate having a starch content of 37.68% in 100 grams seeds. The optimum conditions for alpha-amylase production were pH of 7, temperature of 37°C and C/N ratio of 1:2. Alphaamylase activity was highest during exponential growth at 4-8 hours of fermentation. Protein concentration was highest at 6 hours of fermentation. References Agriculture and Fisheries Information Science, Department of Agriculture, Philippines. Ajayi, I. A. (2008). Comparative study of the chemical composition and mineral element content of Artocarpus heterophyllus and Treculia africana seeds and seed oils. Bioresource Technology, Volume 99, 5125-5129. Anyangwa, E. M., Mapsev, C., Musanage, P. and M. Elemva (1993). The effect and removal of starch in the sugar refining industry. J. Int. Sugar., Volume 95, 210-213.
thermostable, thermophilic, alkaline, and chelator resistant amylase from an alkaliphilic Bacillus sp. Isolate ANT-6. Process Biochem, Volume 38 (10), 1397-1403. Castro, G. R., Biagori, M. D. and F. Sineriz (1999). Studies on alpha-amylase production by Bacillus licheniformis MIR-61. Acta. Biotechnol., Volume 19 (3), 263-272. De-Cordt, S., Vanhoof, K., Hu, J., Maesmans, G., and P. Tobback (1994). The influence of polyalcohols and carbohydrate on the thermostability of alpha-amylase. J. Bioeng., Volume 43 (2), 107-114. de Oliveira A. N., de Oliveira L. A., Andrade J. S., and A. F. C. Junior (2007). Rhizobia amylase production using various starchy substances as carbon substrates. Brazilian Journal of Microbiology, Volume 38, 208-216. Dharani Aiyer, P. V. (2004). Effect of C:N ratio on alpha-amylase production by Bacillus licheniformis SPT 27. African Journal of Biotechnology, Volume 3 (10), 519-522. Ednord, R. and K. Dietrich (1996). Kinetics of starch hydrolysis with Bacillus amyloliquefaciens alphaamylase under high hydrostatic pressure. J. Biotechnol., Volume 48 (11-12), 409414. Emanuilova, E. I. and K. Toda (1984). Alpha-amylase production in batch and continuous culture by Bacillus caldolyticus. Appl. Microbiol. Biotechnol., Volume 19, 301-305.
Ekunsaumi, T. Laboratory Production and Assay of Amylase by Fungi and Bacteria. UW-Washington country. Fukumoto, J., Yamamoto, T., and K. Ichikawa (1951). Crystallization of bacterial saccharogenic amylase and the properties of the cyrstalline amylase. Proc. Jap. Acad., Volume 27, 352-358. Fuwa, H. (1954). A new method for microdetermination of amylase activity by the use of amylose as the substrate. J. Biochem., Volume 41, 583-603. Harger, C., Sprada, D., and E. Hiratsuka (1982). Amilase Fungica. In: Bioquimica das Fermentacoes. 56. Haq, I., Ashraf, H., Zahara, R., and M. A. Qadeer (1998). Biosynthesis of alphaamylase by Bacillus subtilis GCB-12 using agricultural by products as substrates. Biologia, Volume 44 (1&2), 154-163. Haq, I., Ashraf, H., Zahara, R., and M. A. Qadeer (2003). Production of alphaamylase by Bacillus licheniformis using an economical medium. Bioresource Technology , Volume 87, 57-61. Haq, I., Ashraf, H., Iqbal, J.., and M. A. Qadeer (2005). Pearl millet, a source of alpha-amylase production by Bacillus licheniformis. Bioresource Technology , Volume 96, 1201-1204. Hendrickx, M., Tobback, P., Avila, I., and S. Cordt (1994). DSC and proteinbased time-temperature integrators: Case study of alpha-amylase stabilized by polyols and/or sugar. Biotechnol. Bioeng., Volume 4 (7), 859-865.
Ivanova, V., Yankov, D., Kabaivanova, L. and D. Pashkkoulov (2001). Simultaneous biosynthesis and purification of two extra cellular Bacillus hydrolases in aqueous two alpha-amylase. J. Biochem. Eng., Volume 8 (1), 61-81. Kathiresan K. and S. Manivannan (2006). Alpha-amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. African Journal of Biotechnology Volume 5 (10), 829-832. Khajeh, K., Naderi, H., Ranjbar, B., Moosavi, A., and M. Nemat (2001). Chemical modification of lysine residues in Bacillus alpha-amylases: Effect on activity and stability. Enzyme. Microbiol. Technol., Volume 28 (6), 543-549. Krishnan, T. and A. K. Chandra (1982). Effect of oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC 305. Appl. Environ. Microbiol., Volume 44 (2), 270-274. Krishnan, T. and A. K. Chandra (1983). Correlation between alpha-amylase production and sporulation in Bacillus licheniformis CUMC 305 with respect to the effect of some carbohydrates and phenylmethylsulfonyl flouride treatment. Zentralbl Mikrobiol., Volume 138 (6), 475-485. Kumar, S., Singh, A. B., Abidi, A. B., Upadhyah, R. G., and A. Singh (1988). Proximate composition of jackfruit seeds. J. Food Sci. Tech, Volume 25, 308-309. Madigan, Michael T., Martinko, John M., and Jack Parker (2003). Brock
Biology of Microorganisms. 10th Edition. Pearson Education, Inc. 980981. Nickless, D. M., Sobieski, R. J., and S. S. Crupper (2001). Genetic Regulation of Amylase Expression in Bacillus. Bioscene, Volume 27 (4), 27-29. Niziolek S. (1998). Production of extracellular amylolytic enzymes by some species of the genus Bacillus. Acta. Microbiologica. Polonica., Volume 47 (1), 19-29. Nomura, M., Moruo, B., and S. Akabor (1956). Studies on amylase fermentation by Bacillus subtilis. Effect of high concentration of polyetylene glycol on amylase formation by Bacillus subtilis. J. Biochem., Volume 43, 143. Odoemalam, S. A. (2005). Functional Properties of Raw and Heat Processed Jackfruit (Artocarpus heterophyllus). Pakistan Journal of Nutrition, Volume 4 (6), 366-370. Onyeike, E. N., Olungwe, T., and A. A. Uwakwe (1995). Effect of heat treatment and defatting on the proximate composition of some Nigerian local soup thickeners. Food Chem, 53, 173-175. Padmanabhan, S., Ramakrishna, M., Lonsane, B.K., and M. M. Krishnaiah (1992). Enhanced leaching of product at elevated temperatures: Alpha-amylase produced by Bacillus licheniformis M27 in solid state fermentation system. Lett. Appl. Microbiol., Volume 15 (6), 235238. Pandey, A. (1990). Aspects of Fermenter Design for Solid State Fermentation,
Process Biochemistry. Volume 26, 335361. Pandey, A., Nigam P., Soccol C. R., Soccol, V. T., Singh D., and R. Mohan (2000). Advances in microbial amylases. Biotechnol. Appl. Biochem, Volume 31, 135-152. Pandey, A., Webb, C., Soccol, C. R., and C. Larroche (2005). Enzyme Technology. New Delhi: Asiatech Publishers, Inc. 197. Pretorius, S., De-Kock, M. J. Britz, T. J., Potgieter, H. J. and P. M. Lategan (1986). Numerical taxonomy of alphaamylase producing strain of Bacillus species. J. Appl. Bacteriol., Volume 60 (4), 351-360. Pratima, B. and Umender, S. (1989). Production of alpha-amylase in a low cost medium by Bacillus licheniformis TCRC-B13. J. Ferment. Bio. Eng., Volume 67 (6), 422-423. Ramesh, M. V. and B. K. Lonsane (1990). Critical importance of moisture content of the medium in alpha-amylase production by Bacillus licheniformis M27 in a solid-state fermentation system. Appl. Microbiol, Biotechnol., Volume 33 (5), 501-505. Ramesh, M.V. and B. K. Lonsane (1991). Ability of solid state fermentation technique to significantly minimize catabolic repression of alphaamylase production by Bacillus licheniformis M26. Appl. Microbiol. Biotechnol., Volume 35 (5), 591-593. Rehana, F., Venkatasubbaiah, P. and K. Nand (1989). Preliminary studies on the production of thermostable alpha-
amylase by a mesophilic strain of Bacillus licheniformis. Chem. Mikrobiol. Technol. Lebensem., Volume 12 (1), 813. Rothstein, D. M., Devlin, P. E., and R. L. Cate (1986). Expression of alphaamylase in Bacillus licheniformis. American Society for Microbiology, Volume 168 (2), 839-842. Sai Annapurna S. and D. Siva Prasad (1991). Purification of trypsin/ chymotrypsin inhibitor from jackfruit seeds. Journal of the Science of Food and Agriculture, Volume 54 (3), 399411. Saito N. and K. Yamamoto (1975). Regulatory factors affecting alphaamylase production in Bacillus licheniformis. American Society for Microbiology, Volume 121 (3), 848-856. Shukla J. and R. Kar (2006). Potato peel as a solid substrate for thermostable alpha-amylase production by thermophilic Bacillus isolates. World Journal of Microbiology and Biotechnology, 417-422. Smith, B. W. and J. H. Roe (1949). A photometric method for the determination of alpha-amylase in blood and urine with the use of the starchiodine color. J. Biol. Chem., Volume 179, 53-56. Spier, M. R., Vandenberghe L. P. D. S., Woiciehowski A. L., C. R. Soccol (2006). Production and Characterization of Amylases by Aspergillus niger under Solid State Fermentation Using Agro Industrial Products. International Journal of Food Engineering, Volume 2 (3), 6.
Tulyathan V., Tananuwong K., Songjinda P., and N. Jaiboon (2002). Some Physicochemical Properties of Jackfruit (Artocarpus heterophyllus Lam) Seed Flour and Starch. Science Asia, Volume 28, 37-41. Vortruba, J., Emanuilova, E., Kaymakchiev, A. and J. Pazlarova (1984). Kinetics of alpha-amylase production in a continuous culture of Bacillus licheniformis. Folia. Microbiol., Volume 29 (1), 19-22. Weemaes, C., De-Cordt, S., Goossens, K., Ludikhuyze, L., Hedrickx, M., Heremans, K., and P. Tobback (1996). High pressure, thermal, and combined pressure temperature stabilities of alphaamylases from Bacillus species. Biotechnol. Bioeng. Volume 50 (1), 4956. Wilson, J. J. and W. M. Ingledew (1982). Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes. Appl. Env. Microbiol., Volume 44, 301-307.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue reading from where you left off, or restart the preview.