Biochemical Characterization of Alpha-amylase from the Digestive Fluid of Larvae of the Rhino Beetle, Oryctes owariensis
Keywords:
Oryctes owariensis, alpha-amylase, Enzyme, monomeric protein, Energy production, Starch saccharificationAbstract
Alpha-amylase was purified from the digestive fluid of larvae of the Rhino Beetle (Oryctes owariensis) by chromatography on ion-exchanges and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gel. The enzyme was purified 260.20-fold to a specific activity of 128.50 (U/ mg of protein) and an overall yield of 26.22 %. SDS-PAGE revealed a single polypeptide of 53.8 ± 1.2 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography showed that the enzyme was a 50.4 ± 2.2 kDa monomeric protein. The purified enzyme exhibited pH and temperature optima at 7.0 and 55°C, respectively. The alpha-amylase was stable at 37 °C and its pH stability was in the range of 6.6-7.6. This enzyme exhibited a high affinity towards soluble starch with Km values of 0.6 ±0.04. The alpha-amylase activities were stimulated by Ca2+, K+, Mg2+ and Na+ and were inhibited by EDTA, Fe2+ and Cu2+. The analysis of hydrolytic products after soluble starch hydrolysis by alpha-amylase revealed that maltose, maltotriose and maltotetraose were the major products. This indicated that this enzyme can be classified as the alpha-amylase (the endoamylase). From a physiological point of view, alpha-amylase play a fundamental role in energy production for this insect larvae. Consequently, the digestive tract of this larva could be a good source of alpha-amylase for starch saccharification.
References
Henrissat B., Deleury E., Coutinho P.M., Glycogen metabolism loss: a common marker of parasitic behaviour in bacteria, Trends in Genetics, vol. 18, pp. 437-440, 2002.
Da Lage J., Danchin E., and Casane, D., Where do animal α-amylases come from? An interkingdom trip, FEBS Letters, Vol. 581, no. 21, pp. 3927-3935, 2007.
Franco O.L., Rigden D.J., Melo F.R., and Grossi-De-Sa, M.F., Plant α-amylase inhibitors and their interaction with insect α-amylases structure, function and potential for crop protection, European Journal of Biochemistry, Vol. 269, pp. 397-412, 2002.
Souza P.M., and Magalhães, P.O., Application of microbial alpha-amylase in industry. Brazilian Journal of Microbiology, Vol. 41, pp. 850-861, 2010.
Reddy N.S., Nimmagadda A., and Sambasiva Rao, K.R.S., An overview of the microbial alpha-amylase family, African Journal of Biotechnology, Vol. 2,pp. 645-648, 2003.
Rajagopalan G., and Krishnan, C., Alpha-amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate, Bioresource Technology, Vol. 99, pp. 3044-3050, 2008.
Sivaramakrishnan S., Gangadharan D., Nampoothiri K.M., Soccol C.R., and Pandey, A., alpha-amylases from microbial sources, An overview on recent developments, Food Technology and Biotechnology, Vol. 44, pp. 173-184, 2006.
Utong J., Al-Quadan F., and Akel, H., Effect of various growth conditions on extracellular amylase from thermotolerant Bacillus species isolated from hot springs in Jordan, Journal of Biological Sciences, Vol. 6, pp. 621-625, 2006.
Buonocore V., Poerio E., Silvano V., and Tomasi, M., Physical and catalytic properties of alpha-Amylase from Tenebrio molitor L. Larvae, Journal of Biochemistry, Vol. 153, pp. 621-625, 1976.
Baker, J.E., Purification and partial characterization of alpha-amylase alloenzymes from the lesser grain borer, Rhizopertha dominica, Insect Biochemistry, Vol. 21, pp. 303-311, 1991.
Mendiola-Olaya E., Valencia-Jiménez A., Valdes-Rodriguez S., Delano-Frier J., and Branco-Labra, A., Digestive amylase from the larger grain borer, Prostephanus truncatus Horn, Comparative Biochemistry and Physiology, Vol. 126 B, pp. 425-433, 2000.
Zeng F., and Cohen, A.C., Partial characterization of alpha amylase in the salivary glands of Lygus Hesperus and L. lineolaris, Comparative Biochemistry and Physiology, Vol. 126B, pp. 9-16, 2000.
Pelegrini P.B., Murad A.M., Grossi-De-Sa M.F., Mello L.V., Roméiro L.A.S., Noronha E.F., Caldas R.A., and Franco, O.L., Structure and Enzyme Properties of Zabrotes subfasciatus alpha-amylase, Archives of Insect Biochemistry and Physiology, Vol. 61, pp. 77-86, 2006.
Dojnov B., Bozic N., Nenadovic V., Ivanovic J., and Vujcic, J., Purification and properties of midgut amylase isolated from Morimus funereus (Coleoptera: Cerambycidae), Comparative Biochemistry and Physiology, Vol. 149 B, pp. 153-160, 2008.
Dué E.A., Kouadio J.P.E.N., Kouakou H.T., Dabonné S., Niamké S.L. and Kouamé, L.P., Purification and physicochemical properties of alpha amylase from cockroach, Periplaneta americana (LINNAEUS), for starches saccharification, African Journal Biotechnology, Vol. 7, pp. 2707-2716, 2008.
Kouadio E.J.P., Ahipo D.E., Etchian O.A., Shaw J., and Kouamé, L.P., Purification and Characterization of Two Dimeric alpha-amylases from Digestive tract in the tropical house cricket Gryllodes sigillatus (Orthoptera: Gryllidae), Australian Journal Of Basic And Applied Sciences, Vol. 4 no. 10, pp. 5241-5252, 2010.
Vidyasagar P.S.P.V., Hagi M., Abozuhairah R.A., Al-Mohana O.E., and Al-Saihati, A., Impact of mass pheromone trapping on the red palm: Adult population and infested level in date palm gardens of Saudi Arabia, The Planter, Vol. 76, pp. 347-355, 2000.
Hoyt, C.P., Investigations of Rhinoceros Beetles In West Africa, Pacific Science, Vol.17, pp. 444-451, 1963.
Fasoranti J.O., and Abijoye, D.O., Some edible insects of Kwara state Nigeria, American Entomologist, Vol. 39, pp. 113-116, 1993.
Mohan, C., Global Invasive Species Database, Oryctes rhinoceros,Accessed May 17, 2012 from: http://www.issg.org/database/species/ecology.asp?si=173. 2005.
Womeni H.M., Linder M., Tiencheu B., Mbiapo F.T., Villeneuve P., Fanni J., and Parmentier, M., Oils of Oryctes owariensis and Homorocoryphus nitidulus consumed in Cameroom: sources of linoleic acid, Journal of Food Technology, Vol. 7, no.2, pp. 54-58, 2009.
Bernfeld, P., Amylases α and β, In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in enzymology, Academic Press, New York, Vol. 1, pp. 149-158, 1995.
Lowry O.H., Rosebrough N.J., Farr A.L., and Randall, R.J., Protein measurement with the folinphenol reagent, Journal of Biological Chemistry, Vol. 193, pp. 265-275, 1951.
Laemmli, U.K., Cleavage of structural proteins during the assembly of head of bateriophage T-4, Nature, Vol. 227, pp. 680-685, 1970.
Blum, H., Beier H., and Gross, B., Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels, Electrophoresis, Vol. 8, pp. 93-99, 1987.
Sivakumar S., Mohan M., Franco O.L., and Thayumanavan, B., Inhibition of insect pest α-amylases by little and Winger millet inhibitors, Pesticide Biochemistry and Physiology, Vol. 85, pp. 155–160, 2006.
Wisessing A., Engkagul A., Wongpiyasatid A., and Chuwongkomon, K., Purification and Characterization of Callosobruchus maculatus alpha-amylase, Kasetsart Journal (Natural Science), Vol. 42, pp. 240-244, 2008.
Campos F.A.P., Xavier-Filho J., Silva C.P., and Ary, M.B., Resolution and partial characterization of proteinases and alpha-amylases from midguts of larvae of the bruchid beetle Callosobruhus maculates (F.), Comparative Biochemistry and Physiology, Vol. 92B, pp. 51-57, 1989.
Lemos F.J.A., Campos F.A.P., Silva C.P., and Xavier-Filho, J., Proteinases and amylases of larval midgut of Zabrotes subfasciatus reared on cowpea (Vigna unguiculata) seeds, Entomological Experiences Applied, Vol. 56, pp. 219-227, 1990.
Mehrabadi M., and Bandani, A.R., Study of salivary gland α-amylase in wheat bug Eurygaster maura (Hemiptera: Scutelleridae), American Journal of Applied Sciences, Vol. 6, pp. 555-560, 2009.
Cinco-Moroyoqui F.J., Diaz-Malvaez F.L., Alanis-Villa A., Baron-Hoyos J.M., Cardenas-Lopez J.L., Cortez-Rocha M.O., and Wong-Corral, F.J., Isolation and partial characterization of three isoamylase of Rhyzopertha dominca F. (Coleoptera: Bostrichidae), Comparative Biochemistry and Physiology, Vol. 150 B, pp. 153-160, 2008.
Hamilton L.M., Kelly C.T., and Fogarty, W.M., Production and properties of the raw starch-digesting a-amylase of Bacillus sp. IMD 435, Process Biochemistry, Vol. 35, pp. 27–31, 1999.
Ben Abdelmalek-KhedherI. M.C., UdarciF., Limam J.M., Schmitter M.N., Marzouki and Bressollier, P., Purification, characterization, and partial primary sequence of a major-maltotriose producing α-amylase, Sc Amy 43; from Sclerotinia Sclerotiorum, Journal of Microbiology and Biotechnology, Vol. 18, pp. 1555-1563, 2008.
Hagihara H., Igarashi K., Hayashi Y., Endo K., Ikawa-Kitayama K., Ozaki K., Kawal S., and Ito, S., Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38, Applied and Environmental Microbiology, Vol. 67, pp. 1744-1750, 2001.
Murakami S., Nishimoto H., Toyama Y., Shimamoto E., Takenaka S., Kaulpiboon J., Prousoontorn M., Limpaseni T., Pongsawasdi P., and Aoki, K., Purification and characterization of two alkaline, thermotolerant α-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli, Bioscience Biotechnology and Biochemistry, Vol. 71, pp. 2393-2401, 2007.
Duedahl-Olesen L., Kragh K.M., and Zimmermann, W., Purification and characterization of a maltooligosacharide-forming amylase active at hight pH from Bacillus claussii BT-21, Carbohydrate Research, Vol. 329, pp. 97-107, 2000.
Takeuchi A., Shimuzu-Ibuka A., Nishiyama Y., Mura K., Okada S., Tokue C., and Arai, S., Purification and characterization of an alpha-amylase from Pichia burtonii isolated from the traditional starter “Murcha†in Nepal, Bioscience Biotechnology and Biochemistry, Vol. 70, pp. 3019-3024, 2006.
Downloads
Published
Issue
Section
License
- Papers must be submitted on the understanding that they have not been published elsewhere (except in the form of an abstract or as part of a published lecture, review, or thesis) and are not currently under consideration by another journal published by any other publisher.
- It is also the authors responsibility to ensure that the articles emanating from a particular source are submitted with the necessary approval.
- The authors warrant that the paper is original and that he/she is the author of the paper, except for material that is clearly identified as to its original source, with permission notices from the copyright owners where required.
- The authors ensure that all the references carefully and they are accurate in the text as well as in the list of references (and vice versa).
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Attribution-NonCommercial 4.0 International that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
- The journal/publisher is not responsible for subsequent uses of the work. It is the author's responsibility to bring an infringement action if so desired by the author.
