www.sciencejournal.in Vol. 1 No. 1 (2012) ISSN 2320–0421(Print); ISSN 2320–043X(Online) © 2012 DAMA International. All rights reserved. 49 α-AMYLASE FROM SUGARCANE WOOLLY APHID (CERATOVACUNA LANIGERA ZEHNTNER) Padul M.V.*, Chougale A.D.***, Dama L.B. ****, Gadge P.P. *, Shaikh F.K.*, Chopde R.M. ** and Vadane S.S. ** *Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, (M.S.), India. **Post Graduate Department of Biochemistry, New Arts, Commerce and Science College, Ahmednagar, (M.S.), India. ***Mass spectrometry and Proteomics group, Organic Chemistry Division, National Chemical Laboratory, Pune 411 008, (M.S.), India. ****Department of Zoology, D.B.F. Dayanand College of Arts and Science, Solapur-413002, (M.S.), India. (E-Mail: manoharpadul@yahoo.co.in) ABSTRACT Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane which is responsible for the low production of the sugarcane in India. The incidences of pest itself suggest importance of study on this pest. Carbohydrate metabolizing digestive enzyme, α-amylase was studied from this insect. Biochemical characterization of this enzyme and screening of natural inhibitors will prove vital strategies against infestation. As initial step to this goal here we report the detection of α-amylase of Ceratovacuna lanigera by starch agar well diffusion assay. Biochemical parameters like pH, temperature and effect of substrate concentration were studied and compared with α-amylases from other sources. The optimum pH of α-amylase of Ceratovacuna lanigera was found to be pH 8 in contrast to human salivary α-amylases and fungal α-amylases those shows optimum activity at pH 7 and pH 6 respectively. Maximum activity of α-amylase was found to be at 40°C. Protein profile by Native - PAGE showed banding pattern with varied intensities. The α-amylase of Ceratovacuna lanigera detected in the present study needs further exploration for its use in better management of sugarcane woolly aphid. KEY WORDS: α-amylase, Native-PAGE, Sugarcane, woolly aphid. INTRODUCTION Sugar is a universal sweetening agent and sugarcane (Saccharum officinarum L.) is the primary age old source of it. Sugarcane is damaged by about 288 species of insects and non-insets (David and Nandagopal, 1986) and tissue borers, white grubs, white flies, rodents, mealy bugs, pyrilla, scale insects etc. in which Raychaudhuri (1984) listed 17 species of aphids associated with sugarcane. Sugarcane woolly aphid ( Ceratovacuna lanigera Zehnter) is well known but comparatively less studied pest. The sugarcane woolly aphid was first reported from west Bengal in 1958 and later from other parts of India. Recently, in Maharashtra state during July-2002, an epidemic of sugarcane woolly aphid was noticed in Sangli, Kolhapur and Satara districts and later on spread in parts of Solapur, Pune and Ahmednagar districts. The aphid undergoes an anholocyclic life cycle on Poaceae (Joshi and Viraktamath, 2004). This aphid which constitute serious pest of sugarcane is dependent on their α-amylases for metabolism of carbohydrate. A detailed understanding of digestive α-amylases is essential when developing methods of insect pest control. Hence current study deals with the detection and biochemical characterization of α-amylase of the C. lanigera. This information may be exploited for planning the strategies for the better management of sugarcane woolly aphid. MATERIALS AND METHODS Collection of the Ceratovacuna lanigera Ceratovacuna lanigera Zehnter was collected from the affected sugarcane field located at Rahuri, district Ahmednagar. Collection was made in the dry petri plates and preserved in freeze condition until use. Extraction of enzyme Two gram Ceratovacuna lanigera insects were crushed in 10 ml physiological saline, after complete homogenization the homogenate was centrifuged at 15000 rpm for 30 min at 4°C. Clear supernatant was used as source of α-amylase. Concentration of protein was determined by Lowry method (Lowry et al., 1951). Detection of enzyme activity Clear supernatant obtained was screened for α-amylase activity by starch agar plate method. In this method agar plate containing 2 % starch was used. The various combinations of enzyme and buffer poured in wells of the agar plate, incubated at 37°C for 30 min. Plate was stained with iodine solution and zone of clearance (α -amylase activity) was observed visually. Estimation of α-amylase activity The α-amylase activity was determined by measuring the formation of reducing sugars when the crude supernatant was incubated with starch. The standard reaction mixture contained 0.2M sodium phosphate buffer (pH 7.0), 1 % starch and