45 ALGINATE LYASE FROM INDONESIAN Bacillus megaterium S245 SHOWS ACTIVITIES TOWARD POLYMANNURONATE AND POLYGULURONATE Subaryono 1 , Yuwanita Ardilasari 2 , Rosmawaty Peranginangin 1 , Fransisca Rungkat Zakaria 2 , and Maggy Thenawidjaja Suhartono 2* 1 Research and Development Center for Marine and Fisheries Product Competitiveness and Biotechnology Jalan KS Tubun Petamburan VI, Central Jakarta, Indonesia 10260; 2 Departement of Food Science and Technology, Bogor Agriculture University, Jl. Raya Dramaga, Kampus IPB, Bogor, Indonesia 16680 Abstract Screening of alginate lyase producing bacteria associated with seaweed Sargassum crassifolium was carried out, and isolate S245, identified as Bacillus megaterium S245 was found to produce high alginate lyase activity. This research was conducted to evaluate activity of the alginate lyase enzyme at various pHs, temperatures and substrates. Polymannuronate and polyguluronate were used to evaluate substrate specificities. Alginate lyase activity was assayed by analysis of reducing sugar released using the 3,5 dinitrosalicylic acid (DNS) method. The research showed that the activity of alginate lyase was optimum at pH of 7.0 and temperature of 45 0 C. This enzyme was active for both polymannuronate and polyguluronate susbtrates. The V max and K m of this enzyme for polymannuronate and polyguluronate were 200 unit/ml/min and 79.8 mg/ml for polymannuronate substrate and 27.78 unit/ml/min and 13.17 mg/ml for polyguluronate substrate. This enzyme showed unique characteristic in working toward the two substrates. Keywords: alginate lyase, seaweed, Bacillus, kinetic activity, Sargassum sp. Article history: Received: 15 May 2016; Revised: 3 July 2016; Accepted: 25 July 2016 Squalen Bull. of Mar. and Fish. Postharvest and Biotech. 11 (2) 2016, 45-52 www.bbp4b.litbang.kkp.go.id/squalen-bulletin Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology ISSN: 2089-5690 e-ISSN: 2406-9272 *Corresponding author. E-mail: mthenawidjaja@yahoo.com Copyright © 2016, Squalen BMFPB. Accreditation Number: 631/AU2/P2MI-LIPI/03/2015. DOI: http://dx.doi.org/10.15578/squalen.v11i2.250 1. Introduction Alginate lyase or alginase (EC4.2.2.3 and EC4.2.2.11), an enzyme that catalyze the degradation of alginate by a -elimination mechanism has been isolated from various sources, such as soil bacteria, marine bacteria, marine mollusks and crustaceae, brown seaweed and fungi (Guan, Wang & Guo, 2011 b ; Li et al., 2011 a ; Li, Jiang, Matiur, Ling, Akira & Takao, 2012; Subaryono, Peranginangin, Suhartono & Zakaria, 2013). The alginases from alginolytic bacteria, included those of soil and marine bacteria have been isolated from Pseudomonas sp. QD03 (Xiao, Han, Yang, Xin-zhi and Wen-gong, 2006), Pseudoalteromonas atlantica AR06 (Matsushima et al., 2010), Pseudoalteromonas sp. SM0524 (Li et al., 2011 a ), Pseudomonas fluorescens HZJ216 (Li, Jiang, Guan, Wang & Guo, 2011 b ), Stenotrophomas maltophilia KJ-2 (Lee, Choi, Lee & Kim, 2012), Bacillus subtitlis (El Ahwany & Elborai, 2012), and Flavobacterium sp. S20 (Lishuxin, Jungang, Qiang, Hong & Yuguang, 2013). Based on their substrate specificity, alginate lyases are classified into poly (- D-mannuronate) lyases (EC4.2.2.3) and poly (-L- guluronate) lyases (EC 4.2.2.11). Examples of alginate lyases with poly (-D-mannuronate) as substrate are produced from Azotobacter chroococcum (Khanafari & Sepahei, 2007), Azotobacter vinelandii (Gimmestad et al., 2009), Littorina spp (Matiur, Ling, Akira & Takao, 2012), and Pseudomonas aeruginosa (Rasamivaraka, Labtani, Duez & Jaziri, 2015). On the oher hand, alginate lyase from Klebsiella pneumoniae (Baron et al., 1994), Vibrio sp. 510 (Hu, Jiang & Hwang, 2006) and Enterobacter cloacae WD 7 (Prasertsan, Wichienchot, Doelle & Kennedy, 2008), showed specificity toward poly ( -L-guluronate) lyases. Another alginate lyase with the two substrates specificity were produced by P. alginovora strain XO17 and Sphingomonas sp. strain A1. Hashimoto, Miyake, Momma, Kawai and Murata (2000) also reported alginate lyase from soil bacteria Sphingomonas sp.