Enzyme and Microbial Technology 90 (2016) 19–25 Contents lists available at ScienceDirect Enzyme and Microbial Technology j o ur na l ho mepage: www.elsevier.com/locate /emt Overexpression and secretion of AgaA7 from Pseudoalteromonas hodoensis sp. nov in Bacillus subtilis for the depolymerization of agarose Kristine Rose M. Ramos a,1 , Kris Ni˜ no G. Valdehuesa a,1 , Rhudith B. Cabulong a , Llewelyn S. Moron a , Grace M. Nisola a , Soon-Kwang Hong b , Won-Keun Lee b,∗ , Wook-Jin Chung a,∗ a Energy and Environment Fusion Technology Center (E 2 FTC), Department of Energy Science and Technology (DEST), Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin-si, Gyeonggi-do 17058, Republic of Korea b Division of Bioscience and Bioinformatics, Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin-si, Gyeonggi-do 17058, Republic of Korea a r t i c l e i n f o Article history: Received 28 October 2015 Received in revised form 18 February 2016 Accepted 19 April 2016 Available online 21 April 2016 Keywords: Agarase Protein secretion Signal peptide a b s t r a c t Interest in agar or agarose-based pharmaceutical products has driven the search for potent agarolytic enzymes. An extracellular -agarase (AgaA7) recently isolated from Pseudoalteromonas hodoensis sp. nov was expressed in Bacillus subtilis, which was chosen due to its capability to overproduce and secrete functional enzymes. Phenotypic analysis showed that the engineered B. subtilis secreted a functional AgaA7 when fused with the aprE signal peptide (SP) at the amino-terminus. The maximum agarolytic activity was observed during the late logarithmic phase. To further improve the secretion of AgaA7, an expression library of AgaA7 fused to different naturally occurring B. subtilis SPs was created. The amount of AgaA7 secreted by the clones was compared through activity assay, immuno-blot, and purification via affinity chromatography. Although the aprE SP can readily facilitate the secretion of AgaA7, other SPs such as yqgA, pel, and lipA were relatively more efficient. Among these SPs, lipA was the most efficient in improving the secretion of AgaA7.The use of B. subtilis as host for the expression and secretion of agarolytic and other hydrolytic enzymes can be a useful tool in the field of white biotechnology. © 2016 Elsevier Inc. All rights reserved. 1. Introduction Agarose, which is made of alternating units of d-galactose and 3,6-anhydro-l-galactose (l-AHG), is one of the main compo- nents of marine red macroalgae [1]. Its high content (52 wt% dry basis) in Gelidium amansii makes it a viable biomass feedstock of fermentable d-galactose for the synthesis of biofuels and other fine chemicals [1–4]. Furthermore, agar- or agarose-derived com- pounds have various applications in the food, cosmetic, and medical industries [5,6]. Saccharification of agarose into its monomeric units requires several agarolytic enzymes. Agarases are generally classified according to their cleavage pattern. -agarases cleave -1,4 glycosidic linkages producing neoagarooligosaccharides with d- ∗ Corresponding authors. E-mail addresses: wklee@mju.ac.kr (W.-K. Lee), wjc0828@gmail.com (W.-J. Chung). 1 These authors contributed equally to this work. galactose at the reducing end. On the other hand, -agarases cut the -1,3 bonds releasing agarooligosaccharides as products with L-AHG at the reducing end [7,8]. Recently, a thermostable -agarase (AgaA7) was isolated from Pseudoalteromonas hodoensis sp. nov. Molecular and biochemical tests showed that AgaA7 has a molec- ular weight of ∼30–35 kDa and exhibits optimal activity at pH 7.0 and 45 ◦ C. Additionally, AgaA7 has both exo- and endolytic activities that depolymerize agarose into neoagarotetraose, neoa- garohexaose, and neoagarooctaose (Fig. 1) [9]. Majority of the isolated agarases have been cloned in the Gram- negative Escherichia coli [4,5,10,11]. E. coli has been commonly used as an expression host due to its fast growth and expres- sion, high product yield, and ease in genome modification. But problems associated with inclusion bodies formation and protein misfolding during expression and protein purification can occur [12]. The Gram-positive Bacillus subtilis has also been used for the mass production of proteins [13,14]. It is generally regarded as safe (GRAS) and its genetic manipulation tools are well established. The most notable property of B. subtilis is its ability to efficiently secrete functional proteins due to the absence of an outer mem- http://dx.doi.org/10.1016/j.enzmictec.2016.04.009 0141-0229/© 2016 Elsevier Inc. All rights reserved.