Process Biochemistry 50 (2015) 1572–1580 Contents lists available at ScienceDirect Process Biochemistry jo u r n al homep age: www.elsevier.com/locate/procbio Modulation of transglycosylation and improved malto-oligosaccharide synthesis by protein engineering of maltogenic amylase from Bacillus lehensis G1 Nor Hasmaliana Abdul Manas a , Mohd Anuar Jonet b , Abdul Munir Abdul Murad c , Nor Muhammad Mahadi b , Rosli Md. Illias a,∗ a Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia b Comparative Genomics and Genetics Research Centre, Malaysia Genome Institute, Kajang, Selangor, Malaysia c School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia a r t i c l e i n f o Article history: Received 2 April 2015 Received in revised form 4 June 2015 Accepted 8 June 2015 Available online 18 June 2015 Keywords: Maltogenic amylase Transglycosylation Malto-oligosaccharide Site-directed mutagenesis Protein engineering a b s t r a c t Malto-oligosaccharide synthesis using maltogenic amylase often struggles with product re- hydrolyzation. The malto-oligosaccharide synthesis using a maltogenic amylase (MAG1) from Bacillus lehensis G1 was enhanced using a structure-guided protein engineering approach. Mutations decreased the hydrolysis activity of the enzyme and caused various modulations in its transglycosylation properties. W359F, Y377F and M375I mutations caused a reduction in steric interference, an alteration of subsite occupation and an increase in internal flexibility to accommodate longer donor/acceptor molecules for transglycosylation, resulting in an increase in the transglycosylation to hydrolysis ratio of up to 4.0-fold. The increase in active site hydrophobicity that was caused from the W359F and M375I mutations reduced the concentration of maltotriose required for use as a donor/acceptor for transglycosylation to 100 mM and 50 mM, respectively, compared to the 200 mM needed for wild-type. An improvement of the trans- glycosylation to hydrolysis ratio by 4.2-fold was also demonstrated in each of the mutants. Interestingly, a reduction of steric interference and hydrolysis suppression was caused by the Y377F mutation and introduced a synergistic effect to produce malto-oligosaccharides with a higher degree of polymeriza- tion than wild-type. These findings showed that modification of the active site structure imposed various effects on MAG1 activities during malto-oligosaccharide synthesis. © 2015 Published by Elsevier Ltd. 1. Introduction Maltogenic amylase (glucan-1,4-alpha-maltohydrolase EC 3.2.1.133) is an amylolytic enzyme from glycosyl hydrolase family 13 (GH 13) that exhibits multi-substrate specificity and multi-functional catalysis. Unlike typical -amylases, maltogenic amylases and their homologous enzymes (i.e., cyclomaltodextri- nase and neopullulanase) display the highest hydrolytic affinity toward cyclodextrins (CDs), followed by starch and pullulan. In addition to hydrolysis, these enzymes also demonstrate synthesis activity known as transglycosylation to form sugar molecules of various lengths [1]. Due to these unique properties, maltogenic amylase and related enzymes have been the subjects of extensive research. ∗ Corresponding author at: Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. E-mail address: r-rosli@utm.my (R.Md. Illias). Crystal structures of maltogenic amylase and homologous enzymes have been elucidated, thus enabling researchers to under- stand the architecture of the enzyme’s catalytic machinery, which accommodates more distinct types of substrates than typical - amylases and performs two utterly contradictory biochemical reactions. It has been observed that maltogenic amylase pos- sesses an extra N-terminal domain, which is absent in -amylases. This N-terminal domain is responsible for the dimerization of the enzyme through its interactions with an (/) 8 -barrel domain on the partner monomer. Therefore, the dimer interface forms a narrow and deep active site cleft that is responsible for the prefer- ence of the enzyme toward small molecules, such as cyclodextrins, versus starch. On the other hand, it was discovered that extra space is present at the bottom of the active site cleft, which could accommodate small acceptor sugar molecules for transgly- cosylation reactions [2]. From this finding, it is now understood how two different catalytic activities (i.e., hydrolysis and trans- glycosylation) can occur within the same active site of the enzyme. http://dx.doi.org/10.1016/j.procbio.2015.06.005 1359-5113/© 2015 Published by Elsevier Ltd.