AmyJ33, a truncated amylase with improved catalytic properties.

Biochemical and kinetic properties are of special interest for the specific applications of α-amylases in industrial sectors such as textile industries, detergents, biofuels and food among others. Therefore, protein engineering is currently directed towards a continuous demand to improve the properties of amylases and thus meet the specific characteristics for various industrial sectors. In the present work, modular protein engineering was performed to improve the biochemical and kinetic properties of AmyJ33r an α-amylase isolated from Bacillus siamensis JJC33M consisting of five domains, A, B, C, D and E (SBD) (Montor-Antonio et al. in 3 Biotech 7:336, 2017. https://doi.org/10.1007/s13205-017-0954-8 ). AmyJ33r is not active on native starch, only showing activity on gelatinized starch. At the C-terminal, AmyJ33r has a starch binding domain (SBD, domain E) belonging to the CBM26 family. In this study, four truncated versions were constructed and expressed in E. coli (AmyJ33-AB, AmyJ33-ABC, AmyJ33-ABCD, and SBD) to determine the role of the A, B, C, D, and E domains in the biochemical behavior of AmyJ33r on starch. Biochemical and kinetic characterization of the truncated versions showed that domain C is essential for catalysis; domain D improved enzyme activity at alkaline pH values, is also involved negatively in thermostability at 40, 50, and 60 °C and its presence favored the production of maltooligosaccharides with a higher degree of polymerization (DP4). E domain have interaction with raw starch, also the deletion of E domain (SBD) favors the affinity for the substrate while the deletion of D domain increased enzyme kcat at the time of product release. In conclusion, AmyJ33-ABC has better kinetic parameters than AmyJ33-ABCD and AmyJ33r, but is less stable than these two enzymes.

Effect of differential processing of the native and recombinant α-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties.

AmyJ33, an α-amylase isolated from Bacillus amyloliquefaciens JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.