Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

Abstract

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A ␣-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 ␣-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation. Starch is an excellent carbon and energy source for many microorganisms, which employ a dedicated set of proteins for extracellular hydrolysis of this polysaccharide, uptake of shorter oligosaccharides into the cell, and further degradation into glucose. Most studies on degradation of starch by microbial enzymes have focused on soluble starch. This has resulted in the identification and characterization of a large variety of enzymes cleaving either ␣(1¡4) or ␣(1¡6) linkages in amylose and amylopectin. Most of these enzymes belong to the glycoside hydrolase 13 (GH13) family (1). Sequence diversity is such that, at the moment, the GH13 family contains a total of 40 subfamilies (1). Most of the new members in subfamilies are identified in DNA sequencing projects, and biochemical information about the activity and specificity of these potentially new enzymes is highly lagging.Many plants produce starch in a granular form for the storage of carbohydrates. The crystallinity of such granules varies with the plant source. Potato starch granules have a relatively high degree of crystallinity, making them notoriously resistant to bacterial and fungal degradation (2-4). Nevertheless, some microorganisms have been reported to employ enzymes that are able to digest granular starch (5, 6).Amylases found to be involved in granular starch degradation are often multidomain enzymes that include one or more carbohydrate binding modules (CBMs), which aid in the binding of the enzyme to the granular substrate (7-10).In previous work, various bacteria able to grow on potato starch granules as a carbon source were isolated, and their enzymatic degradation mechanism was evaluated. Initially, this resulted in the identification of an enzyme mechanism involving peeling off layer afte...

Raw starch degrading α-amylases: an unsolved riddle

“…Formation of pores on starch granules and activity on raw starch per se was a direct consequence of presence of the CBM25 domains, while it was possible to exclude the direct effect of the FNIII domains on substrate binding or enzyme activity. The 300 amino acid C-terminal tail seems to have functional role since its presence affected the size of pores significantly, whereas its absence clearly showed decrease in pores size on starch granules [88]. This novel SBD is a new CBM, the first representative of family CBM74 that undoubtedly assists MaAmyA in raw starch degradation [89].…”

“…Peculiar α-amylase MaAmyA from Microbacterium aurum B8.A belongs to subfamily GH13_32 is forming pores in starch granules [88]. M. aurum strain B8.A was isolated from the sludge of a potato starch-processing factory [58].…”

The evolutionary origin and possible functional roles of FNIII domains in two Microbacterium aurum B8.A granular starch degrading enzymes, and in other carbohydrate acting enzymes

“…Only in a small number of CBM families (13 out of 66 families), the majority of the CBM members is not located at one of the protein Figure 3. Alignment of all 24 bacterial FNIII domains explicitly reported in literature [2,4,6,15,16,34,36,[38][39][40][41][42][43], in carbohydrate acting enzymes. Sequences were extracted as described in the Methods section, except for the two "FNIII-like domains" from C. thermocellum CbhA (GenBank: CAA56918.1), which were not recognized as such by the domain annotation tools, and were therefore extracted manually [15].…”

“…MaAmyA (148 kDa) carries two carbohydrate binding modules (CBM25), four fibronectin type III (FNIII) domains and one CBM74 ( Fig. 1) [2,3]. Characterization of MaAmyA mutant proteins with C-terminal deletions of various lengths showed that the CBM25 domains are essential for the ability of this enzyme to degrade raw starch.…”

“…We used both bioinformatics approaches as well as the available literature to analyse the possible roles of FNIII domains in carbohydrate acting enzymes. [2], MaAmyB [3] and the general domain organization of GH13_42 members. Numbers refer to the number of amino acids in the protein.…”