Glucoamylase 1 from Aspergillus niger is an economically important enzyme in many industrial processes. It hydrolyses granular starch and comprises two distinct domains, a catalytic and a starchbinding domain (SBD). We have transformed A. niger with an expression vector for the secretion of SBD for physico-chemical studies. This was achieved by introducing into the glucoamylase gene a short sequence encoding an endoproteolytic cleavage recognition site such that free SBD was secreted at yields up to 200 mg/l. Free SBD was also obtained by proteolytic digestion of full-length glucoamylase 1. Electrospray mass spectroscopy was used to determine the carbohydrate content of both SBDs. It revealed that the engineered one is more glycosylated: an average of three mannose residues compared to one for the proteolytically derived SBD. Sequencing results also suggest partial glycosylation for the three Thr residues involved (510, 51 1, 513). It is probable that the engineered SBD represents the true glycosylation level of the SBD in native glucoamylase. Binding of b-cyclodextrin to the SBD was investigated. It was found that the stoichiometry and the spectral perturbation of Trp residues were identical for both SBDs, but the engineered SBD bound less strongly to the ligand. At high concentrations of p-cyclodextrin relative to the estimated Kd values, the maximum absorbance changes were identical. The observed difference at low P-cyclodextrin levels was probably due to the higher level of glycosylation of the expressed SBD. We conclude that the proteolytically derived and expressed starch binding domains both bind 2 mol /I-cyclodextrinlmol protein, but that the pattern of glycosylation and strength of binding are different.Keywords: glucoamylase 1 ; starch-binding domain ; Aspergillus niger; heterologous expression ; carbohydrate binding.Glucoamylase 1 (GI : 1,4-<1-D-ghCan glucohydrolase, EC 3.2.1.3) from Aspergillus niger catalyses the release of p-D-glucose from the non-reducing ends of starch and related polysaccharides and oligosaccharides. It comprises two domains : one is a catalytic domain (residues 1-470) and the other (residues 509-616) is responsible for binding granular starch. They are linked by a region rich in 0-glycosylated Ser and Thr (residues 471 -508). The starch-binding domain (SBD) has been studied following its purification after proteolytic cleavage from fulllength glucoamylase (Belshaw and Williamson, 1990). It is known that free SBD retains binding activity against granular starch, can unfold reversibly and contains two binding sites for p-cyclodextrin. By proteolysis, yields of SBD are low becauseCorrespondence to
'H and "N NMR resonance assignments of the granular starch-binding domain (SBD) of glucoamylase from Aspergillus riiger have been made by multi-dimensional homonuclear and heteronuclear NMR techniques. Secondary structure analysis based on chemical shifts, 'H-'H NOES, coupling constants and backbone amide exchange data indicates the presence of a well-defined /J-sheet structure. This consists of one parallel and five antiparallel pairs of /I-strands forming two /I-sheets. Cis-trans isomerisation of proline residues and 0-glycosylation of threonine residues are observed and compared between the proteolytically derived SBD fragment and the recombinant protein. Structural features of the SBD in solution were compared to the X-ray crystal structure of a homologous domain of cyclodextrin glycosyltransferase from Bacillus circulans. There are some differences in the locations of the start and end of /J-strands but overall the two structures are very similar. This study will form the basis for the structure determination of the granular SBD and of its complexes.Keywords : glucoamylase 1 : starch-binding domain : Aspergillus niger; NMR ; secondary structure.Glucoamylase 1 (G1; 1,4-a-D-ghcan glucohydrolase) from Aspergillus niger is an exo-acting enzyme which catalyses the hydrolysis of a-D-glucosidic bonds of starch and other polysaccharides to produce ,fi-D-glucose. Overall, G I consists of a catalytic domain (residues 1 -470), an 0-glycosylated linker (residues 471 -508) and a granular starch-binding domain (SBD; residues 509-616) (Svensson el al., 1983).Many starch-degrading and related enzymes are known including a-and /J-amylares, cyclodextrin glycosyltransferase (CGT) and pullulanase from various sources such as animals, plants, bacteria and fungi (Svensson, 1988;Jespersen et al., 1991). G1 has been sequenced from several fungal and yeast sources but of these, the two Aspergillus species, A. niger (Svensson et al., 1983) and A. awanzori (Nunberg et al., 1984), have been the most studied and their amino acid sequences are identical. Of these various enzymes. a number of them, including G I , /J-amylase and CGT from fungal and bacterial sources,
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