Expression in Aspergillus niger of the Starch‐Binding Domain of Glucoamylase

Abstract

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

The starch‐binding domain from glucoamylase disrupts the structure of starch

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Recent Advances in Microbial Raw Starch Degrading Enzymes

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Two Novel, Putatively Cell Wall-Associated and Glycosylphosphatidylinositol-Anchored α-Glucanotransferase Enzymes ofAspergillus niger

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