FAIR-CT96-5025 Modification of the specificity of starch-degrading enzymes by genetic fusions with the starch binding domain of glucoamylase

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FAIR Area 2.2 - Bioprocessing : FAIR Marie Curie Research Training Grants : Fine Chemicals

Objectives:

Starch from cereal seeds is a raw material used by industries that produce ethanol, sugar syrups and many other starch-based derivatives. Starch consists of two major components: amylose (mainly alpha l,4-linked D-glucose residues) and amylopectin (containing both alpha 1,4- and alpha 1,6-linked D-glucose residues). The relative content of amylose and amylopectin in starch varies with the source. Due to the importance of starch, sugars and other products derived from it, cereal seeds are one of the most important industrial raw amterials derived from agriculture and starch degrading enzymes are among the most important industrial enzymes. Glucoamylase and alpha amylase are widely used in industrial applications, e.g. for the production of glucose and various alcoholic beverages. Glucoamylase has the ability to digest raw starch working synergistically with amylase. The aim of this project was to enhance activity and binding of barley alpha-amylase on insoluble starch by fusion of the glucoamylase starch binding domain (SBD) from A. niger to the C- terminal end of the protein.

Activities and Results:

The work involved the construction of the AMYI-SBD gene by genetic engineering, cloning of the fusion gene as well as the AMYI cDNA in an expression vector and transformation in a heterologous organism. This was followed by production and purification of the corresponding recombinant proteins and biochemical characterisation of the purified enzymes. An in-frame fusion of the entire barley alpha-amylase I isozyme (AMY I) encoding sequence to the 3' end of the A. niger glucoamylase region coding for the starch binding domain via the full length glucoamylase linker was made by overlap extension PCR. The resulting fusion gene as well as the cDNA encoding AMYI isozyme have been expressed in A. niger under the control of the A. nidulans glyceraldehyde-3-phosphate dehydrogenase promoter. Both recombinant AMY I and AMY I -SBD were effectively secreted in A. niger when using the AMYI native signal sequence. Polyclonal antibodies, raised against barley alpha-amylase 2 isozyme and cross-reacting with AMYI, recognised the recombinant AMYI as well as the AMYI-SBD fusion protein. The N-terminal sequence of recombinant AMYI and AMYI-SBD (His-Gln-Val-Leu-Phe), was identical to that of AMY I from malt, indicating native-like processing of AMY I signal sequence in A. niger. Both recombinant enzymes were purified to homogeneity from the culture supernatant by a two-step process involving affinity chromatography followed by ion exchange chromatography, allowing further biochemical characterisation.

Conclusions:

The purified recombinant AMY I was structurally identical to the mature malt protein according to the following criteria:

• N-terminal amino acid sequence
• electrophoretic mobility
• immunoreactivity and ESMS.

Furthermore the recombinant and native barley alpha-amylases closely resembled each other in terms of enzymatic activity on insoluble and soluble starch as well as on 2-chloro-4-nitrophenol beta-D-maltoheptaoside. The purified recombinant fusion protein and reAMY1 displayed the same specific activity on pNPG7 and on potato soluble starch. Genetic engineering enabled the production of an active fusion protein between barley alpha-amylase and the starch binding domain of A. niger glucoamylase. Further characterisation of the fusion protein in terms of activity and binding oil insoluble corn starch took place.

Keywords: Starch, glucoamylase, amylase

Contacts

Scientific Supervisor

• Gary WILLIAMSON / Institute of Food Research Norwich Laboratory / UNITED KINGDOM