Expression of the α‐galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha

Publisher Website
Google Scholar
Abstract
The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the α‐galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 μm plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the α‐galactosidase is controlled by the methanol‐regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the α‐galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active α‐galactosidase enzyme was efficiently secreted (>85%) and after methanol induction, the expression level was 42 mg/l. Amino‐terminal sequencing of the purified α‐galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted α‐galactosidase was glycosylated and had a sugar content of 9·5%. The specific activity of the α‐galactosidase produced by H. polymorpha was 38 U mg−1 compared to 100 U mg−1 for guar α‐galactosidase. Deglycosylation of the H. polymorpha α‐galactosidase restored the specific activity completely.