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Expression of the α‐galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha
Author(s) -
Fellinger Arthur J.,
Verbakel John M. A.,
Veale Rosemary A.,
Sudbery Peter E.,
Bom Isaac J.,
Overbeeke Nico,
Verrips C. Theo
Publication year - 1991
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.320070505
Subject(s) - cyamopsis , biology , invertase , expression vector , biochemistry , saccharomyces cerevisiae , yeast , signal peptide , heterologous expression , guar , beta galactosidase , enzyme , plasmid , escherichia coli , microbiology and biotechnology , gene , recombinant dna
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.