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High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts.

Abstract：

:(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals. Limitations in enzyme supply from natural resources were overcome by production of the enzyme in the microbial host systems Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. Expression of Hnl in the prokaryotic system led to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein were obtained. Highest yields were obtained in a high cell density batch fermentation of a P. pastoris transformant that expressed heterologous Hnl to about 50% of the soluble cytosolic protein. At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product was obtained. Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies were not successful. Immunofluorescence microscopy studies indicated that the secretion-directed heterologous Hnl protein accumulated in the plasma membrane forming aggregated clusters of inactive protein.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Hasslacher M,Schall M,Hayn M,Bona R,Rumbold K,Lückl J,Griengl H,Kohlwein SD,Schwab H

doi

10.1006/prep.1997.0765

subject

Has Abstract

pub_date

1997-10-01 00:00:00

pages

61-71

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(97)90765-8

journal_volume

11

pub_type

杂志文章

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