The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts.
Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.
Yurimoto
Hiroya
H
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Yamane
Maiko
M
Kikuchi
Yoshimi
Y
Matsui
Hiroshi
H
Kato
Nobuo
N
Sakai
Yasuyoshi
Y
D002175:Candida / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000378:metabolism; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000502:physiology*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D011503:Transglutaminases / Q000378:metabolism*
D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D011503:Transglutaminases