BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Enhancement of thermostability of fungal deglycating
enzymes by directed evolution
Received: 27 November 2007 / Revised: 8 January 2008 / Accepted: 10 January 2008 / Published online: 1 February 2008
Abstract Fructosyl peptide oxidases are valuable for the
determination of glycoproteins such as hemoglobin A1c.
For practical use in clinical diagnosis, we applied directed
evolution to improve the thermostability of these enzymes.
After two rounds of random mutagenesis and high-
throughput screening, six thermostabilizing amino acid
substitutions were identified. Therefore, site-directed and
cassette mutageneses were applied to combine these six
stabilizing mutations. The simultaneous mutants showed
that the stabilizing effect of the amino acid replacement was
cumulative. The sextuple mutant enzyme, R94K/G184D/
F265L/N272D/H302R/H388Y, had a half-life of thermal
inactivation at 50°C that was 79.8-fold longer than that of
the parental fructosyl peptide oxidase. The thermostable
variants also showed increased tolerance to digestion by a
protease. The sextuple mutant enzyme did not lose its
activity on incubation with neutral protease, while the wild-
type enzyme almost completely lost its activity. Further-
more, three amino acid substitutions were introduced into
another fructosyl peptide oxidase with a different substrate
specificity. The half-life of inactivation at 50°C was 3.61-
fold longer than that of the parent enzyme. These
engineered fructosyl peptide oxidases will be useful for
industrial application to clinical diagnosis.
Keywords Directed evolution
Enzymatic measurement of hemoglobin A1c
Fructosyl amino acid oxidase
Fructosyl peptide oxidase
Hemoglobin A1c (HbA1c) is the major glycohemoglobin
species in blood cells and accepted as the most important
marker in the diagnosis of diabetes mellitus (Bunn et al.
1978; Kobold et al. 1997). HbA1c is defined as hemoglobin
glycated at the N-terminal valine residue of the β-subunits.
In human blood, hemoglobin is readily glycated at this
valine residue in an irreversible manner (Shapiro et al.
1980); therefore, the amount of HbA1c is believed to reflect
the average blood glucose level during the past few months
(Bunn et al. 1978). More recently, to derive average blood
glucose value from HbA1c results, an international study
has been initiated to document this relationship (Consensus
Committee 2007). For the routine measurement of HbA1c,
methods such as high-performance liquid chromatography
(HPLC), immunoassays, isoelectric focusing, and electro-
phoresis have been proposed (Halwachs-Baumann et al.
1997; John et al. 1993). The HPLC and immunoassay
methods are used routinely in clinical laboratories. However,
HPLC requires a specialized system and a long run time. The
immunoassay method can measure many samples in a short
time, but reproducibility is not good. Therefore, researchers
have awaited a new method that solves these problems. An
enzymatic method would fulfill such requirements.
Fructosyl amino acid oxidase (FAOX) catalyzes the
oxidative deglycation of glycated amino acids to produce
the corresponding amino acids, glucosone, and hydrogen
peroxide (Horiuchi et al. 1989). FAOXs have been isolated
from Corynebacterium sp., Aspergillus sp., Penicillium sp.,
Fusarium sp., and Pichia sp. (Horiuchi et al. 1989;
Takahashi et al. 1997; Yoshida et al. 1995; Sode et al.
2001). Bacterial FAOXs had sequence similarity with an
opine-catabolizing enzyme in Agrobacterium and were
active against α-glycated amino acids such as fructosyl
Appl Microbiol Biotechnol (2008) 78:775–781
K. Hirokawa (*)
Research and Development Division, Kikkoman,
Noda City, Chiba Prefecture 278-0037, Japan