1 Introduction
Amadori compounds are formed from reducing sugars, such as glucose, and
an amine by a non-enzymatic glycation reaction. In addition to food
samples, this reaction takes place also in vivo, i.e. proteins of blood
such as albumin and hemoglobin that are undergone the glycation based on
glucose concentration in blood. HemoglobinA1c (HbA1c) is produced by
stable connection of glucose to the N-terminal section of the hemoglobin
β-subunit and took into consideration as a key indication for the
lasting control of the glycemic state of diabetic persons. The average
level of glucose in blood is reflected by HbA1c concentrations in the
stages corresponding to the protein half-life [1, 2]. In comparison
to other assay procedures, the newly developed HbA1c enzymatic assay has
been considered as a popular method because of easy management on
customary auto-analyzers and low cost. Two HbA1c enzymatic assay: i)
HbA1c is denatured by detergents and digested by protease to produce the
fructosyl dipeptide part, fructosyl valyl histidine (F-ValHis), and ii)
the generation of glucosone, valyl histidine (ValHis) and hydrogen
peroxide (H2O2) by the reaction of the
released F-ValHis with Fructosyl peptide oxidase (FPOX) [3]. FPOX
belongs to the flavo-enzymes family and catalyzes the oxidative
deglycation of F-ValHis as a substrate derived from HbA1c by proteolytic
digestion [4]. FPOX which acts specifically on glycated proteins has
a great potential for using as a diagnostic enzyme for diabetes mellitus
[5]. Most penicillium enzymes exhibit high activities toward
F-ValHis [6]. Also, FPOX from Eupenicillium terrenum shows
high activity toward FruValHis [5, 7] and is expected to be
applicable in HbA1c measurement [8].E.coli as an expression
system for production of recombinant protein has several advantages
including, high expression level, fast growth in an inexpensive culture
medium, simplicity of genetic manipulation, well known genetics, and,
availability of various strains [9, 10]. However, there are several
limitations in producing recombinant proteins by E. coli such as
low biological activity, complicated downstream processing, low
solubility and stability of the product. Moreover, recombinant proteins
are largely formed as inclusion bodies, in form of inactive and
insoluble molecules, and so, need refolding in vitro [10]. Although,
the considerable capacity of Bacillus species for secretion of
protein is well known, but, the use of Bacillus for the
production of heterologous protein often have contains limitations
[11]. For improvement these drawbacks different strategies have been
applied to resolve protein secretion in E. coli. These approaches
include using of different signal peptides [12], periplasmic and
extracellular expression of protein [13] and, conditions
optimization of expression [14].Theperiplasmic space creates an
oxidative location that is suitable for the formation of disulphide
bond, protein stability and folding [15, 16]. The use of signal tags
can mediate the recombinant proteins to the periplasmic space [17].
In this study, we investigated the methods for decreasing the formation
of FPOX inclusion bodies in E. coli and secretion of the enzyme
into periplasmic space using PelB signal peptide.