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.