Desirability function for optimization of the synthesis of high-panose isomaltooligosaccharides from maltose catalyzed by a novel commercial enzyme preparation from Aspergillus niger

Highlights

• Side transglycosylation activity of six commercial enzymes was tested with maltose.

• Cellulase DS catalyzes the synthesis of high-panose isomaltooligosaccharides.

• An overall desirability function was used to optimize the response variables.

• Panose kinetic profile was apparently resistant to hydrolysis over reaction time.

Abstract

Isomaltooligosaccharides (IMOs) have wide applications as functional food ingredients, but their increasing demand has intensified the need to produce IMOs enriched with a specific oligosaccharide. In this study, the synthesis of high-panose IMOs catalyzed by the secondary transglycosylation activity of Cellulase DS (commercial enzyme preparation) was optimized using an overall desirability function, considering the initial maltose concentration (364–500 g/L) and enzyme-to-substrate (E/S) ratio (10–14 U_T/g) as study variables according to a central composite rotatable design with three response variables (yield, specific productivity and overall selectivity for panose).

Under the optimal conditions, a yield of 0.43 g/g, specific productivity of 0.28 × 10⁻² g/U_T.h and overall selectivity of 4.04 g/g were achieved at 11 h of reaction time when the initial maltose concentration and E/S ratio were set at 456 g/L and 10.6 U_T/g, respectively. The overall selectivity was 29.9 % higher than that obtained with the commercial α-glucosidase Transglucosidase L 'Amano', and the panose composition was approximately 2.8 times that of the commercial formulation from FUJIFILM Wako Pure Chemical Co.

The set of data obtained regarding the transglycosylation activity of Cellulase DS offers an attractive alternative for obtaining high-panose IMOs.

Introduction

Isomaltooligosaccharides (IMOs) are a mixture of glucose oligomers consisting of a chain of two to five glucosyl units, which are linked by α-(1,6) glycosidic bonds. Most commercial IMOs are enzymatically manufactured from starch and thus may additionally contain one or more α-(1,4) glycosidic bonds [1,2]. IMOs mainly consist of isomaltose (α-d-Glc-(1→6)-α-d-Glc), panose (α-d-Glc-(1→6)-α-d-Glc-(1→4)-α-d-Glc), and isomaltotriose (α-d-Glc-(1→6)-α-d-Glc-(1→6)-α-d-Glc) [1]. The properties of IMOs together with effective marketing have made these compounds one of the most demanded groups of oligosaccharides in the emerging prebiotic market; unlike maltooligosaccharides such as maltotriose (α-d-Glc-(1→4)-α-d-Glc-(1→4)-α-d-Glc), they are reported to have bifidogenic activity [3], and health claims have been made about their prebiotic effects [4]. In this context, the synthesis of IMOs is an attractive field of research because it is an important economic topic due to the increasing demand experienced by the food and pharmaceutical industries [4,5].

α-Glucosidase (EC 3.2.1.20) catalyzes the hydrolysis of α-glycosidic bonds at the nonreducing end of the substrate but can also catalyze them at high substrate concentrations through the double-displacement mechanism (Fig. 1) [6]. The primary role of maltose is to donate and accept glycosyl units during the synthesis of IMOs. Thus, the transglycosylation of maltose occurs via the cleavage of the α-(1,4) glycosidic bond and subsequent transfer of the glycosyl unit onto the 6-OH group of an acceptor other than water, for example, maltose or an isomaltooligosaccharide.

Efficient production alternatives have recently been reported for IMOs. The simultaneous saccharification and transglycosylation to produce IMOs from starch allows an increase in yield and productivity compared to the conventional process [7]. In addition, the development of immobilized biocatalysts enables the application of continuous processes as well as the recovery of the enzyme after conversion, exhibiting a significantly higher recycling stability and thus reducing the cost of producing IMOs [8,9]. However, there have been few studies on the development of reliable processes for the large-scale production of IMOs enriched with a specific saccharide. In particular, high-panose-isomaltose syrups are attractive for manufacturing in the food and pharmaceutical industries [10,11].

One strategy for obtaining panose-rich IMO syrups is through a kinetically controlled reaction (transglycosylation). Thus, it is essential to select the appropriate enzyme for the successful production of IMOs in terms of yield, specific productivity and overall selectivity for panose [12,13]. Among the various α-glucosidases, those obtained from filamentous fungi show high levels of transglycosylation activity and regiospecific transglycolytic synthesis of IMOs [14]. Amano Enzyme, Inc. (Nagoya, Japan) supplies a commercial enzyme called Transglucosidase L ‘Amano’ from Aspergillus niger, and this enzyme is widely used in the production of IMOs from starch/maltose [5,15]. However, obtaining IMOs with another composition requires new commercial enzyme preparations with desirable activities, which offer economic and technical advantages [16].

In view of producing panose at an industrial scale, some studies have optimized the synthesis of panose from sucrose and maltose by dextransucrase (EC 2.4.1.5) [10,11]. However, this strategy requires pure maltose, which acts as a glycosyl acceptor; thus, this process is more expensive. In addition, the formation of byproducts such as fructose, dextran and higher oligosaccharides cannot be avoided, resulting in a more complex and expensive purification [11]. Therefore, the production of panose-rich syrups from maltose-rich starch hydrolysates has great potential in the industry that will mainly depend on the kinetic characteristics of α-glucosidase and a rational selection of the reaction conditions [13], which they must first be tested with maltose.

Most studies have focused on increasing the yield using a high concentration of maltose because it favors transglycosylation over hydrolysis [12,17,18], although this approach may lead to substrate inhibition [[19], [20], [21]] and thus decrease the volumetric productivity. Other operational variables such as temperature, pH and enzyme concentration have been shown to have little or no influence on the yield of IMOs [1,12,[17], [18], [19]]. The temperature and enzyme concentration influence the synthesis and hydrolysis rates and thus impact the volumetric productivity and overall selectivity for panose since IMOs are also potential substrates for α-glucosidases [1,7,18,19].

The total yield and volumetric productivity of IMOs show high values in the early reaction stages, but a large amount of residual maltose is detected [19,20], which can be transformed into panose as the reaction progresses. Therefore, there is a conflict between these two output parameters and the overall selectivity for panose because the yield and productivity are objective functions to be maximized, but the composition of IMOs varies, and as a result, panose is transformed into isomaltose [22]. Thus, the significantly influencing reaction conditions must be determined using an optimization technique subject to the required process specifications.

No systematic research has been conducted to optimize the synthesis of IMOs consisting largely of panose at high yields and specific productivities from maltose as a substrate. The objective of the present study was to optimize the synthesis of high-panose IMOs from maltose catalyzed by the secondary transglycosylation activity of a commercial enzyme preparation using an overall desirability function and considering the initial substrate concentration and enzyme-to-substrate (E/S) ratio as study factors.