One-pot production of maltoheptaose (DP7) from starch by sequential addition of cyclodextrin glucotransferase and cyclomaltodextrinase

Highlights

• Maltoheptaose (DP7) has been synthesized from soluble starch in a one-pot cascade reaction.

• The sequential addition of the enzymes exhibited a higher conversion ratio over simultaneous addition with 5-fold.

• The maximum production of DP7 was achieved in 1 h reaction time with 1 U/g GaCGT and 1 U/g BsCD in a sequential addition.

• The optimal condition converted 30 g/L soluble starch to 5.4 g/L DP7 in 1 h reaction time with a conversion ratio of 16 %.

Abstract

Maltodextrins (dextrins) are glucose chains normally produced by starch hydrolysis. Maltodextrins are characterized by their degree of polymerization (DP), which indicates the average number of glucose units per chain. Maltoheptaose (DP7), also known as amyloheptaose, is one of the maltodextrin mixtures widely used in foods, cosmetics, and pharmaceutical industries. Recently, the enzymatic synthesis of DP7 has attracted considerable attention, owing to its considerable advantages over chemical methods. In this work, we have designed a one-pot cascade reaction bio-synthesis starting from soluble starch to produce a specific degree of polymerization (DP7). The reaction system was catalyzed by cyclodextrin glucotransferase (GaCGT) from Gracilibacillus alcaliphilus SK51.001CGTase (transglycosylation/cyclization reaction) and cyclomaltodextrinase (BsCD) from Bacillus sphaericus E-244CDase (ring-opening reaction). The one-pot cascade reaction exhibited an optimum temperature of 30 °C and pH 7.0, and the addition of Ca²⁺ enhanced the maltoheptaose production. The optimum enzyme units for the one-pot cascade reaction were 80 U/g of GaCGT and 1 U/g of BsCD. However, the sequential addition of the enzymes exhibited a 5-fold higher conversion rate over simultaneous addition. The one-pot cascade reaction converted 30 g/L of soluble starch to 5.4 g/L of maltoheptaose in 1 h reaction time with a conversion rate of 16 %.

Introduction

Maltodextrins are partially hydrolyzed starch products in which D -glucose units are linked by α-1,4 glycosidic linkage. Maltodextrins are characterized by the degree of polymerization (DP), which refers to the average number of glucose units per chain. Maltodextrins are available commercially based on dextrose equivalent (DE), which is a measure of reducing sugar percentages in the maltodextrin relative to the percentage in glucose (100 %). Dextrose’s equivalent value is calculated as 100/DP. Dextrins have lower DP, which means they have higher DE [1]. Maltodextrins are nonsweet nutritive saccharide polymers. Maltodextrins are used as a bulking agent, spray drying aid, texture provider, film former, sport beverage, as well as enteral and parenteral products. The maltodextrin market is experiencing fast-paced growth owing to high demand from infant food, bakery items, ice creams, confectioneries, dairy products, sports drinks, and various food products. The global maltodextrin market stood at about 4.2 million MT in 2020, higher by 6% from a year ago (https://www.expertmarketresearch.com/reports/maltodextrin-market). The latest report by Value Market Research expects the Global Maltodextrin Market to reach USD 5.8 billion by 2025. It was valued at USD 3.6 billion in 2018. The report foresees a 7% Compound annual growth rate (CAGR) from 2019 to 2025 (https://www.valuemarketresearch.com/report/maltodextrin-market).

Maltoheptaose (DP7), (α-(1→4)-D-gluco-heptaose), is a maltooligosaccharide containing seven glucose units. Maltoheptaose is known as amyloheptaose, and it has been used as an activator of phosphorylase B to prepare heptulose-2-phosphate. Maltoheptaose is used as a substrate in the studies of α-amylase transglycosylation activity [2,3]. At present, MOSs with a specific degree of polymerization, including maltotriose, maltotetraose, and maltoheptaose, have a specific application, such as fast energy supply, anti-starch aging, and lowering osmotic pressure. Compared with that of maltotriose and maltotetraose, maltoheptaose has a lower osmotic pressure, higher viscosity, better moisturizing effect, and more robust film-forming performance, so that it has been widely applied in food, medicine, cosmetics, and other fields [4].

Recently, researchers have discovered that maltoheptaose serves as an outstanding encapsulation agent for aroma compounds [5]. Maltoheptaose is naturally found in tiny amounts in potato, corn, and wheat starch. Traditionally, maltoheptaose is produced by liquefaction of starch with maltooligosaccharide-forming amylases, followed by additional purification steps [6]. However, numerous purification steps are needed to obtain highly pure maltoheptaose [7].

Two or multi-enzymatic cascade reactions are crucial for industrial process development, such as synthesizing foods, cosmetics, nutritional compounds, and pharmaceutical industries [8]. Many strategies have been widely reported to construct two or multienzyme structures [9]. Inside the living cells, there are many enzymatic cascade reactions in a large number of metabolic pathways. These cascade reactions occur inside the living cells [10]. Some enzymes catalyze multi-reaction steps (cascade reactions), inducing more than one sequential process without isolating the intermediates [11]. One of the oldest multi-enzymatic reactions is fermentation using native microorganisms for fermentation, such as vinegar production or beer brewing [12].

Cyclomaltodextrin glucanotransferase (CGTase) and cyclomaltodextrinase (CDase) might be involved in starch and maltose metabolism in Pyrococcus furiosus [13]. Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is one of the α-amylase family members (family 13). CGTase can hydrolyze starch into cyclodextrins, which can be further hydrolyzed into the corresponding dextrose equivalent (DP) using cyclomaltodextrinase (CDase). CGTase catalyzes three transglycosylation reactions through a double displacement mechanism involving a covalent enzyme–intermediate complex. These transglycosylations are disproportionation, cyclization, and coupling. Cyclization is a cleavage of an α-glycosidic bond in starch and subsequent formation of a cyclodextrin [14].

Cyclomaltodextrinase (CDase, EC 3.2.1.54) is an enzyme, which hydrolyzes cyclodextrins (CDs) efficiently. Cyclodextrins (CDs) are glucose units’ oligomers in a cyclic way with α-D-(1,4)-glycosidic bond linkages [15]. Cyclomaltodextrinase contains around 130 residues at its N-terminus, which are proposed to be part of the catalytic domain. There are around 70 residues at C-terminus, which cannot be found in α-amylases [15]. Cyclomaltodextrinase has been genetically characterized from bacterial sources. The first characterization of a CDase was from Bacillus macerans [16], Cyclomaltodextrinase from different sources, such as B. coagulans [17], Clostridium thermohydrosulfuricum 39E [18], alkalophilic Bacillus sp. [19], B. sphaericus E-244 [20], Bacillus sp. I-5 [21], Flavobacterium sp. [22], and B. sphaericus ATCC7055 [23] have been described. Most of these CDases from different bacterial sources have an optimal temperature of around 50 °C and produce mainly maltotriose and maltose from cyclomaltodextrin.

Starch is a renewable, cheap, and abundant natural material, which has been used as a resource for the synthesis of valuable products by microbial fermentation or enzyme hydrolysis. Starch can be hydrolyzed to maltooligosaccharides by maltooligosaccharide-forming amylases [24,25]. The composition of hydrolyzed starch is complex; it contains oligosaccharides and maltooligosaccharides with different degrees of polymerization. This probably happened because of the lack of product and substrate specificity of maltooligosaccharide-forming amylases. In this case, the production of a specific degree of polymerization is quite challenging to achieve.

In this study, a one-pot cascade reaction was designed to produce maltoheptaose starting from starch using cyclomaltodextrin glucanotransferase (GaCGT) and cyclomaltodextrinase (BsCD) (Fig. 1). This technique of maltoheptaose production is different from the traditional production method of maltoheptaose, which is enzymatically hydrolysis of starch using α-amylase. To the best of our knowledge, no research has been done to produce maltoheptaose in a one-pot cascade reaction starting from soluble starch.