Maltooligosaccharides Research Articles

Synergistic action of novel maltohexaose-forming amylase and branching enzyme improves the enzymatic conversion of starch to specific maltooligosaccharide

As attractive functional ingredients, maltooligosaccharides (MOS) are typically prepared by controlled enzymatic hydrolysis of starch. However, the random attack mode of amylase often leads to discrete product distribution, thereby reducing yields and purities. In this study, a novel glycoside hydrolase family 13 amylase AmyEs from marine myxobacteria Enhygromyxa salina was identified efficient maltohexaose (G6)-forming ability (40 %, w/w). By deciphering external chain length, we found that the high density of α-1,6-branching points benefits the G6 formation of AmyEs with high purity (71–82 %), indicating the substrate selectivity of AmyEs toward high-branched starch. Based on this, asynchronous conversion strategy was designed to enhance specific MOS yield from corn starch by exploiting branching enzymes and AmyEs, and the purity and yield of G6 respectively increased by 9.5 % and 5 % compared to single AmyEs treatment. Our results demonstrate that combinatorial catalysis of MOS-forming amylases and branching enzymes provides a favorable industrial preparation of specific MOS.

Improvement of combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase by functionalization of cross-linker for maltooligosaccharides synthesis

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1 (Combi-CLEAs-CM) were successfully developed to synthesis maltooligosaccharides (MOS). Yet, the poor cross-linking performance between chitosan (cross-linker) and enzymes resulting low activity recovery and catalytic efficiency. In this study, we proposed the functionalization of cross-linkers with the integration of computational analysis to study the influences of different functional group on cross-linkers in combi-CLEAs development. From in-silico analysis, O-carboxymethyl chitosan (OCMCS) with the highest binding affinity toward both enzymes was chosen and showed alignment with the experimental result, in which OCMCS was synthesized as cross-linker to develop improved activity recovery of Combi-CLEAs-CM-ocmcs (74 %). The thermal stability and deactivation energy (205.86 kJ/mol) of Combi-CLEAs-CM-ocmcs were found to be higher than Combi-CLEAs-CM (192.59 kJ/mol). The introduction of longer side chain of carboxymethyl group led to a more flexible structure of Combi-CLEAs-CM-ocmcs. This alteration significantly reduced the Km value of Combi-CLEAs-CM-ocmcs by about 3.64-fold and resulted in a greater Kcat/Km (3.63-fold higher) as compared to Combi-CLEAs-CM. Moreover, Combi-CLEAs-CM-ocmcs improved the reusability with retained >50 % of activity while Combi-CLEAs-CM only 36.18 % after five cycles. Finally, maximum MOS production (777.46 mg/g) was obtained by Combi-CLEAs-CM-ocmcs after optimization using response surface methodology.

Taste of common prebiotic oligosaccharides: impact of molecular structure.

Prebiotic oligosaccharides are naturally occurring nondigestible carbohydrates with demonstrated health benefits. They are also a chemically diverse class of nutrients, offering an opportunity to investigate the impact of molecular structure on oligosaccharide taste perception. Accordingly, a relevant question is whether these compounds are detected by the human gustatory system, and if so, whether they elicit sweet or "starchy" taste. Here, in 3 psychophysical experiments, we investigated the taste perception of 3 commercially popular prebiotics [fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS)] in highly pure form. Each of these classes of prebiotics differs in the type of glycosyl residue, and position and type of bond between those residues. In experiments I and II, participants were asked to discriminate a total of 9 stimuli [FOS, GOS, XOS; degree of polymerization (DP) of 2, 3, 4] prepared at 75mM in the presence and absence of lactisole, a sweet receptor antagonist. We found that all 9 compounds were detectable (P < 0.05). We also found that GOS and XOS DP 4 were discriminable even with lactisole, suggesting that their detection was not via the canonical sweet receptor. Accordingly, in experiment III, the taste of GOS and XOS DP 4 were directly compared with that of MOS (maltooligosaccharides) DP 4-6, which has been reported to elicit "starchy" taste. We found that GOS and MOS were perceived similarly although narrowly discriminable, while XOS was easily discriminable from both GOS and MOS. The current findings suggest that the molecular structure of oligosaccharides impacts their taste perception in humans.

Identification and Characterization of Novel Malto-Oligosaccharide-Forming Amylase AmyCf from Cystobacter sp. Strain CF23.

Malto-oligosaccharides (MOSs) from starch conversion is advantageous for food and pharmaceutical applications. In this study, an efficient malto-oligosaccharide-forming α-amylase AmyCf was identified from myxobacter Cystobacter sp. strain CF23. AmyCf is composed of 417 amino acids with N-terminal 41 amino acids as the signal peptide, and conserved glycoside hydrolase family 13 (GH13) catalytic module and predicted C-terminal domain with β-sheet structure are also identified. Phylogenetic and functional analysis demonstrated that AmyCf is a novel member of GH13_6 subfamily. The special activity of AmyCf toward soluble starch and raw wheat starch is 9249 U/mg and 11 U/mg, respectively. AmyCf has broad substrate specificity toward different types of starches without requiring Ca2+. Under ideal circumstances of 60 °C and pH 7.0, AmyCf hydrolyzes gelatinized starch into maltose and maltotriose and maltotetraose as the main hydrolytic products with more than 80% purity, while maltose and maltotriose are mainly produced from the hydrolysis of raw wheat starch with more than 95% purity. The potential applicability of AmyCf in starch processing is highlighted by its capacity to convert gelatinized starch and raw starch granules into MOSs. This enzymatic conversion technique shows promise for the low-temperature enzymatic conversion of raw starch.

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase from Bacillus lehensis G1 for maltooligosaccharides synthesis

Maltooligosaccharides (MOS) are functional oligosaccharides that can be synthesized through enzymatic cascade reaction between cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1. To address the problems of low operational stability and non-reusability of free enzymes, both enzymes were co-immobilized as combined cross-linked enzyme aggregates (Combi-CLEAs-CM) with incorporation of bovine serum albumin (BSA) and Tween 80 (Combi-CLEAs-CM-add). Combi-CLEAs-CM and Combi-CLEAs-CM-add showed activity recoveries of 54.12 % and 69.44 %, respectively after optimization. Combi-CLEAs-CM-add showed higher thermal stability at higher temperatures (40 °C) with longer half-life (46.20 min) as compared to those of free enzymes (36.67 min) and Combi-CLEAs-CM (41.51 min). Both combi-CLEAs also exhibited higher pH stability over pH 5 to pH 9, and displayed excellent reusability with >50 % of initial activity retained after four cycles. The reduction in Km value of about 22.80 % and 1.76-fold increase in starch hydrolysis in comparison to Combi-CLEAs-CM attested the improvement of enzyme-substrate interaction by Tween 80 and pores formation by BSA in Combi-CLEAs-CM-add. The improved product specificity of Combi-CLEAs-CM-add also produced the highest yield of MOS (492 mg/g) after 3 h. Therefore, Combi-CLEAs-CM-add with ease of preparation, excellent reusability and high operational stability is believed to be highly efficacious biocatalyst for MOS production.

Maltooligosaccharides: Properties, Production and Applications

Maltooligosaccharides (MOS) are homooligosaccharides that consist of 3-10 glucose molecules linked by α-1,4 glycosidic bonds. As they have physiological functions, they are commonly used as ingredients in nutritional products and functional foods. Many researchers have investigated the potential applications of MOS and their derivatives in the pharmaceutical industry. In this review, we summarized the properties and methods of fabricating MOS and their derivatives, including sulfated and non-sulfated alkylMOS. For preparing MOS, different enzymatic strategies have been proposed by various researchers, using α-amylases, maltooligosaccharide-forming amylases, or glycosyltransferases as effective biocatalysts. Many researchers have focused on using immobilized biocatalysts and downstream processes for MOS production. This review also provides an overview of the current challenges and future trends of MOS production.

A novel maltooligosaccharide-forming α-amylase from Bacillus cereus and its application in the preparation of maltopentaose product

Maltooligosaccharides (MOS) have become a prominent market in the pharma and food industries due to their excellent bio-preservative and prebiotic properties. However, producing specific maltooligosaccharides like maltopentaose (G5) is relatively tedious and expensive. Here, a novel maltooligosaccharide-forming α-amylase from Bacillus cereus (BcMFAse) was characterized and exhibited a specific activity of 1965.0 ± 9.7 U/mg. The BcMFAse principally produced G5 by hydrolyzing soluble starch, with a maximum G5 yield of 215.8 ± 5.9 g/kg starch, accounting for 63.8 % of the saccharide mixture. The enzymatic hydrolysate was purified by AB-8 macroporous resin column chromatography. Briefly, 1.0 mL adsorbate containing 486.6 g/L total saccharides with deionized water elution at a flow rate of 0.7 mL/min and column temperature of 55 °C. Afterward, G5 was enriched up to 95.4 % purity, and 72.2 % was recovered in solution. The proposed process was simulated by SuperPro Designer software, and the remarkable economic results included a payback period of 0.08 years and an internal rate of return of 294.6 %. This study represented that BcMFAse could be a promising candidate for applications in the food and pharma fields. It first provided a high-efficiency process for producing > 90 % pure G5, which was economically attractive on an industrial scale.

Development of Fermented Rice Water to Improve the Quality of Garaetteok, a Traditional Korean Rice Cake.

In the rice processing industry, wastewater is an inevitable by-product of rice washing. To increase the utilization of washed rice water (WRW), seven types of fermented washed rice water (FWRW) were prepared using lactic acid bacteria (LAB) and carbohydrate hydrolase. The total concentration of small maltooligosaccharides (MOSs) in the amyloglucosidase (AMG) treatment groups was about ten times higher than in the untreated groups. After 6 h of fermentation, six of the seven FWRW samples reached a pH of 4 due to the increased concentration of organic acids and could, therefore, be used as food acidity regulators. To confirm the applicability of FWRW, the traditional Korean rice cake garaetteok was prepared with FWRW and stored at 4 °C for 5 days. A texture profile analysis (TPA) revealed that the hardness of garaetteok treated with FWRW was significantly lower than that of untreated garaetteok following storage. Differential scanning calorimetry (DSC) showed that FWRW retarded the retrogradation of garaetteok during storage. The addition of FWRW using Lactobacillus reuteri with an AMG group was particularly effective for inhibiting microbial activity in garaetteok during storage. These results suggest that FWRW using AMG-added L. reuteri can be used as a novel food additive for improving the quality of traditional Korean starch foods and could also reduce the volume of waste WRW.

Comparative analysis of prebiotic effects of four oligosaccharides using in vitro gut model: digestibility, microbiome, and metabolome changes.

Fructooligosaccharides (FOS), Ad-fructooligosaccharides (Ad-FOS), resistant maltodextrin (RMD), and maltooligosaccharides (MOS) are commercially available prebiotic oligosaccharides. In this study, the effects of prebiotics on the human gut microbial ecosystem were evaluated using an in vitro gut model. FOS and Ad-FOS showed tolerance to digestion, whereas RMD and MOS showed moderate digestion by digestive enzymes. In in vitro fecal fermentation, Bifidobacterium spp. increased in the following order: FOS, Ad-FOS, MOS, and RMD, whereas Bacteroides spp. increased in RMD medium. Bacteroides xylanisolvens exhibited cross-feeding by enabling the growth of other beneficial bacteria during co-culture in RMD medium. In metabolome analysis, total short-chain fatty acids (SCFAs) were highly produced in the following order: RMD, FOS, MOS, and Ad-FOS; acetate in the order of FOS, MOS/RMD, and Ad-FOS; butyrate in the order of RMD, MOS, FOS, and Ad-FOS; and propionate only in RMD. In addition, the conversion of betaine to trimethylamine was rarely affected in the following order: MOS, RMD, FOS, and Ad-FOS. Lastly, the four oligosaccharides inhibited the adhesion of pathogenic Escherichia coli to human epithelial cells to a similar extent. The comparative analysis results obtained in this study will provide comprehensive information of these substances to manufacturers and customers.

Exploring the novel mechanistic aspects of function of a hyperthermophile two‐site exo‐amylase‐cum‐glucanotransferase displaying substrate versatility

We have recently described an exo‐amylase‐cum‐glucanotransferase, PfuAmyGT, from Pyrococcus furiosuswhich uses disproportionation to generate a pool of glucose and small malto‐oligosaccharides (MOGs) from starch, or from any single MOG (or a combination of MOGs). PfuAmyGT appears to use a combination of exo‐amylase action and excision‐and‐transfer of glucose from donor to acceptor MOGs, using a unique and novel mechanism involving two catalytically‐active sites (i.e., separate donor and acceptor sites, with a loop transferring the excised glucose) in place of the more common single site (at which the donated glucose is excised, and left, by the departing donor MOG, for being picked up by the acceptor MOG). We demonstrate that there are five residues (three glutamates and two aspartates) that are essential for activity, suggesting that different acidic side chains act at the donor and acceptor sites. We also demonstrate that PfuAmyGT acts upon substrates possessing different monomeric units and different types of glycosidic bonds (e.g., pectin, xylan or cellulose), indicating an unprecedented level of versatility, given that only one other glycosyl hydrolase (a neo‐pullulanase) has previously been reported to act upon more than one type of glycosidic bond (alpha‐1,4 and alpha‐1,6). Our bioinformatics and biochemical analyses suggest that the donor site of PfuAmyGT is processive, while the acceptor site releases and rebinds MOGs in each duty cycle. We have identified a total of six residues in the vicinity of a catalytic aspartate (D362) that potentially function to give rise to the said substrate versatility. Four of the six residues are also catalytically important for the functioning of PfuAmyGT on different substrates.

Immobilization of maltogenic amylase in alginate-chitosan beads for improved enzyme retention and stability

Maltogenic amylase (Mag1) is a potent enzyme that hydrolyzes the glycosidic bond of polysaccharides to produce malto-oligosaccharides (MOS). However, the Mag1 enzyme has poor stability and reusability, leading to inefficient MOS production. Enzyme immobilization is a promising method to solve the enzyme stability problem. Entrapment and encapsulation technique was used in this study to immobilize Mag1 because of high biocompatibility and prevention of enzyme degradation, hence lesser loss of enzymatic activity. Chitosan was used as a coating membrane on the alginate matrix, preventing enzyme leaching from the beads. Mag1 entrapped in alginate-chitosan beads showed better performance compared to alginate beads in terms of thermostability, reusability and enzyme retention. Alginate-chitosan beads showed improvement of temperature stability of approximately 35%, 30% and 20% at a respective temperature of 30 °C, 40 °C and 50 °C. Reusability analysis showed immobilized Mag1 can be used up to at least eight cycles with retained activity of 80% and 70% from its initial activity for alginate-chitosan and alginate beads respectively. Enzyme leakage percentage in alginate-chitosan was 7-21%, while that in alginate was 12-35%. The overall findings envisage the promising application of alginate-chitosan beads immobilized Mag1 as a biocatalyst for MOS synthesis.

Exploring the novel mechanistic aspects of function of a hyperthermophile two-site exo-amylase-cum-glucanotransferase displaying substrate versatility

We have recently described an exo-amylase-cum-glucanotransferase, PfuAmyGT, from Pyrococcus furiosus which uses disproportionation to generate a pool of glucose and small malto-oligosaccharides (MOGs) from starch, or from any single MOG (or a combination of MOGs). PfuAmyGT appears to use a combination of exo-amylase action and excision-and-transfer of glucose from donor to acceptor MOGs, using a unique and novel mechanism involving two catalytically-active sites (i.e., separate donor and acceptor sites, with a loop transferring the excised glucose) in place of the more common single site (at which the donated glucose is excised, and left, by the departing donor MOG, for being picked up by the acceptor MOG).

Taste perception of cyclic oligosaccharides: α, β, and γ cyclodextrins.

Oligosaccharides, a subclass of complex carbohydrates, occur both naturally in foods and as a result of oral starch digestion. We have previously shown that humans can taste maltooligosaccharides (MOS) and that their detection is independent of the canonical sweet taste receptor. While MOSs most commonly occur in a linear form, they can also exist in cyclic structures, referred to as cyclodextrins (CD). The aim of this study was to investigate how the structure of the MOS backbone (i.e. cyclic form) and the size (i.e. degree of polymerization; DP) affect their taste perception. We tested taste detection of cyclodextrins with DP of 6, 7, and 8 (i.e. α-, β-, and γ-CD, respectively) in the presence and absence of lactisole, a sweet receptor antagonist. We found that subjects could detect the taste of cyclodextrins in aqueous solutions at a significant level (P < 0.05), but were not able to detect them in the presence of lactisole (P > 0.05). These findings suggest that the cyclodextrins, unlike their linear analogs, are ligands of the human sweet taste receptor, hT1R2/hT1R3. Study findings are discussed in terms of how chemical structures may contribute to tastes of saccharides.

Efficient hydrolysis of raw starch by a maltohexaose-forming α-amylase from Corallococcus sp. EGB

The process of raw starch conversion has attracted extensive attention in recent years due to its high energy conversion efficiency and saving of operational costs. In this study, extensive hydrolysis of raw starch by α-amylase AmyM from Corallococcus sp. EGB was investigated with robust substrate and products tolerance. AmyM showed broad substrate specificity toward raw starches with different types, and kept active and stable over a wide range of temperatures (30–70 °C) and pH values (5.0–9.0) and recycling. The Vmax values of AmyM towards wheat and corn raw starch were 1111 and 741 μmol/min/mg without depending on Ca2+, respectively. SEM analysis showed that AmyM hydrolysis results in the formation of porous and creviced surfaces around the starch granules peripheries. The cost-effective conversion of raw starch for the production of malto-oligosaccharides (MOSs) was identified. AmyM achieved high hydrolysis degree of 90% for 30% wheat and corn raw starch after 20 h at 45 °C with 5 U per mg of raw starch, resulting in formation of 70% MOSs and 30% dextrin. During this process, maltose (G2), maltotriose (G3) and maltotetraose (G4) were produced synchronously with G9 and G11, indicating the potential application in enzymatic conversion of raw starch under low temperature.

Novel Malto‐Oligosaccharide‐Producing Amylase AmyAc from Archangium sp. Strain AC19 and Its Catalytic Properties

AbstractFunctional maltooligosaccharides (MOSs) derived from starch have potential applications in the food industry and medicine due to their specific functional properties. In this study, a novel maltose (G2) and maltotriose (G3)‐producing amylase AmyAc from Archangium sp. strain AC19 is identified. AmyAc is a protein of 874 amino acids, with an N‐terminal signal peptide as well as hydrolase family 13 (GH13) catalytic and C‐terminal binding modules. However, its sequence is not highly similar to any of the reported starch hydrolases. The Vmax of AmyAc is 124 µmol min−1 mg−1 under optimal conditions of 50 °C and pH 7.0. AmyAc catalyzes the conversion of MOSs and soluble starch into maltose and maltotriose as the end‐product with more than 85% purity. AmyAc initially produces G2, G3, and oligosaccharides with even units, following by the release of G2 and oligosaccharides with odd units during later stages of hydrolysis, indicating a distinct catalytic selectivity. The production of MOSs from starch by AmyAc is of significant interest due to potential applications in starch processing.

Synergetic modification of waxy maize starch by dual-enzyme to lower the in vitro digestibility through modulating molecular structure and malto-oligosaccharide content

Cyclodextrinase (CDase) and cyclodextrin glucosyltransferase (CGTase) were synergistically used to provide a novel enzymatic method in lowing in vitro digestibility of waxy maize starch. The molecular structure, malto-oligosaccharide composition, and digestibility properties of the generated products were investigated. The molecular weight was reduced to 0.3 × 105 g/mol and 0.2 × 105 g/mol by simultaneous and sequential treatment with CDase and CGTase, while the highest proportion of chains with degree of polymerization (DP) < 13 was obtained by simultaneous treatment. The resistant starch contents were increased to 27.5% and 36.9% by simultaneous and sequential treatments respectively. Dual-enzyme treatment significantly promoted the content of malto-oligosaccharides (MOSs) by hydrolyzing cyclodextrins from CGTase with CDase. However, the replacement of cyclodextrins by MOSs did not obviously influence the digestibility of the products. The starch digestion kinetics further revealed the hydrolysis pattern of these two enzymes on the starch hydrolysate. It was proved that the starch digestibility could be lowered by modulating the molecular structure and beneficial MOSs content by this dual-enzyme treatment.

Synergistic action of cyclodextrin glucanotransferase and maltogenic amylase improves the bioconversion of starch to malto-oligosaccharides

Overwhelming consumer consciousness for healthier food has led to high market demand for functional oligosaccharides. These functional sugars can be extracted from various sources, especially starch, through enzymatic approaches. Maltogenic amylases are able to synthesise malto-oligosaccharides (MOS) from starch. However, the use of maltogenic amylase alone presents several limitations such as low specificity towards starch and low productivity of MOS. Cyclodextrin glucanotransferase (CGTase) acts on starch to produce cyclodextrins. Herein, asynchronous and synchronous conversion strategies were used to enhance the MOS yield from soluble starch by exploiting maltogenic amylase and CGTase reactions. Factors including pH, temperature, enzyme loading and enzymes ratio that affect MOS production were investigated. The combination of CGTase and maltogenic amylase (MAG1) from Bacillus lehensis G1 produced the highest MOS yield (307.9 mg.g−1). The synchronous strategy exhibited a 2.1-fold higher MOS production compared to the asynchronous approach (146.8 mg.g−1). Kinetic studies revealed that the catalytic efficiency of the synchronous approach was ∼800-fold (1746.1 mL.g−1. h−1) higher than that of MAG1 alone (18.5 mL.g−1. h−1). The high production of MOS and catalytic efficiency of the reaction demonstrate that the combinatorial catalysis of MAG1 and CGTase is very promising and provides a foundation for industrial-scale production of MOS.

Analysis of malto-oligosaccharides and related metabolites in rice endosperm during development.

Linear glucans with degree of polymerization of up to 23 were detected in rice endosperm at the very early developmental stage of endosperm and considered to play an important role in the de novo synthesis of branched glucans. Little is known concerning the contribution of malto-oligosaccharides (MOS) and longer linear glucans to the starch biosynthesis in cereal endosperm. In the present study, the changes in the amount of major metabolic intermediates including MOS and linear glucans with a degree of polymerization (DP) of ≤ 9 and ≥ 10, respectively, in rice endosperm were measured during the development. Significant amounts of linear glucans of at least DP23 were present in the endosperm at 3 and 5days after pollination (DAP), whereas most MOS of DP up to 8 were detected in the endosperm throughout the development up to 20 DAP. It was also found that a significant amount of simple sugars such as sucrose, glucose, and fructose, and organic acids such as malic acid, citric acid, and succinic acid were present in the developing endosperm. Although the levels of metabolites are not directly related to the extent of the metabolic flux, the present results suggest that MOS and linear glucans as well as these sugars and organic acids are involved in starch biosynthesis of rice endosperm. It is thought that linear glucans might play a role in starch biosynthesis in rice endosperm, presumably as the precursor for the subsequent synthesis of branched glucans involved in the initiation process that is possibly active in the endosperm at the very early developmental stage.

Entrapment of porous cross-linked enzyme aggregates of maltogenic amylase from Bacillus lehensis G1 into calcium alginate for maltooligosaccharides synthesis

Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (β-CD) (Km = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.