Maltooligosaccharide-forming Amylase Research Articles

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.

Enzymatic Approaches for Structuring Starch to Improve Functionality.

Starch is one of the most abundant renewable biopolymers in nature and is the main constituent in the human diet and a raw material for the food industry. Native starches are limited in most industrial applications and often tailored by structural modification to enhance desirable attributes, minimize undesirable attributes, or create new attributes. Enzymatic approaches for structuring starch have become of interest to the food industry precisely because the reactions minimize the formation of undesirable by-products and coproducts and are therefore considered environmentally friendly methods for producing clean-label starches with better behavioral characteristics. Starches with improved functionalities for various applications are produced via enzyme hydrolysis and transfer reactions. Use of novel, multifunctional, starch-active enzymes to alter the structures of amylose and/or amylopectin molecules, and thus alter the starch's physiochemical attributes in a predictable and controllable manner, has been explored. This review provides state-of-the-art information on exploiting glycosyl transferases and glycosyl hydrolases for structuring starch to improve its functionalities. The characteristics of starch-active enzymes (including branching enzymes, amylomaltases, GH70 α-transglycosylases, amylosucrases, maltogenic amylases, cyclomaltodextrinases, neopullulanases, and maltooligosaccharide-forming amylases), structure-functionality-driven processing strategies, novel conversion products, and potential industrial applications are discussed.

Starch-Binding Domain Modulates the Specificity of Maltopentaose Production at Moderate Temperatures.

Maltooligosaccharide-forming amylases (MFAs) hydrolyze starch into maltooligosaccharides with a defined degree of polymerization. However, the enzymatic mechanism underlying the product specificity remains partially understood. Here, we show that Saccharophagus degradans MFA (SdMFA) contains a noncatalytic starch-binding domain (SBD), which belongs to the carbohydrate-binding module family 20 and enables modulation of the product specificity. Removal of SBD from SdMFA resulted in a 3.5-fold lower production of the target maltopentaose. Conversely, appending SBD to another MFA from Bacillus megaterium improved the specificity for maltopentaose. SdMFA exhibited a higher level of exo-action and greater product specificity when reacting with amylopectin than with amylose. Our structural analysis and molecular dynamics simulation suggested that SBD could promote the recognition of nonreducing ends of substrates and delivery of the substrate chain to a groove end toward the active site in the catalytic domain. Furthermore, we demonstrate that a moderate temperature could mediate SBD to interact with the substrate with loose affinity, which facilitates the substrate to slide toward the active site. Together, our study reveals the structural and conditional bases for the specificity of MFAs, providing generalizable strategies to engineer MFAs and optimize the biosynthesis of maltooligosaccharides.

Fusion of maltooligosaccharide-forming amylases from two origins for the improvement of maltopentaose synthesis

Maltopentaose-forming amylases are promising enzymes for their ability to hydrolyze starch and produce functional maltooligosaccharides. Two maltopentaose-forming amylase genes from Bacillus megaterium (BmMFA) and Saccharophagus degradans (SdMFA) were expressed heterologously and their characteristics were analyzed. BmMFA has substantial thermostability and SdMFA owns superior product specificity. The carbohydrate-binding module of SdMFA was fused with BmMFA and the fused protein showed ideal enzymatic properties and displayed potential for industrial production of maltopentaose. Under the optimized conditions, the final product containing 47.41% maltopentaose was obtained with a conversion rate of 92.67% from starch. This study provides a novel strategy for the directed modification of MFAses through protein fusion approach.

Structure-Based Engineering of a Maltooligosaccharide-Forming Amylase To Enhance Product Specificity.

Maltooligosaccharide-forming amylases (MFAses) are promising tools for a variety of food industry applications because they convert starch into functional maltooligosaccharides. The MFAse from Bacillus stearothermophilus STB04 (BstMFAse) is a thermostable enzyme that preferentially produces maltopentaose and maltohexaose. An X-ray crystal structure of the BstMFAse-acarbose complex suggested that mutation of glycine 109 would increase its maltohexaose specificity. Using site-directed mutagenesis, glycine 109 was replaced with several different amino acids. Mutant-containing asparagine (G109N), aspartic acid (G109D), and phenylalanine (G109F) produced 36.1, 42.4, and 39.0% maltohexaose from starch, respectively, which was greater than that produced by the wild-type (32.9%). These mutants also exhibited substantially altered oligosaccharide hydrolysis patterns in favor of maltohexaose production. Homology models suggested that the mutants form extra interactions with the substrate at subsite -6, which were responsible for the enhanced maltohexaose specificity of BstMFAse. The results of this study support the proposition that binding of the substrate's nonreducing end in the nonreducing end-subsite of the MFAse active center plays a crucial role in its product specificity.

Insights into the thermostability and product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04

Analyze the thermostability, mode of action, and product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) from the biochemical and structural point of view. Using three-dimensional co-crystal structure of Bst-MFA with acarbose as a guide, experiments were performed to analyze the thermostability, mode of action and product specificity of Bst-MFA. The results showed that the Ca2+-Ca2+-Ca2+ metal triad of Bst-MFA is responsible for its high thermostability. Multiple substrate binding modes, rather than one productive binding mode determined by non-reducing end recognition, are in accordance with an endo-type mode of action. Significant interactions between subsites -5 and -6 and glucosyl residues at the non-reducing end explain the maltopentaose(G5) and maltohexaose(G6) specificity of Bst-MFA. Bst-MFA is a thermostable enzyme that preferentially produces G5 and G6, with an endo-type mode. The understanding of structure-function relationships provides the foundation for future efforts to the modification of Bst-MFA.

Structure of maltotetraose-forming amylase from Pseudomonas saccharophila STB07 provides insights into its product specificity

The maltooligosaccharide-forming amylases (MFAses) degrade starch into maltooligosaccharides which potentially benefit human diet and grow popular in food processing, but little has been studied about their product specificity and structures. We focused on this topic and provide evidence through an X-ray crystal structure of the maltotetraose (G4)-forming amylase from Pseudomonas saccharophila STB07 (MFAps), as well as co-crystal structures of MFAps with G4 and with pseudo-maltoheptaose (pseudo-G7) determined at up to 1.1 Å resolution. G4 and pseudo-G7 occupy active cleft subsites −4 to −1 and −4 to +3 respectively. Binding induces conformational changes in the active sites except Asp193, working as the base catalyst. Comparison of the MFAps structure with those of other α-amylases revealed obvious differences in the loop structures providing dominant interactions between protein and substrate in the non-reducing side of the active sites cleft. These structures at the non-reducing end may govern the G4 specificity of MFAps and also be relevant to its exo-type action pattern.

Importance of Trp139 in the product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04.

The maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) randomly cleaves the α-1,4 glycosidic linkages of starch to produce predominantly maltopentaose and maltohexaose. The three-dimensional co-crystal structure of Bst-MFA with acarbose highlighted the stacking interactions between Trp139 and the substrate in subsites - 5 and - 6. Interactions like this are thought to play a critical role in maltopentaose/maltohexaose production. A site-directed mutagenesis approach was used to test this hypothesis. Replacement of Trp139 by alanine, leucine, or tyrosine dramatically increased maltopentaose production and reduced maltohexaose production. Oligosaccharide degradation indicated that these mutants also enhance productive binding of the substrate aglycone, leading to a high maltopentaose yield. Therefore, the aromatic stacking between Trp139 and substrate is suggested to control product specificity and the oligosaccharide cleavage pattern.

Crystal structure of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04

To better understand structure-function relationships, an X-ray crystal structure of the maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) with bound acarbose has been determined at 2.2 Å. The structure revealed a classical three-domain fold stabilized by four calcium ions, in which CaI–CaIII form an unprecedented linear metal triad in the interior of domain B. Catalytic residues are deduced to be two aspartic acids and one glutamic acid (Asp234, Glu264, Asp331), and the acarbose is bound to surrounding amino acid residues, mainly through extensive hydrogen bonds. Furthermore, analysis of the structure indicates the existence of at least 8 subsites in Bst-MFA, six glycone sites (−6, −5, −4, −3, −2, −1) and two aglycone sites (+1, +2). Subsite +3 remains to be further explored. Sugar-binding subsites contribute to further presentation of the oligosaccharide-binding mode, which explains the product specificity of Bst-MFA to some extent. In addition, we propose a mechanism by which maltooligosaccharide-forming amylases produce particular maltooligosaccharide products, a result different from that seen with typical α-amylases. Finally, the three-dimensional structure of Bst-MFA complexed with acarbose provides the basis for further studies, designed to increase product specificity.

A novel maltooligosaccharide-forming amylase from Bacillus stearothermophilus

A novel maltooligosaccharide-forming amylase (MFAse) from Bacillus stearothermophilus (Bst-MFAse) was heterologously expressed in B. subtilis and purified by hydrophobic and ion-exchange chromatographies. Bst-MFAse, an approximately 58 kDa monomer, could effectively hydrolyze linear α-glucan with DP > 6 and slightly hydrolyze maltohexaose. In addition, Bst-MFAse was proved to majorly produce maltopentaose and maltohexaose from starch and categorized as a maltopentaose-forming amylase. The action pattern was analyzed using amylopectin and amylose as substrate, showing that Bst-MFAse could rapidly reduce iodine blue value. Thus, Bst-MFAse was categorized as both a maltopentaose-forming amylase and an endo-amylase. During the initial period (2 h) of amylolysis, Bst-MFAse mainly cleaved amylopectin (18.7%) than amylose (14.0%). However, the final conversion rate (24 h) of amylose (68.8%) was slightly higher than that of amylopectin (64.8%). Bst-MFAse may have great potential applications in industry due to its highly specific activity, unique substrate specificity, and end-type product pattern.

Calcium and sodium ions synergistically enhance the thermostability of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04

Maltooligosaccharide-forming amylases (MFAses) are promising tools for a variety of food industry applications because of their ability to hydrolyze starch into maltooligosaccharides. However, high thermostability is a key requirement for enzymes used in these applications. In this work, we investigated the effect of Ca2+ and Na+ on the thermostability of an MFAse from Bacillus stearothermophilus (Bst-MFAse). The results showed that Ca2+ and Na+ synergistically prolong the half-life of Bst-MFAse. The most significant improvement, which preserved 71.1% of initial activity after incubation at 80 °C for 180 min, was achieved by adding 10 mM Ca2+ and 40 mM Na+ simultaneously. The increase in Bst-MFAse thermostability imparted by the addition of Ca2+ and Na+ may be associated with an important Ca2+–Na+–Ca2+ triad structure. This study provides an effective way to enhance the thermostability of Bst-MFAse and related enzymes.

Starch-Modifying Enzymes.

Starch is a carbohydrate polymer found abundantly on earth. It is synthesized in plants as a short-term storage compound for respiration in the leaves and for long-term storage in the tubers, seeds and roots of plants. A wide variety of enzymes modify or convert starch into various products. The classes of enzymes that act on starch include endoamylases, exoamylases, debranching enzymes and transferases. Starch-modifying enzymes of microbial origin are utilized in a wide variety of industrial applications. Alkaline-active amylases are diverse in terms of optimum reaction conditions, substrate and product specificity. Amylases that are active at lower temperatures and alkaline conditions are most suited for detergent formulation. Other notable starch-modifying enzymes from alkaliphiles include maltooligosaccharide-forming amylases and cyclodextrin glycosyltransferases (CGTases), which produce a variety of maltooligosaccharides and cyclodextrins, respectively. Such compounds are used in the food, fine chemical, pharmaceutical and cosmetic industries, among others. Alkaline-active amylases are also applicable in the paper, textile and leather industries and also in bioremediation and alkaline waste water treatment. Their application in these fields is further enhanced through stabilization and improving their specificity and catalytic action by employing nanotechnology and genetic engineering. Graphical Abstract *Alkaline alpha-amylase AmyK from Bacillus sp. KSM-1378. Shirai T, Igarashi K, Ozawa T, Hagihara H, Kobayashi T, Ozaki K, Ito S (2007) Proteins 66:600-610. Source: Protein Data Bank in Europe (PDBe).

A Maltooligosaccharide-Forming Amylase Gene fromBrachybacteriumsp. Strain LB25: Cloning and Expression inEscherichia coli

Brachybacterium sp. strain LB25 produces a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents. The enzyme hydrolyzed starch to produce maltotriose primarily. The structural gene encoding the amylase from strain LB25 was cloned and sequenced. The amino acid sequence of the product showed significant similarity (45 to 49%) to amylases from the genus Streptomyces. The amylase gene was expressed in Escherichia coli, but the specific activity of the recombinant amylase was lower than that of the amylase purified from strain LB25.

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0-8.5 and active at pH 6.0-7.5. The optimum temperature at pH 7.0 was 35 degrees C in the presence of 5 mM CaCl(2). The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.

Immobilization of alpha-amylase produced by Bacillus circulans GRS 313

A maltooligosaccharide-forming amylase from B circulans GRS 313 was immobilized by entrapment in calcium alginate beads. The immobilized activity was affected by the size of the bead and bead size of 2mm was found to be most effective for hydrolysis. Kinetics constants, Km and Vmax were estimated and were found to be affected by the bead size. The catalytic activity of the enzyme was studied in presence of various starchy residues and metal ions. HgCl2, CuSO4 and FeCl3 caused inhibition of the enzyme. The reaction conditions, pH and temperature, was optimized using response surface methodology. At the optimum pH and temperature of 4.9 and 57ºC, the apparent activity was 25.6U/g of beads, resulting in almost 2-fold increase in activity. The immobilized enzyme showed a high operational stability by retaining almost 85% of the initial activity after seventh use.

Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313.

A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48 degrees C and 4.9, respectively. The enzyme was stable up to 60 degrees C and its pH stability was in the range of 5.0-8.0. The Km and Vmax of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, Ea, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a Ki of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a Ki of 9.5 mg/ml. The enzyme was inhibited by Hg2+, Mn2+, Fe3+ and Cu2+ and enhanced by Co2+ and Mg2+. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate.

Enhanced production of amylase by optimization of nutritional constituents using response surface methodology

Response surface methodology was employed to study the cumulative interactive effect of the macronutrients of the media and to optimize their concentration to enhance the production of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313. A 2 3 factorial central composite experimental design was used to study the combined effect of the nutritional constituents like soybean meal, yeast extract and wheat bran. The p-value of the coefficient for linear effect of soybean meal concentration was found to be 0.081, suggesting that this was the principal experimental variable having the greatest effect on the production of maltooligosaccharide-forming amylase. The optimal combination of the media constituents for amylase production from the contour plots were: soybean meal =4.84 g/100 ml , yeast extract =1.58 g/100 ml , wheat bran =2.84 g/100 ml . The optimization of the media increased the amylase yield by 1.25 times.