Cyclodextrin Glycosyltransferase Research Articles

Modification of Bacillus clarkii γ-Cyclodextrin Glycosyltransferase and Addition of Complexing Agents to Increase γ-Cyclodextrin Production.

γ-Cyclodextrin (γ-CD), a cyclic oligosaccharide containing eight glucose units linked by α-1,4-glycosidic bonds, can be produced from starch using cyclodextrin glycosyltransferase (CGTase). Unfortunately, this enzymatic process produces mixtures of α-, β-, and γ-CD. In this study, amino acid residues in the subsite -3 (T47 and F91) and the central subsite (Y186) of Bacillus clarkii γ-CGTase were modified to improve the γ-CD production. The cyclization activities and product specificities of mutants T47H and F91W were similar to those of the wild-type. The cyclization activities of mutants F91N and F91L were significantly greater than those of the wild-type but their γ-CD product specificities were lower. Finally, the central subsite mutant Y186W displayed a γ-CD specificity (94.6%) significantly greater than that of the wild-type (77.1%). To maximize the γ-CD yield, the effects of added complexing agents were investigated. Among the cyclic complexing agents tested, low-boiling cyclododecanone was the smallest that precipitated with γ-CD. When cyclododecanone was used with Y186W, the total CD yield reached 72.6%, and 96.6% of the product was γ-CD. These results, which represent the highest γ-CD yield ever reported, may provide a way to improve large-scale γ-CD preparation and expand the uses of γ-CD in the future.

Effect of added psyllium and food enzymes on quality attributes and shelf life of chickpea-based gluten-free bread

A 24 full factorial design with four center points was used to investigate the effects of chickpea flour (CF), psyllium (PSY), cyclodextrin glycosyltransferase (CGTase), and transglutaminase (TGase) on dough Mixolab® parameters and fresh and stored gluten-free bread (GFB) physical properties. Results show that CF and PSY have the greatest effects on the investigated variables. CF increases the loaf-specific volume and crumb firmness values of fresh GFB, effects of increased starch stability (C4) and tendency to starch retrogradation (C5). Both PSY and PSY- CF interactions reduce the loaf-specific volume and increase the crumb firmness of GFB during storage, effects of an increased initial consistency (C1). CF - CGTase interaction reduced crumb firmness during storage, and TGase had no effect. High CF-levels (75 and 100 g/100 g flour weight basis, fwb) combined with low PSY-levels (4.5 and 5.5 g/100 g fwb) resulted in favorable dough consistency for increasing loaf volume and crumb softness. Results also show that the combination of 75 CF and 5.5 PSY (g/100 g fwb) produces a GFB with good physical properties and appearance, reaching values comparable to commercially available fresh and stored GFB.

Genomic sequencing of Gracilibacillus dipsosauri reveals key properties of a salt-tolerant α-amylase.

Gracilibacillus dipsosauri is a moderately-halophilic Gram-positive bacterium which forms an extracellular α-amylase that is induced by starch, repressed by D-glucose, and active in 2.0M KCl. Previous studies showed that while enzyme activity could be measured with the synthetic substrate 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3), other assays were inconsistent and the protein showed aberrant mobility during nondenaturing gel electrophoresis. To clarify the properties of this enzyme, the genome of G. dipsosauri was sequenced and was found to be 4.19Mb in size with an overall G+C content of 36.9%. A gene encoding an α-amylase composed of 691 amino acids was identified. The protein was a member of the glycosyl hydrolase 13 family, which had a molecular mass of 77,396 daltons and a pI of 4.39 due to an unusually large number of aspartate and glutamate residues (95/691 or 13.7%). BLAST analysis of the amino acid sequence revealed significant matches to other proteins with cyclodextrin glycosyltransferase activity. Partial purification of the protein from G. dipsosauri showed that fractions catalyzing the hydrolysis of CNPG3 and p-nitrophenyl-D-maltoheptoside also catalyzed the formation of β-cyclodextrin but not α-cyclodextrin or γ-cyclodextrin. Formation of β-cyclodextrin was not stimulated by high salt concentrations but did occur with rice, potato, wheat, and corn starches and amylopectin. These studies explain the unusual features of the α-amylase from G. dipsosauri and indicate it should be classified as EC 2.4.1.19. The availability of the complete genomic sequence of G. dipsosauri will provide the basis for studies on other enzymes from this halophile which may be useful for biotechnology.

Byproducts from cassava industry: alternative substrates for cyclodextrin glycosyltransferase production by alkalophilic Bacillus trypoxylicola SM–02

Bioproductos de la industria de la yuca: sustratos alternativos para la producción de ciclodextrina glucosiltransferasa por alcalófilo Bacillus trypoxylicola SM-02 En el presente trabajo estudiamos el uso licor de maíz fermentado (LMF), harina de cáscara de yuca (HCY) y aguas residuales de yuca para la producción de ciclodextina glicosiltransferasa (CGTase) por un nuevo aislado alcalófilo de Bacillus trypoxylicola SM-02 en fermentación sumergida. Los experimentos se realizaron por Diseño Central Compuesto Rotativo 22 totalizando 11 ensayos. La mayor actividad enzimática de 352.53 U/mL se obtuvo con 1.5 g de HCY y 0.6 g de LMF. La temperatura y el pH óptimos fueron 55 ºC y pH 8.0, respectivamente. CGTase mostró una actividad relativa superior al 50% durante 120 min. a la temperatura óptima. Solo el CaCl2 mostró actividad positiva para CGTasa. Los resultados apuntaron a un buen potencial de B. trypoxylicola SM-02 para la producción de CGTasa usando substratos residuales. In the present work was studied the use of cassava peel flour (CPF), corn steep liquor (CSL), and cassava wastewater as substrates to produce cyclodextrin glycosyltransferase (CGTase) from a new alkalophilic isolate of Bacillus trypoxylicola SM-02 by submerged fermentation. The experiments were performed as a Central Composite Design 22, totalizing 11 assays. An enzymatic activity of 352.53 U/mL was obtained using 1.5 g of CPF and 0.6 g of CSL. The optimum temperature and pH of CGTase was 55 °C and 8.0, respectively. The CGTase depicted a relative activity of more than 50% for 120 min at the optimum temperature. The only salt that positively influenced the CGTase activity was CaCl2. The results are indicative of a potential role of B. trypoxylicola SM-02 in the production of CGTase using residual substrates.

Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity.

Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, β-, and γ-CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased Km value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the -5, -6, and -7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths.IMPORTANCE The low water solubility of sophoricoside seriously limits its applications in the food and pharmaceutical industries. Long-chain glycosylated sophoricosides show greatly improved water solubility. Here, the product specificity of cyclodextrin glycosyltransferase (CGTase) for long-chain glycosylated sophoricosides was significantly affected by pH. Our results reveal the pH-regulated mechanism of the glycosylated product specificity of CGTase. This work adds to our understanding of the synthesis of long-chain glycosylated sophoricosides and provides guidance for exploring related product specificity of CGTase based on pH regulation.

Structural and property characterization of corn starch modified by cyclodextrin glycosyltransferase and specific cyclodextrinase

Enzymatic modification is generally applied to enhance desirable attributes of starch. In this study, β-cyclodextrin glycosyltransferase (β-CGTase) together with a specific cyclodextrinase (CDase) was used to modify corn starch. For single β-CGTase treatment, the molecular weight (Mw) was reduced to 36.7 × 105 g/mol and 7.7 × 105 g/mol for 1 h and 6 h reaction, and the debranched samples had a high proportion of chains with degree of polymerization (DP) < 13, indicating an apparent depolymerization effect. CDase could hardly act on starch, while it had a high preference for cyclodextrin hydrolysis and produced mainly maltotetraose with β-CGTase. There was a synergistic effect between β-CGTase and CDase in starch degradation. Interestingly, the utilization of dual-enzyme significantly promoted the retrogradation inhibition property of starch, reducing the retrogradation enthalpy from 5.65 J/g to 1.42 J/g. It was proved that β-CGTase and CDase had a great prospect in the future application.

The characterization and evaluation of the synthesis of large-ring cyclodextrins (CD9-CD22) and α-tocopherol with enhanced thermal stability.

Large-ring cyclodextrins LR-CDs (CD9–CD22) were obtained from rice starch using cyclodextrin glycosyltransferase (CGTase), and were used as a wall material for embedding α-tocopherol. Complexes of α-tocopherol and LR-CDs were prepared by co-precipitation. A molar ratio of α-tocopherol/LR-CD of 1 : 2 showed the highest encapsulation efficiency. The surface morphology of the complex particles was observed to vary from irregular flakes to the formation of smaller clusters of particles using scanning electron microscopy (SEM). Based on 1H NMR and FT-IR observations, the inclusion complexes exhibited significant chemical shifts of 0.3 ppm and decreased peak signals. In addition, thermal analysis showed that the microcapsules improved the thermostability of the α-tocopherols. Antioxidant activity analysis proved the stability of α-tocopherol during storage. This study could serve as a reference for the more effective use of LR-CDs as wall materials.

Production of the Enzyme Cyclodextrin Glycosyltransferase Using Different Fermentation Techniques

Cyclodextrins produced by cyclodextrin glycosyltransferase (CGTase) are widely used in the pharmaceutical industry to improve the solubility of drug substances as well as protect them against oxidation. The use of this enzyme in the cosmetics industry is also significant. CGTase is an enzyme that belongs to the α-amylase family, which is part of the group of non-Leloir glycosyltransferases. Enzyme-catalysed transglycosylation reactions may involve cyclization, coupling and disproportionation processes. The enzyme CGTase is mostly used to produce cyclodextrins (CDs). CGTase can produce α-, β- and γ-CDs during transglycosylation reactions, depending on the number of glucopyranose units involved (6, 7 or 8). The enzyme CGTase can also be used for enzymatic bioconversion, e.g., in the development of alternative sweeteners, where the bitter aftertaste of the product is reduced during the enzymatic bioconversion of steviol glycosides, thereby obtaining an even sweeter and more advantageous material. In our research, the enzyme CGTase was produced using different fermentation techniques to compare the activity and amount of CGTase produced by each process and optimize the subsequently planned scale-up. In our studies, the strain DSM 13 of Bacillus licheniformis was used, which produced CGTase extracellularly. During the experiments the batch, fed-batch and semi-continuous fermentation techniques were compared in terms of enzymatic production. All cultivation processes were carried out in a desktop lab scale fermenter.

Improved production of cyclodextrin glycosyltransferase from Bacillus stearothermophilus NO2 in Escherichia coli via directed evolution

Cyclodextrin glycosyltransferases (CGTases) are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. The CGTase from Bacillus stearothermophilus NO2 possesses excellent catalytic properties but suffers from low production in E. coli. In this study, directed evolution was used to create three point mutants (I631T, I641T and K647E) that were produced in E. coli with shake-flask yields 1.7-, 2.1-, and 2.2-fold higher than that of wild-type, respectively. The wild-type and K647E were then produced in a 3-L fermenter. The CGTase activity of the K647E (1904 U mL-1) was 2.0-fold higher than that of the wild-type. The K647E fermentation supernatant could be used directly to prepare 2-O-α-D-glucopyranosyl-L-ascorbic acid, reducing the costs associated with its production. Structural modeling of the three mutants suggested that hydrophilicity, hydrogen bonding, and negative charge may be responsible for their improved production. Since K647 is conserved in the CGTase family, the corresponding residues in the CGTases from Bacillus circulans 251, Paenibacillus macerans, and Anaerobranca gottschalkii were changed to glutamic acid. Productions of the resulting K647E mutants were 2.0-, 1.5-, and 1.0-fold higher than those of their respective wild-types. Electrostatic protein surface analysis suggested that mutations occurring at low negative surface charge may increase CGTase production.

Recovery of starch from cassava bagasse for cyclodextrin production by sequential treatment with α-amylase and cyclodextrin glycosyltransferase

Abstract This work aimed at the enzymatic recovery of starch from cassava bagasse using α-amylase and the production of cyclodextrins (CDs) by cyclodextrin glycosyltransferase (CGTase). Cassava bagasse containing 50% (w/w) starch (dry basis) was hydrolyzed by α-amylase until a maximum starch recovery as a maltooligosaccharide solution. Afterwards, this solution was used for the production of CDs using CGTase as biocatalyst. Optimal conditions for starch extraction using α-amylase were 60 °C and 0.15 mL enzyme/kg dry bagasse, with 93% starch recovery. Several operational parameters were evaluated to optimize the CD yields, such as starch dextrose equivalent (DE), temperature and CGTase loading. The starch DE did not significantly influence the production of CDs, resulting in a similar yield for all experiments (43%). The highest productivity of CDs (93.6 mg of CDs per enzyme Unit) was obtained using an enzyme loading of 2.5 U/g starch for 1 h at 60 °C and pH 6.0. After 6 h of reaction at 60 °C and pH 6.0, the CD yield reached a value of 50%. These findings did indicate that cassava bagasse is a promising feedstock for the production of CDs using this bi-enzymatic system.

Biosynthesis of methyl glucoside and its antibacterial activity against Staphylococcus aureus and Escherichia coli

In this study, the methyl glucosides (MGn) was synthesized using β-cyclodextrin (β-CD) and methanol through the coupling reaction of recombinant cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11. The optimal condition for the synthesis of MGn consisted of an incubation of 1.5% (w/v) β-CD and 500 U/mL of CGTase in a water/methanol solution containing 30% (v/v) methanol at pH 6.0, 50 °C for 120 h. Upon analysis by TLC, at least three MG products were observed. The molecular weight of the main transferred product was 217.08 Da; this value was in accordance with methyl monoglucoside (MG1). MG1 was produced and prepared on a large scale and subsequently purified by preparative TLC. The combined 1H- and 13C-NMR analysis confirmed that the structure of MG1 was methyl-α-d-glucopyranoside. In addition, MG1 showed emulsification activity and stability in its formation in water and n-hexadecane. The antibacterial activity of MG1 was also determined by agar disc diffusion method. The results found that the MG1 (1, 5 and 10 mg/disc) showed antibacterial activity against E. coli ATCC 25922 only, with inhibition zones of 28, 38 and 40 mm, respectively. The MIC values (mg/mL) of MG1 against S. aureus ATCC 25923 and E. coli ATCC 25922 were found to be 20.00 and 0.63, while MBC values (mg/mL) were 40.00 and 0.63, respectively.

Cyclodextrin Glycosyltransferase-Catalyzed Synthesis of Pinoresinol-α-D-glucoside Having Antioxidant and Anti-Inflammatory Activities

Enzymatic synthesis of pinoresinol-α-glucosides (PGs) was performed through the transglycosylation reaction catalyzed by the recombinant cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11 using β-cyclodextrin as the glycosyl donor and pinoresinol as the acceptor molecule. Incubation of 0.5% (wt/vol) β-cyclodextrin with 1.5% (wt/vol) pinoresinol (P) and 80.0 U/mL of CGTase in 20 mM of Tris–HCl buffer (pH 9.0) at 50oC for 60 h was optimal for PGs synthesis. Under these conditions, two PG transfer products with molecular weights of 544 and 682 Da corresponding to pinoresinol monoglucoside (PG1-I and PG1-II) and pinoresinol diglucoside (PG2), respectively, were detected by TLC and MS. The structures of PG1 and PG2 were confirmed as pinoresinol-α-D-glucopyranoside and pinoresinol-α-D-diglucopyranoside, respectively, by 1H-NMR analysis. The free-radical scavenging and anti-inflammatory activities of PG1 and PG2 were reduced as compared with the original P using α,α-diphenyl-β-picrylhydrazyl radical scavenging reactions and the β-glucuronidase inhibition assay. The loss of antioxidant and anti-inflammatory activities might result from the addition of glucose in the position 4-OH of P that may have disturbed its electron rotation. In vivo, PGs are converted to Ps before they are absorbed and so loss of activity may be minimal. Interestingly, the α-glycosylated compounds which showed the change of physicochemical properties such as increase of solubility and sweetness could promote a positive effect on the bioavailability of original P.

Optimization of defined medium for recombinant Komagataella phaffii expressing cyclodextrin glycosyltransferase

The cyclodextrin glycosyltransferase (CGTase) is an important enzyme for cyclodextrin (CD) production, and is also widely used in the biotechnology, food, and pharmaceuticals industries. Secretory CGTase production by recombinant Komagataella phaffii using defined medium is a promising approach because of low cost, less impurity protein. It was found that no CGTase was expressed using traditional defined medium (basal salt medium [BSM]) because of pH value decreasing significantly. CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5-7.0. β-CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium. These results showed that it was necessary to establish recombinant K. phaffii-based special defined medium although the same host cell used for different heterologous protein expression.

RQ3, A Natural Rebaudioside D Isomer, Was Obtained from Glucosylation of Rebaudioside A Catalyzed by the CGTase Toruzyme 3.0 L.

In this study, a monoglucosyl rebaudioside A product was isolated from the mixture of glucosylated rebaudioside A obtained from the most reported and industrial used cyclodextrin glycosyl transferase, Toruzyme 3.0 L (CGTase, Toruzyme 3.0 L). The molecular structure of the monoglucosyl rebaudioside A was characterized using LC-MS/MS and methylation analysis combined with 1D and 2D NMR, indicating that it is 13-[(2-O-(3-α-O-D-glucopyranosyl)-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (also known as RQ3, which naturally exists in Stevia extract as an isomer of rebaudioside D). This study may help to further understand the reaction mechanism of glucosylation of steviol glycoside assisted by Toruzyme 3.0 L in the aspect of molecule linkage pattern, and also benefit theapplication of the glucosylated rebaudiosides.

Variants at position 603 of the CGTase from Bacillus circulans STB01 for reducing product inhibition

Inhibition of cyclodextrin glycosyltransferases (CGTases) by their product cyclodextrins limits the efficiency of cyclodextrin production. In an effort to produce variants with good activity but reduced product inhibition, six mutants were constructed at position 603 of the CGTase from Bacillus circulans STB01, which exhibits mixed-type product inhibition. In a kinetic analysis, N603I showed reduced noncompetitive inhibition while N603K, N603H and N603R showed increased noncompetitive inhibition. Unexpectedly, N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition. Noncompetitive product inhibition is closely related to the interaction between the cyclodextrin and the enzyme in maltose binding site 2 (MBS2). Structural models led to the suggestion that there is increased interaction between maltose binding sites 1 and 2 in mutants N603E and N603D, which may have led to the unexpected results. N603D exhibited a 23.9% greater cyclodextrin yield per gram of enzyme than the wild-type, suggesting it has potential for industrial use. Further reductions in product inhibition may be gained through studies of maltose binding site interactions.

Cyclodextrin glucosyltransferase immobilization on polydopamine-coated Fe3O4 nanoparticles in the presence of polyethyleneimine for efficient β-cyclodextrin production

An enzymatic approach to β-cyclodextrin synthesis attracts the attention of food engineers and chemists because of the broad applications of β-cyclodextrin in the food industry and the demand for a green process for β-cyclodextrin production. Achieving a high recovery of cyclodextrin glycosyltransferase through immobilization and a high proportion of β-cyclodextrin in a product mixture of cyclodextrins would improve the purification process of β-cyclodextrin. In this study, a recombinant cyclodextrin glycosyltransferase (CGTase) was immobilized on polydopamine-Fe3O4 (PDA-Fe3O4) nanoparticles in the presence of polyethyleneimine (PEI). After optimization of the preparation of the PDA-Fe3O4 nanoparticles and the CGTase immobilization step, 77.9% of the CGTase was recovered. The immobilized CGTase exhibited good thermal and operational stabilities, and gave a ratio of 88.9% β-cyclodextrin when the reaction temperature and kinetic parameters were optimized. Immobilized CGTase was recycled nine times and retained 19.1% β-activity and 0% γ-activity. This study has established a reliable approach to the high recovery of CGTase during efficient β-cyclodextrin production.

An Efficient One‐Pot Enzymatic Synthesis of Cardiac Glycosides with Varied Sugar Chain Lengths

AbstractAn efficient one‐pot enzymatic synthesis of cardiac glycosides with varied sugar chain lengths (average yield &gt;80%) was carried out through sequential glycosylation using a steroid glycosyltransferase UGT74AN3 from Catharanthus roseus and a cyclodextrin glycosyltransferase from Bacillus licheniformis. A series of cardiac glycosides were obtained and showed enhanced affinity and selectivity for inhibition of the α2 isoform of Na+/K+‐ATPase. These findings demonstrate the significant potential of the one‐pot biocatalytic approach in the synthesis of diverse cardiac glycosides as potentially safer cardiotonic agents.magnified image

Cover Image, Volume 94, Issue 5

The cover image is based on the Research Article Cross‐linked enzyme aggregates of recombinant cyclodextrin glycosyltransferase for high‐purity β‐cyclodextrin production by Jianguo Zhang et al., DOI: 10.1002/jctb.5912. image

Cross‐linked enzyme aggregates of recombinant cyclodextrin glycosyltransferase for high‐purity β‐cyclodextrin production

AbstractBACKGROUNDHigh‐purity α‐, β‐, or γ‐cyclodextrin (CD) production using cyclodextrin glycosyltransferase (CGTase) as biocatalyst to cyclize starch is advantageous owing to its low cost of purification and product specificity. Several approaches have been investigated to enhance product specificity of CGTase, such as new CGTase isolation, CGTase gene (cgt) mutation, and chemical addition.RESULTSRecombinant CGTase was cross‐linked by glutaraldehyde to obtain cross‐linked enzyme aggregates of recombinant CGTase (CLEA‐CGTase) at 85 °C because free CGTase showed high product specificity at 85 °C. The CLEA‐CGTase was prepared under the conditions 75 U mL−1 CGTase with 0.1% (v/v) glutaraldehyde for 10 min at 85 °C after optimization first, and produced a high proportion of β‐CD from soluble starch at 50 °C in the conversion system. The conversion process was carried out with 10 to 200 U mL−1 CLEA‐CGTase, resulting in 100% β‐CD from soluble starch after 420 min conversion of the potato starch solution at 50 °C. And the proportion of β‐CD was still higher than 90% when 8000 U mL−1 CLEA‐CGTase was added.CONCLUSIONSCLEA technology maintained CGTase conformation of 85 °C, and produced a high proportion of β‐CD at 50 °C. This research showed the CLEA technology kept the conformation of enzyme for specific product production. © 2018 Society of Chemical Industry

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).