Identification of the sequence motif of glycoside hydrolase 13 family members

A bioinformatics analysis of sequences of enzymes of the glycoside hydrolase (GH) 13 family members such as α-amylase, cyclodextrin glycosyltransferase (CGTase), branching enzyme and cyclomaltodextrinase has been carried out in order to find out the sequence motifs that govern the reactions specificities of these enzymes by using hidden Markov model (HMM) profile. This analysis suggests the existence of such sequence motifs and residues of these motifs constituting the -1 to +3 catalytic subsites of the enzyme. Hence, by introducing mutations in the residues of these four subsites, one can change the reaction specificities of the enzymes. In general it has been observed that α -amylase sequence motif have low sequence conservation than rest of the motifs of the GH13 family members.     

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.     

Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity

The enzymes from the alpha-amylase family all share a similar alpha-retaining catalytic mechanism but can have different reaction and product specificities. One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 A X-ray structures of E257A/D229A CGTase in complex with maltoheptaose and maltohexaose. Both sugars are bound at the donor subsites of the active site and the acceptor subsites are empty. These structures mimic a reaction stage in which a covalent enzyme-sugar intermediate awaits binding of an acceptor molecule. Comparison of these structures with CGTase-substrate and CGTase-product complexes reveals three different conformational states for the CGTase active site that are characterized by different orientations of the centrally located residue Tyr 195. In the maltoheptaose and maltohexaose-complexed conformation, CGTase hinders binding of an acceptor sugar at subsite +1, which suggests an induced-fit mechanism that could explain the transglycosylation activity of CGTase. In addition, the maltoheptaose and maltohexaose complexes give insight into the cyclodextrin size specificity of CGTases, since they precede alpha-cyclodextrin (six glucoses) and beta-cyclodextrin (seven glucoses) formation, respectively. Both ligands show conformational differences at specific sugar binding subsites, suggesting that these determine cyclodextrin product size specificity, which is confirmed by site-directed mutagenesis experiments.     

Enzymatic synthesis of α-glucosyl-timosaponin BII catalyzed by the extremely thermophilic enzyme: Toruzyme 3.0L

Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of ¹³C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1→4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100 °C. In addition, we also found that the α-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as γ-amylase (GH15 family), had no such activity under the same reaction conditions.     

The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 implications for product inhibition and product specificity.

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) is used for the industrial production of cyclodextrins. Its application, however, is hampered by the limited cyclodextrin product specificity and the strong inhibitory effect of cyclodextrins on CGTase activity. Recent structural studies have identified Arg47 in the Bacillus circulans strain 251 CGTase as an active-site residue interacting with cyclodextrins, but not with linear oligosaccharides. Arg47 thus may specifically affect CGTase reactions with cyclic substrates or products. Here we show that mutations in Arg47 (to Leu or Gln) indeed have a negative effect on the cyclization and coupling activities; Arg47 specifically stabilizes the oligosaccharide chain in the transition state for these reactions. As a result, the mutant proteins display a shift in product specificity towards formation of larger cyclodextrins. As expected, both mutants also showed lower affinities for cyclodextrins in the coupling reaction, and a reduced competitive (product) inhibition of the disproportionation reaction by cyclodextrins. Both mutants also provide valuable information about the processes taking place during cyclodextrin production assays. Mutant Arg47-->Leu displayed an increased hydrolyzing activity, causing accumulation of increasing amounts of short oligosaccharides in the reaction mixture, which resulted in lower final amounts of cyclodextrins produced from starch. Interestingly, mutant Arg47-->Gln displayed an increased ratio of cyclization/coupling and a decreased hydrolyzing activity. Due to the decreased coupling activity, which especially affects the production of larger cyclodextrins, this CGTase variant produced the various cyclodextrins in a stable ratio in time. This feature is very promising for the industrial application of CGTase enzymes with improved product specificity.     

Carbohydrate-Binding Module–Cyclodextrin Glycosyltransferase Fusion Enables Efficient Synthesis of 2-O-d-Glucopyranosyl-l-Ascorbic Acid with Soluble Starch as the Glycosyl Donor

In this study, we achieved the efficient synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) from soluble starch by fusing a carbohydrate-binding module (CBM) from Alkalimonas amylolytica α-amylase (CBM_Amy) to cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans. One fusion enzyme, CGT-CBM_Amy, was constructed by fusing the CBM_Amy to the C-terminal region of CGTase, and the other fusion enzyme, CGTΔE-CBM_Amy, was obtained by replacing the E domain of CGTase with CBM_Amy. The two fusion enzymes were then used to synthesize AA-2G from soluble starch as a cheap and easily soluble glycosyl donor. Under the optimal conditions, the AA-2G yields produced using CGTΔE-CBM_Amy and CGT-CBM_Amy were 2.01 g/liter and 3.03 g/liter, respectively, which were 3.94- and 5.94-fold of the yield from the wild-type CGTase (0.51 g/liter). The reaction kinetics of the two fusion enzymes were analyzed and modeled to confirm the enhanced specificity toward soluble starch. It was also found that, compared to the wild-type CGTase, the two fusion enzymes had relatively high hydrolysis and disproportionation activities, factors that favor AA-2G synthesis. Finally, it was speculated that the enhancement of soluble starch specificity may be related to the changes of substrate binding ability and the substrate binding sites between the CBM and the starch granule.     

Molecular cloning and characterization of a novel γ-CGTase from alkalophilic Bacillus sp.

We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme β-/γ-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of β-/γ-CGTase. CGTase825-6 produced γ-cyclodextrin (CD) as the main product, but degradation of γ-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between γ-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 β-/γ-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for γ-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce γ-CD and to study the differences of producing mechanisms between γ-CD and β-CD.     

Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the alpha-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 micromol min(-1) mg(-1)); acarbose is a strong inhibitor of CGTase and of most other alpha-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of alpha-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the alpha-amylase family, changed ATase into an enzyme with 4-alpha-glucanotransferase activity (3.4 micromol min(-1) mg(-1)). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 micromol min(-1) mg(-1)).     

Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans

We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the α-1,6-glucosidic linkage of isomaltooligosaccharides from their non-reducing ends to produce α-glucose. By using the mutant of catalytic acid Glu236→Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 Å resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (β/α)₈-barrel in domain A, which is common to the α-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites −1 to +2. The environment of the glucose residue at subsite −1 is similar to the environment of this residue in the α-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite −1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile α-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile α-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the α-amylase structure from Bacillus subtilis. The comparison with the α-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of α-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes.     

Synthesis and glycosidase inhibitory activity of novel (2-phenyl-4H-benzopyrimedo[2,1-b]-thiazol-4-yliden)acetonitrile derivatives

A series of (2-phenyl-4H-benzopyrimodo[2,1-b][1,3]thiazol-4-yliden-4-yliden)acetonitrile derivatives have been prepared by ring transformation reaction of 4-(methylthio)-2-oxo-6-aryl-2H-pyrane-3-carbonitriles. The yield of ring transformation product is moderate to good. Furthermore the glycosidase inhibitory activities were tested by using α-amylase and α-glucosidase pancreatic, intestinal and liver enzymes, responsible for hyperglycemia in type II diabetes. The results revealed that all compounds exhibit significant glycosidase inhibitory activity.     

Measurement of α-amylase and glucoamylase activities produced during fermentation

A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α-d-glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.     

Bifunctional inhibitor of α-amylase/trypsin from wheat grain

A trypsin inhibitor, isolated from whole-wheat grain (Triticum aestivum L.) by the method of biospecific chromatography on trypsin-Sepharose, was potent in inhibiting human salivary α-amylase. The bifunctional α-amylase/trypsin inhibitor was characterized by a narrow specificity for other α-amylases and proteinases. The high thermostability of the inhibitor was lost in the presence of SH group-reducing agents. The inhibitor-trypsin complex retained its activity against α-amylase. The inhibitor—α-amylase complex was active against trypsin. Studies of the enzyme kinetics demonstrated that the inhibition of α-amylase and trypsin was noncompetitive. Our results suggest the existence of two independent active sites responsible for the interaction with the enzymes.     

Trehalose production with a new enzymatic system from Sulfolobus solfataricus KM1

An amylolytic activity that converts soluble starch to α,α-trehalose (trehalose) was found in the cell homogenate of the hyperthermophilic, acidophilic archaeum Sulfolobus solfataricus KM1. Two enzymes, a glycosyltransferase and an α-amylase, which are essential for this activity, were purified to homogeneity. A glycosyltransferase catalyzed the conversion of maltooligosaccharides to glycosyltrehaloses and an α-amylase catalyzed the hydrolysis of glycosyltrehaloses to trehalose. The glycosyltransferase transferred an oligomer segment of maltooligosaccharide to the C1–OH position of glucose, located at the reducing end of the maltooligosaccharide, to produce a glycosyltrehalose having an α-1,1 linkage. The α-amylase hydrolyzed only the α-1,4 glucosidic linkage adjacent to the trehalose unit of the glycosyltrehaloses. Their activities were maximal at 70–80°C and 70–85°C, with high thermostability, respectively. The genes encoding for both enzymes were cloned and expressed in Escherichia coli. The regions highly conserved in α-amylase family exist in the amino acid sequences of these enzymes. A new process for trehalose production from starch was developed using the purified enzymes. The yield of trehalose from starch was 81.5% using these two enzymes. This review describes our efforts to reveal in detail the characters of these enzymes involved in practical trehalose production.     

Molecular basis for the specific binding of different α-amylase inhibitors from Phaseolus vulgaris seeds to the active site of α-amylase

In search of a possible mechanism of inhibition which might be responsible for the different specificities of the three isoforms of the bean (Phaseolus vulgaris) α-amylase inhibitor α-AI1, α-AI2 and α-AIL (EC 3.2.1.1), the two isoforms α-AI2 and α-AIL were modelled from the atomic co-ordinates of α-AI1 in the α-AI1/PPA complex and docking experiments were performed with pig pancreatic α-amylase (PPA) and the modelled amylase from Zabrotes subfasciatus (ZSA). The modelled α-AI2 penetrates without any steric hindrance in the substrate cleft of both enzymes but the possible hydrogen bonds between PPA and α-AI2 seem too few to maintain the stability of the complex. α-AIL, which differs from α-AI1 and α-AI2 by the absence of post-translational proteolytic cleavage and the occurrence of two additional loops of fifteen and six residues, creates steric clashes with PPA and ZSA that prevent its penetration into the substrate cleft of the enzyme. Docking experiments explain at the molecular level the specificity of α-amylase inhibitor isoforms towards enzymes of different origins. In addition, they explain why, according to its unprocessed and more bulky character, α-AIL was previously shown to be inactive on all α-amylases assayed. In fact, this last isoform is now considered as an evolutionary intermediate between phytohaemagglutinins, arcelins and α-amylase inhibitors.     

The Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

BackgroundOriginally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1 → 4) linkages in maltodextrin/starch and the synthesis of consecutive (α1 → 6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD.MethodsThe purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies.ResultsThe A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1 → 6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1 → 4) and (α1 → 6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose.ConclusionsIn view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes.General significanceThis study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.     

Isolation,identification and in silico analysis of alpha-amylase gene of Aspergillus niger strain CSA35 obtained from cassava undergoing spoilage

In this investigation, a gene (CDF_Amyl) encoding extracellular α-amylase in Aspergillus niger strain CSA35 associated with cassava spoilage was amplified using specific primers and characterized in silico. The gene had a partial nucleotide sequence of 968 bp and encoded a protein of 222 aa residues with a molecular weight and isoelectric point of 25.13 kDa and 4.17, respectively. Its catalytic site was located in the active site domain. BLASTp analysis showed that the protein primary sequence of the α-amylase gene had 98% and 99% homologies with the α-amylase of A. niger and A. oryzae RIB40, respectively. The gene is more closely related to α-amylase genes from fungi than to bacterial, plant, or animal α-amylase genes. Restriction mapping of the gene showed it can be digested with restriction enzymes like NcoI, PstI, SmaI, and BcLI among others but not with EcoRI and EcoRV. Its protein product had a hydrophobicity score of ? 0.43 but no transmembrane helix. The CDF_Amyl protein was subcellularly localized in the secretory pathway, an indication of its release into extracellular space after secretion. Also, the 3D structure of the CDF-Amyl protein was barrel-shaped with domains characteristic of α-amylases. The encoded α-amylase Vmax is 6.90 U/mg protein and Km is 6.70 mg/ml. It was concluded that the unique characteristics of the CDF_Amyl gene and its deduced protein could find applications in biotechnological, food and pharmaceutical industries where cloning and further modification of this gene would be required for product development and improvement.     

Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.     

Mutations at subsite −3 in cyclodextrin glycosyltransferase from <Emphasis Type="Italic">Paenibacillus macerans</Emphasis> enhancing α-cyclodextrin specificity

A major disadvantage of cyclodextrin production is the limited cyclodextrin product specificity of cyclodextrin glycosyltransferase (CGTase). Here, we described mutations of Asp372 and Tyr89 at subsite −3 in the CGTase from Paenibacillus macerans strain JFB05-01. The results showed that Asp372 and Tyr89 played important roles in cyclodextrin product specificity of CGTase. The replacement of Asp372 by lysine and Tyr89 by aspartic acid, asparagine, lysine, and arginine resulted in a shift in specificity towards the production of α-cyclodextrin, which was most apparent for the mutants D372K and Y89R. Furthermore, the changes in cyclodextrin product specificity for the single mutants D372K and Y89R could be combined in the double mutant D372K/Y89R, which displayed a 1.5-fold increase in the production of α-cyclodextrin, with a concomitant 43% decrease in the production of β-cyclodextrin when compared to the wild-type CGTase. Thus, the D372K and Y89R single and double mutants were much more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The enhanced α-cyclodextrin specificity of these mutants might be a result of stabilizing the bent conformation of the intermediate in the cyclization reaction.     

Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases

The actions of cyclomaltodextrin glucanotransferases (CGTase; EC 2.4.1.19) from alkalophilic Bacillus sp. strain A2-5a (A2-5a CGTase), Bacillus macerans (Bmac CGTase), and Bacillus stearothermophilus (Bste CGTase) on amylose were investigated. All three enzymes produced large cyclic alpha-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses. However, the rates of this conversion differed among the three enzymes. The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount of each cycloamylose from CD6 to CD31 (CDn, a cycloamylose with a degree of polymerization of n). A2-5a CGTase produced 10 times more CD7, while Bmac CGTase produced 34 times more CD6 than other cycloamyloses. Bste CGTase produced 12 and 3 times more CD6 and CD7 than other cycloamyloses, respectively. The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated, and we found that CD7 and CD8 are extremely poor substrates for both hydrolytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate. By repeating these cyclization and linearization reactions, the larger cycloamyloses initially produced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8. These three enzymes also differ in their hydrolytic activities, which seem to accelerate the conversion of larger cycloamyloses into smaller cycloamyloses.     

Microbial α-amylase: A biomolecular overview

α-Amylase is an important amylolytic enzyme participating in hydrolysis of starch, the most common carbohydrate in nature. Compared to plant and animal origins, microbial α-amylase is the most popular source of industrial α-amylase. As such, high productive and favourable α-amylases for wider range of applications are highly sought after demands. The expression of α-amylase is regulated by its structural gene, amyR, DegU-P, PrsA lipoprotein, cutinase and other similar flanking genes, components of gene expression regulatory systems, molecular chaperones and enzymes. Moreover, the characteristics of α-amylase are closely related to the structures of constitutive domains and conserved regions, particularly the functional regions such as Ca²⁺-binding sites, non-catalytic carbohydrate-binding modules and surface-binding sites. Recent production of α-amylase based on genetic engineering and academic researches focused on mechanisms of catalysis greatly benefit from these biomolecular studies. Despite rapid developments, no reviews have systematically summarized these fundamental biomolecular studies. This review outlines microbial α-amylase at gene and structure levels by covering these significant aspects. The computer analytical tools are also reported, especially frequently used databases. A deeper understanding of the biomolecular basis of microbial α-amylase will significantly pave greater opportunities for industrial α-amylase and open our minds towards its related or even other enzymes.