Subsite mapping of enzymes. Application of the depolymerase computer model to two alpha-amylases.

In the preceding paper (Allen and Thoma, 1976) we developed a depolymerase computer model, which uses a minimization routine to establish a subsite map for a depolymerase. In the present paper we show how the model is applied to experimental data for two alpha-amylases. Michaelis parameters and bond-cleavage frequencies for substrates of chain lengths up to twelve glucosyl units have been reported for Bacillus amyloliquefaciens, and a subsite map has been proposed for this enzyme [Thoma et al. (1971) J. Biol. Chem. 246, 5621-5635]. By applying the computer model to the experimental data, we have arrived at a ten-subsite map. We find that a significant improvement in this map is achieved by allowing the hydrolytic rate coefficient to vary as a function of the number of occupied subsites comprising the enzyme-binding region. The bond-cleavage frequencies, the enzyme is found to have eight subsites. A partial subsite map is arrived at, but the entire binding region cannot be mapped because Michaelis parameters are complicated by transglycosylation reactions. The hydrolytic rate coefficients for this enzyme are not constant.     

Monte Carlo simulation of the α-amylolysis of amylopectin potato starch. 2. α-amylolysis of amylopectin

A model is presented that describes all the saccharides that are produced during the hydrolysis of starch by an -amylase. Potato amylopectin, the substrate of the hydrolysis reaction, was modeled in a computer matrix. The four different subsite maps presented in literature for -amylase originating from Bacillus amyloliquefaciens were used to describe the hydrolysis reaction in a Monte Carlo simulation. The saccharide composition predicted by the model was evaluated with experimental values. Overall, the model predictions were acceptable, but no single subsite map gave the best predictions for all saccharides produced. The influence of an (16) linkage on the rate of hydrolysis of nearby (14) linkages by the -amylase was evaluated using various inhibition constants. For all the subsites considered the use of inhibition constants led to an improvement in the predictions (a decrease of residual sum of squares), indicating the validity of inhibition constants as such. As without inhibition constants, no single subsite map gave the best fit for all saccharides. The possibility of generating a hypothetical subsite map by fitting was therefore investigated. With a genetic algorithm it was possible to construct hypothetical subsite maps (with inhibition constants) that gave further improvements in the average prediction for all saccharides. The advantage of this type of modeling over a regular fit is the additional information about all the saccharides produced during hydrolysis, including the ones that are difficult to measure experimentally.     

Wedtrilosides A and B,two new diterpenoid glycosides from the leaves of Wedelia trilobata (L.) Hitchc. with α-amylase and α-glucosidase inhibitory activities

In the ongoing research to find new diabetes constituents from the genus Wedelia, the chemical constituent of Wedelia trilobata leaves, a Vietnamese medicinal plant species used to treat type 2 diabetes mellitus, was selected for detailed investigation. From a methanolic extract, two new ent-kaurane diterpenoids, wedtrilosides A and B (1 and 2), along with five known metabolites (3–7), were isolated from W. trilobata. The chemical structures of (1–7) were assigned via spectroscopic techniques (IR, 1D, 2D NMR and HR-QTOF-MS data) and chemical methods. The isolates were evaluated for α-amylase and α-glucosidase inhibitory activities compared to the clinical drug acarbose. Among them, compounds 4, 6, and 7 showed the most potent against α-glucosidase enzyme with IC₅₀ values of 27.54 ± 1.12, 173.78 ± 2.37, and 190.40 ± 2.01 μg/mL. While moderate inhibitory effect against α-amylase was observed with compounds 6 and 7 (with IC₅₀ = 181.97 ± 2.62 and 52.08 ± 0.56 μg/mL, respectively). The results suggested that the antidiabetic properties from the leaves of W. trilobata are not simply a result of each isolated compound, but are due to other factors such as the accessibility of polyphenolic groups to α-amylase and α-glucosidase activities.     

Cloning of a foreign gene coding for alpha-amylase in Bacillus subtilis.

Foreign DNA has been introduced into the genome of bacteriophage Ø3T, producing a specialized transducing bacteriophage containing the genetic information encoding α-amylase from $\text{Bacillus}\text{amyloliquefaciens}$H. Genetic and physical studies demonstrated that the gene(s) is inserted into the bacteriophage genome. These bacteriophage carrying the gene(s) encoding α-amylase lysogenized and replicated in $\text{Bacillus}\text{subtilis}$ with normal efficiency. In these lysogens, the gene(s) encoding α-amylase appears to map near the bacteriophage attachment site rather than the chromosomal $\text{amy}$E locus. This method of construction of specialized bacteriophage should be applicable to the cloning of other genes for which no primary selection exists.     

In vitro and in silico elucidation of antidiabetic and anti-inflammatory activities of bioactive compounds from Momordica charantia L.

Bitter melon (Momordica charantia) has been used to manage diabetes and related conditions in various parts of the world. In the present study, ten compounds were isolated from acetone and methanol extracts of bitter melon. The chemical structures of compounds were unambiguously elucidated by 1D, 2D NMR, and high-resolution mass spectra. Identified compounds 1–7 exhibited significant inhibition of α-amylase and moderate inhibition of α-glucosidase activities. Momordicoside G and gentisic acid 5-O-β-d-xyloside showed the highest inhibition of α-amylase (70.5%), and α-glucosidase (56.4%), respectively. Furthermore, molecular docking studies of isolated compounds 1–7 were able to bind to the active sites of both enzymes. Additionally, the isolated compounds 1–7 significantly attenuated lipopolysaccharide (LPS)-induced inflammation, downregulating the expression of pro-inflammatory markers NF-κB, INOS, IL-6, IL-1β, TNF-α, and Cox-2 in murine macrophage RAW 264.7 cells. One phenolic derivative, gentisic acid 5-O-β-d-xyloside, was isolated and identified for the first time from bitter melon, and significantly suppressed the expression of Cox-2 and IL-6 compared to the LPS-treated group. α-Amylase and α-glucosidase are targets of anti-diabetes drugs, our findings suggest that compounds purified from bitter melon may have potential to use as functional food ingredients for the prevention of type 2 diabetes and related inflammatory conditions.     

Subsite mapping of enzymes. Depolymerase computer modelling.

We have developed a depolymerase computer model that uses a minimization routine. The model is designed so that, given experimental bond-cleavage frequencies for oligomeric substrates and experimental Michaelis parameters as a function of substrate chain length, the optimum subsite map is generated. The minimized sum of the weighted-squared residuals of the experimental and calculated data is used as a criterion of the goodness-of-fit for the optimized subsite map. The application of the minimization procedure to subsite mapping is explored through the use of simulated data. A procedure is developed whereby the minimization model can be used to determine the number of subsites in the enzymic binding region and to locate the position of the catalytic amino acids among these subsites. The degree of propagation of experimental variance into the subsite-binding energies is estimated. The question of whether hydrolytic rate coefficients are constant or a function of the number of filled subsites is examined.     

Characteristics of an aqueous two-phase system for α-amylase production

The characteristics of an aqueous two-phase system for the overproduction of extracellular enzyme through α-amylase fermentation by Bacillus amyloliquefaciens were investigated. With higher molecular weight of polyethylene glycol (PEG) or lower molecular weight of dextran, the partition coefficient of α-amylase was increased. α-Amylase biosynthesis was increased when PEG 6000 was included in the medium compared to the medium without PEG. Phosphate addition to the PEG-dextran system improved the partition coefficient of α-amylase, but deactivated α-amylase severely. By using sodium sulfate instead of phosphate, α-amylase deactivation was negligible, and high partitioning of the enzyme in the top phase was obtained.     

Trehalose-6-phosphate synthetase activity in extracts of Dictyostelium discoideum.

Trehalose-6-phosphate (T-6-P) synthetase activity in extracts of Dictyostelium discoideum has been reexamined in an effort to resolve discrepancies between the results of previous studies (R. Roth and M. Sussman (1966). Biochim. Biophys. Acta, 122, 225; K. A. Killick and B. E. Wright (1972). J. Biol. Chem., 247, 2967). We find that T-6-P synthetase is not cold sensitive as reported by Killick and Wright (1972), is not present in bacterial-grown vegetative cells (though subject to some modulation by other nutritional conditions), and is not in our hands unmasked or activated by ammonium sulfate fractionation. We conclude that the pattern of T-6-P synthetase accumulation and disappearance during fruiting body construction in D. discoideum is as originally described by R. Roth and M. Sussman (1968). J. Biol. Chem., 243, 5081) and confirmed elsewhere (P. C. Newell et al. (1972). J. Mol. Biol., 63, 373; R. W. Brackenbury et al. (1974). J. Mol. Biol., 90, 529; B. D. Hames and J. M. Ashworth (1974). Biochem. J., 142, 301).     

The hyperthermophilic α-amylase from <Emphasis Type="Italic">Thermococcus</Emphasis> sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct explanation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial processes.     

Synthesis of the glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan, which serve as a hexosamine acceptor for enzymatic glycosyl transfer

Betaglycan, also known as TGF-β type III receptor, is a membrane-anchored proteoglycan, which has two glycosaminoglycan (GAG) attachment sites (López-Casillas, F.; Payne, H. M.; Andres, J. L.; Massagué, J. J.Cell Biol.1994, 124, 557-568). Chondroitin sulfate (CS) or heparan sulfate (HS) can attach to the first site, Ser⁵³⁵, whereas only CS attaches to the second, Ser⁵⁴⁶. Although the mechanism behind the assembly of CS and HS is not fully understood, it has been reported that the assembly of HS requires not only a cluster of acidic residues but also hydrophobic residues located near the Ser-Gly attachment sites (Esko, J. D. Zhang, L. Curr. Opin. Struct. Biol.1996, 6, 663-670). To further understand the effects of amino acids close to the Ser residues of the GAG-attachment sites on the glycosyltransferases, two tetraosyl peptides derived from the CS attachment sites of betaglycan, GlcA-Gal-Gal-Xyl-SerGlyAspAsnGly (1) and GlcA-Gal-Gal-Xyl-SerGlyAspAsnGlyPheProGly (2), were synthesized, and used as donor substrates for β1,4-N-acetylgalactosaminyltransferase-I (β4GalNAcT-I) and α1,4-N-acetylglucosaminyltransferase-I (α4GlcNAcT-I). Both the chemically synthesized linkage region tetrasaccharides were far better acceptors for β4GalNAcT-I than for α4GlcNAcT-I in vitro, although they also showed appreciable acceptor activity for α4GlcNAcT-I.     

Accumulation of messenger RNA for extracellular enzymes as a general phenomenon in Bacillus amyloiquefaciens

Recently it has been established that in harvested Bacillus amyloliquefaciens there exists a pool of messenger RNA which can support de novo extracellular protease production for 80 minutes Both, McInnes, Hanlon, May &; Elliott 1972. The present study shows that this is not peculiar to protease; the other two extracellular enzymes under study, α-amylase and ribonuclease, show similar characteristics. Synthesis of all three extracellular enzymes is insensitive to rifampicin and actinomycin D for extended time periods after cessation of RNA and cellular protein synthesis.     

Enhanced extracellular production of α-amylase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by optimization of regulatory elements and over-expression of PrsA lipoprotein

α-Amylase was used as a heterologous model protein to investigate the effects of promoters, signal peptides and over-expression of an extra-cytoplasmic molecular chaperone, PrsA lipoprotein, on enhancing the secretion of α-amylase in Bacillus subtilis. Four promoters and six signal peptides were compared, successively, and the highest yield of α-amylase was achieved under the promotion mediated by P_AprE, a strong constitutive promoter, and secretion by SP_nprE, a signal peptide from B. subtilis. Moreover, under conditions of overexpressed PrsA lipoprotein, the secretion production and activity of α-amylase increased to 2.5-fold. The performance of the recombinant B. subtilis 1A751PL31 was evaluated with a fed-batch fermentation in a 7.5 l fermentor. Optimization of regulatory elements and over-expression of PrsA lipoprotein had a significant effect on enhancing the production of α-amylase in B. subtilis.     

1,4-Butanediol diglycidyl ether coupling of carbohydrates to sepharose: Affinity adsorbents for lectins and glycosidases

The use of 1,4-butanediol diglyeidyl ether to couple carbohydrate ligands to Sepharose [Sundberg, L., and Porath, J. (1975) J. Chromatogr. 90, 87; Vretblad, P. (1976) Biochim. Biophys. Acta. 434, 169] has been explored with a number of carbohydrate derivatives, such as amino sugars (N-acetylglucosamine, N-acetylgalactosamine, nonacetylated short oligomers of glucosamine), methyl glycosides (O-methyl-α-mannoside and O-methyl-β-galactoside), di- and oligosaccharides (lactose and stachyose), and polysaccharides (yeast mannan and dextrin). Using model reactions it has been established that all but the reducing sugars are sufficiently stable to withstand the alkaline conditions required for the coupling reaction, and that the primary alcohol at the C₆ position of the sugars is the primary site for the coupling reaction. In the case of glucosamine, the amino group is six times more reactive than is the 6-hydroxy group. The following affinity adsorbents have been tested and found useful in the purification of lectins and glycosidases: a yeast mannan adsorbent binds jack bean α-mannosidase (0.15 mg/ml of adsorbent) and concanavalin A (0.5 mg/ml); a dextrin adsorbent binds bovine pancreatic α-amylase (2 mg/ml of adsorbent); a lactose adsorbent binds peanut lectin (5 mg/ml of adsorbent); a (GlcNAc)_3–4 adsorbent binds wheat germ lectin (50 mg/ml of adsorbent); and, as previously reported [Vretblad, P. (1976) Biochim. Biophys. Acta. 434, 169], a GlcNAc adsorbent binds wheat germ lectin (10 mg/ml of adsorbent) and a GalNAc adsorbent binds soybean lectin (18 mg/ml of adsorbent).     

Synthesis, α-amylase inhibitory potential and molecular docking study of indole derivatives

In search of potent α-amylase inhibitor we have synthesized eighteen indole analogs (1–18), characterized by NMR and HR-EIMS and screened for α-amylase inhibitory activity. All analogs exhibited a variable degree of α-amylase inhibition with IC₅₀ values ranging between 2.031 ± 0.11 and 2.633 ± 0.05 μM when compared with standard acarbose having IC₅₀ values 1.927 ± 0.17 μM. All compounds showed good α-amylase inhibition. Compound 14 was found to be the most potent analog among the series. Structure-activity relationship has been established for all compounds mainly based on bringing about the difference of substituents on phenyl ring. To understand the binding interaction of the most active analogs molecular docking study was performed.     

Modeling of a new tubercular maltosyl transferase,GlgE, study of its binding sites and virtual screening

Recently maltosyl transferase of Mycobacterium tuberculosis (mtb GlgE) belonging to α-amylase family has been identified as a potential drug target. Despite its importance, its three dimensional (3D) structure is unavailable. In this study we have modeled its 3D homo-dimeric structure using its homologue in Streptomyces ceolicolor (stp GlgE) as the template. Its monomer consists of five domains and four inserts, out of which two inserts are unique to mtb GlgE. It also has three binding cavities. One primary (pbs) and two secondary (sbs1 and sbs2), with one unique insert appearing within sbs2. Investigation of its homo-dimeric model revealed the presence of a disulphide bridge between Cys-29 of both the chains which is absent in stp GlgE. Virtual screening with known substrates and substrate analogues of α-amylase family proteins indicated better binding of maltose to sbs1 than pbs. Among all computationally screened substrates 3-O-Alpha-d-Glucopyranosyl-d-Fructose (OTU) docked with best binding affinity to pbs. Interaction of known inhibitors of α-amylase family proteins from CHEMBL is also studied. This reveals for the first time the unique 3D structure of mtb GlgE and provides insights into its active sites and substrate binding affinities. This may help in developing new anti-tubercular drugs and its analogues.     

The effect of glucose on the expression of a clonedBacillus amyloliquefaciens α-amylase gene in strains ofBacillus subtilis

Bacillus subtilis strain 1A297 was shown to relieve the glucose repression of a clonedB. amyloliquefaciens -amylase gene carried on the hybrid plasmid pVC102 without affecting its temporal activation. However, glucose repression of -amylase occurred when pVC102, was introduced intoB. subtilis strain 1A289. Glucose repression was relieved by -methyl-d-glucoside, an analog of glucose that blocks its uptake. The relief of glucose repression in 1A297 did not act at the level of plasmid copy number. As 1A297 was capable of exerting glucose repression on a homologous chromosomally encoded gene, it is postulated that the putativetrans-acting product involved in glucose repression inB. subtilis (Nicholson and Chambliss, 1986, J. Bacteriol. 165:663–670) is altered in strain 1A297 and does not recognize theB. amyloliquefaciens -amylase gene.     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

α-Amylase production by Bacillus amyloliquefaciens in a membrane recycle bioreactor

A strain of Bacillus amyloliquefaciens was cultivated in a membrane recycle bioreactor for the production of an extracellular -amylase. Continuous cultivations of B. amyloliquefaciens in the recycle fermentor led to higher cell mass and volumetric productivities than the ones obtained in batch or chemostat cultivations; the -amylase activities were lower than in the batch mode but significantly higher than in the chemostat mode. The operation of the membrane recycle bioreactor was sometimes disturbed by high broth viscosity leading to a stronger fouling of the membrane.