Characterization of alpha-amylase and pullulanase activities of Clostridium thermohydrosulfuricum. Evidence for a novel thermostable amylase.
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ABSTRACT:
SUBMITTER: Melasniemi H
PROVIDER: S-EPMC1148257 | biostudies-other | 1987 Aug
REPOSITORIES: biostudies-other
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Project description:Clostridium thermohydrosulfuricum mutant Z 21-109 produced intracellular thermostable pullulanase and glucoamylase activities. The glucoamylase activity was inactivated by treating C. thermohydrosulfuricum cells with 10% (v/v) propan-1-ol at 85 degrees C in the presence of 5 mM-CaCl2. Pullulanase activity was selectively solubilized from cells by treatment with detergent and lipase. The solubilized pullulanase was purified by treatment with streptomycin sulphate and (NH4)2SO4 and by DEAE-Sephacel, octyl-Sepharose and pullulan-Sepharose chromatography. Pullulanase was purified 3511-fold and displayed homogeneity on SDS/polyacrylamide-gel electrophoresis. The pullulanase was a monomeric glycoprotein with an apparent Mr of about 136,500, and it displayed a pI of 5.9. The enzyme was enriched in both acidic and hydrophobic amino acids. The purified pullulanase was stable and optimally active at 90 degrees C. The optimum pH for activity and pH-stability ranges were 5.0-5.5 and 3.0-5.0 respectively. The enzyme was inhibited by cyclodextrins, EDTA and N-bromosuccinimide, but not by p-chloromercuribenzoate and acarbose. The pullulanase displayed a relative substrate specificity for hydrolysis of pullulan (100%) versus 75% for glycogen and 50% for soluble starch. The apparent Km, Vmax. and Kcat. values for enzyme activity on pullulan at 60 degrees C were 0.675 mg/ml, 122.5 mumol of reducing sugar formed/min per mg of protein and 16,240 min-1 respectively. The novel properties of this extremely thermostable pullulanase are discussed in relation to other purified starch-debranching enzymes.
| S-EPMC1149150 | biostudies-other
Project description:An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined. The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa beta-amylase. The beta-amylase was enriched in both acidic and hydrophobic amino acids. The pure enzyme displayed an isoelectric point of 5.1 and a pH activity optimum of 5.5. The optimum temperature for beta-amylase activity was 75 degrees C, and enzyme thermostability at 80 degrees C was enhanced by substrate and Ca2+ addition. The beta-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by alpha- and beta-cyclodextrins. The enzyme displayed kcat. and Km values for boiled soluble starch of 400,000 min-1 per mol and 1.68 mg/ml, respectively. The enzyme was antigenically distinct from plant beta-amylases.
| S-EPMC1135158 | biostudies-other
Project description:Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.
| S-EPMC9945562 | biostudies-literature
Project description:The novel alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum E 101-69 was purified as two forms (I and II) from culture medium, by using gel filtration in 6 M-guanidine hydrochloride as the final step. Renatured alpha-amylase-pullulanase I and II had apparent Mr values of 370,000 +/- 85,000 and 330,000 +/- 85,000 respectively, as determined by native polyacrylamide-gradient-gel electrophoresis. Both forms appear to be dimers of two similar subunits, with Mr values of 190,000 +/- 30,000 for enzyme I and 180,000 +/- 30,000 for enzyme II according to SDS/polyacrylamide-gradient-gel electrophoresis. The two forms had similar amino acid compositions, the same N-terminal sequence (Glu-Ile-Asp-Thr-Ala-Pro-Ala-Ile) and the same pI of 4.25. Both forms contained sugars having mobilities identical with those of rhamnose, glucose, galactose and mannose. The amount of neutral hexoses relative to protein was 11-12% (w/w) for both forms.
| S-EPMC1148928 | biostudies-other
Project description:The re-investigation of a methanolic extract of Salvia africana-lutea collected from the Cape Floristic Region, South Africa (SA), afforded four new abietane diterpenes, namely 19-acetoxy-12-methoxycarnosic acid (1), 3?-acetoxy-7?-methoxyrosmanol (2), 19-acetoxy-7?-methoxyrosmanol (3), 19-acetoxy-12-methoxy carnosol (4), and two known named clinopodiolides A (5), and B (6), in addition to four known triterpenes, oleanolic, and ursolic acids (7, 8), 11,12-dehydroursolic acid lactone (9) and ?-amyrin (10). The chemical structural elucidation of the isolated compounds was determined on the basis of one and two dimensional nuclear magnetic resonance (1D and 2D NMR), high-resolution mass spectrometry (HRMS), ultra violet (UV), fourier transform infrared (IR), in comparison with literature data. The in vitro bio-evaluation against alpha-glucosidase showed strong inhibitory activities of 8, 10, and 7, with the half inhibitory concentration (IC50) values of 11.3 ± 1.0, 17.1 ± 1.0 and 22.9 ± 2.0 µg/mL, respectively, while 7 demonstrated the strongest in vitro alpha-amylase inhibitory activity among the tested compounds with IC50 of 12.5 ± 0.7 µg/mL. Additionally, some of the compounds showed significant antioxidant capacities. In conclusion, the methanolic extract of S. africana-lutea is a rich source of terpenoids, especially abietane diterpenes, with strong antioxidant and anti-diabetic activities that can be helpful to modulate the redox status of the body and could therefore be an excellent candidate for the prevention of the development of diabetes, a disease where oxidase stress plays an important role.
| S-EPMC6827013 | biostudies-literature
Project description:This study aimed to identify thermo-stable pullulanase-producing bacteria in soil samples of potato fields and food-producing companies. Pullulan agar medium was used to screen 17 bacterial strains, which were incubated at 65 °C. The isolate with the maximum activity (375U/ml) was selected and recognized as Geobacillus stearothermophilus ADM-11 by morphological, biochemical characterization, and 16S rRNA gene sequencing. The pullulanase production required optimum pH of 7 and temperature of 75 °C, respectively. The electrophoresis of purified pullulanase on SDS-polyacrylamide gel revealed 83 kDa of a molecular weight that is active at 70 °C and pH 7.0. It was also stable at 90 °C but its activity was decreased by 10 % at 100 °C. The action of pullulanase was increased and stabilized by Ca+2 among the metal ions. Beta and gamma-cyclodextrins inhibited enzyme activity while ethylenediaminetetraacetate (EDTA) and phenylmethylsulfonyl fluoride (PMSF) have no significant effect on pullulanase activity. A full-length pullulanase gene was amplified from G. stearothermophilus ADM-11 using genomic DNA 2.1 kb of PCR product which was then purified and ligated in the cloning vector pTZ57R using the TA cloning technique. Colony PCR confirmed cloning on the positive clones after the pullulanase gene had been ligated and subjected to restriction digestion. It revealed 74 % similarity with the reported pullulanase gene from Geobacillus sp. 44C. The thermostability of pullulanase and its ability to degrade raw pullulan may therefore have wide-scale applications in starch processing, the detergent business, and new biotechnological applications.
| S-EPMC10792974 | biostudies-literature
Project description:A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved. These include an active center and common substrate-binding sites of various amylases. In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times. The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter. Purification of AapT from this recombinant E. coli was performed, and it was shown that the aapT gene product exhibits both alpha-amylase and pullulanase activities with one active site. The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively. Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch. Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. The enzyme also hydrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9).
| S-EPMC201885 | biostudies-literature
Project description:In this study, preliminary screening revealed that of 134 desert soil actinobacterial isolates, only 43 isolates produced amylase. Among these, an isolate DA7-7, which was identified as Streptomyces fragilis DA7-7, showed a prominent zone of clearance and significant amount of α-amylase production. The pre-optimization studies showed varying physicochemical and nutrients properties of the medium influenced the enzyme production significantly. Consequently, central composite design was employed with the selected variables (pH, temperature, dextrose, and peptone) for α-amylase production. The optimum fermentation conditions were 3.07% dextrose, 1.085% peptone, pH 6.0, and incubation temperature 27.27 °C. The predicted optimum α-amylase activity was 991.82 U/mL/min, which was similar to the experimental amylase activity of 973.5 U/mL/min. The crude α-amylase produced by S. fragilis DA7-7 was purified with ammonium sulfate precipitation, followed by gel filtration chromatography, and the estimated molecular mass was 51 kDa. The purified α-amylase was stable under the following conditions: pH (4-9), temperature (40-80 °C), NaCl (1-4 M), and detergents (1-10 mM). The Km and Vmax values of enzyme were found to be 0.624 mU/mg and 0.836 mg/mL, respectively.
| S-EPMC5614901 | biostudies-literature
Project description:Industrial demands for enzymes that are stable in a broad range of conditions are increasing. Such enzymes, one of which is α-amylase, could be produced by extremophiles. This study reports a thermostable α-amylase produced by a newly isolated Geobacillus sp. nov. from a geothermal area. The phylogenetic analysis of the 16S rRNA gene showed that the isolate formed a separate branch with 95% homology to Geobacillus sp. After precipitation using ammonium sulphate followed by ion-exchange chromatography, the enzyme produced a specific activity of 25.1 (U/mg) with a purity of 6.5-fold of the crude extract. The molecular weight of the enzyme was approximately 12.2 kDa. The optimum activity was observed at 75 °C and pH 8. The activity increased in the presence of Ba2+ and Fe2+ but decreased in the presence of K+ and Mg2+. Ca2+ and Mn2+ increased the activity slightly. The activity completely diminished with the addition of Cu2+. EDTA and PMSF also sharply reduced enzyme activity. Although the stability was moderate, the low molecular weight could be an important feature for its future applications.
| S-EPMC6667821 | biostudies-literature
Project description:BackgroundChinese Nong-flavor (NF) liquor is continuously and stably produced by solid-state fermentation technology for 1000 years, resulting in enrichment of special microbial community and enzymes system in its starter. Based on traditional culture-dependent methods, these functional enzymes are hardly obtained. According to our previous metatranscriptomic analysis, which identifies plenty of thermostable carbohydrate-active enzymes in NF liquor starter, the aim of this study is to provide a direct and efficient way to mine these thermostable enzymes.ResultsIn present study, an alpha-amylase (NFAmy13A) gene, which showed the highest expression level of enzymes in starch degradation at high temperature stage (62 °C), was directly obtained by functional metatranscriptomics from Chinese Nong-flavor liquor starter and expressed in Pichia pastoris. NFAmy13A had a typical signal peptide and shared the highest sequence identity of 64% with α-amylase from Aspergillus niger. The recombinant enzyme of NFAmy13A showed an optimal pH at 5.0-5.5 and optimal temperature at 60 °C. NFAmy13A was activated and stabilized by Ca2+, and its half-lives at 60 and 70 °C were improved significantly from 1.5 and 0.4 h to 16 and 0.7 h, respectively, in the presence of 10 mM CaCl2. Meanwhile, Hg2+, Co2+ and SDS largely inhibited its activity. NFAmy13A showed the maximum activity on amylopectin, followed by various starches, amylose, glycogen, and pullulan, and its specificity activity on amylopectin was 200.4 U/mg. Moreover, this α-amylase efficiently hydrolyzed starches (from corn, wheat, and potato) at high concentrations up to 15 mg/ml.ConclusionsThis study provides a direct way to mine active enzymes from man-made environment of NF liquor starter, by which a fungal thermostable α-amylase (NFAmy13A) is successfully obtained. The good characteristics of NFAmy13A in degrading starch at high temperature are consistent with its pivotal role in solid-state fermentation of NF liquor brewing. This work would stimulate mining more enzymes from NF liquor starter and studying their potentially synergistic roles in NF liquor brewing, thus paving the way toward the optimization of liquor production and improvement of liquor quality in future.
| S-EPMC5822527 | biostudies-literature