scholarly journals Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome

2020 ◽  
Author(s):  
Pashupati Bhandari ◽  
Jeffrey P. Tingley ◽  
D. Wade Abbott ◽  
Janet E. Hill
Keyword(s):  
Microbial Community ◽  
Phylogenetic Analyses ◽  
Anaerobic Bacteria ◽  
Glycoside Hydrolase Family ◽  
Vaginal Microbiome ◽  
Multistep Process ◽  
Ph Range ◽  
Culture Supernatants ◽  
Layer Chromatography

AbstractGardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, a dysbiosis in which lactobacilli dominant microbial community is replaced with mixed aerobic and anaerobic bacteria including Gardnerella species. The co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, particularly glycogen present in the vaginal environment, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up by bacteria requires the combined activities of several enzymes collectively known as “amylases”. In the present study, glycogen degrading abilities of Gardnerella spp. were assessed. We found that Gardnerella spp. isolates and filtered culture supernatants had amylase activity. Phylogenetic analyses predicted conserved Glycoside Hydrolase family 13 (GH13) members among Gardnerella spp. including a putative α-glucosidase. The gene for this enzyme was cloned and expressed, and recombinant protein was purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides over a broad pH range (4 - 8) with an optimum activity at pH 6-7. Glucose was released from maltose, maltotriose and maltopentose, however, no products were detected on thin layer chromatography (TLC) when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce a secreted α-glucosidase enzyme that can contribute to the complex and multistep process of glycogen breakdown by degrading smaller oligosaccharides into glucose, contributing to the pool of nutrients available to the vaginal microbiota.

scholarly journals Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome

2021 ◽  
Author(s):  
Pashupati Bhandari ◽  
Jeffrey P. Tingley ◽  
David R. J. Palmer ◽  
D. Wade Abbott ◽  
Janet E. Hill
Keyword(s):  
Microbial Community ◽  
Bacterial Vaginosis ◽  
Glycogen Metabolism ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Vaginal Microbiome ◽  
Sexually Transmitted ◽  
Multistep Process ◽  
Mixed Bacteria

Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, in which a lactobacilli dominant community is replaced with mixed bacteria including Gardnerella species. Co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, including glycogen, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 genomes demonstrated that a putative α-glucosidase-encoding sequence, CG400_06090, was conserved in all Gardnerella spp. The predicted α-glucosidase enzyme was expressed, purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides with maximum activity at pH 7. K m , k cat and k cat /K m values for the substrate 4-nitrophenyl α- d -glucopyranoside were 8.3 μM, 0.96 min −1 and 0.11 μM −1 min −1 , respectively. Glucose was released from maltose, maltotriose, maltotetraose and maltopentaose, but no products were detected when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an α-glucosidase enzyme that may contribute to the multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides. 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 carbon source available to vaginal bacteria. The significance of our research is characterizing the activity of an enzyme conserved in Gardnerella species that likely contributes to the ability of these bacteria to utilize glycogen.

scholarly journals Characterization of Microbial Community Structure in Gulf of Mexico Gas Hydrates: Comparative Analysis of DNA- and RNA-Derived Clone Libraries

2005 ◽  
Vol 71 (6) ◽  
pp. 3235-3247 ◽  
Author(s):  
Heath J. Mills ◽  
Robert J. Martinez ◽  
Sandra Story ◽  
Patricia A. Sobecky
Keyword(s):  
Phylogenetic Analysis ◽  
Microbial Community ◽  
Gulf Of Mexico ◽  
Gas Hydrate ◽  
Phylogenetic Analyses ◽  
Active Fraction ◽  
Clone Libraries ◽  
Dna And Rna ◽  
Rna And Dna

ABSTRACT The characterization of microbial assemblages within solid gas hydrate, especially those that may be physiologically active under in situ hydrate conditions, is essential to gain a better understanding of the effects and contributions of microbial activities in Gulf of Mexico (GoM) hydrate ecosystems. In this study, the composition of the Bacteria and Archaea communities was determined by 16S rRNA phylogenetic analyses of clone libraries derived from RNA and DNA extracted from sediment-entrained hydrate (SEH) and interior hydrate (IH). The hydrate was recovered from an exposed mound located in the northern GoM continental slope with a hydrate chipper designed for use on the manned-submersible Johnson Sea Link (water depth, 550 m). Previous geochemical analyses indicated that there was increased metabolic activity in the SEH compared to the IH layer (B. N. Orcutt, A. Boetius, S. K. Lugo, I. R. Macdonald, V. A. Samarkin, and S. Joye, Chem. Geol. 205:239-251). Phylogenetic analysis of RNA- and DNA-derived clones indicated that there was greater diversity in the SEH libraries than in the IH libraries. A majority of the clones obtained from the metabolically active fraction of the microbial community were most closely related to putative sulfate-reducing bacteria and anaerobic methane-oxidizing archaea. Several novel bacterial and archaeal phylotypes for which there were no previously identified closely related cultured isolates were detected in the RNA- and DNA-derived clone libraries. This study was the first phylogenetic analysis of the metabolically active fraction of the microbial community extant in the distinct SEH and IH layers of GoM gas hydrate.

scholarly journals Isolation Detection and Characterization of Syringolin A produced from the Probiotic Strain Bacillus Cereus Isolated from Donkey Milk

2018 ◽  
Vol 1 (2) ◽  
Author(s):  
Rashmi D ◽  
Sharmila T ◽  
Sushama Patil ◽  
Onkar Apine ◽  
Srinivas Sistla ◽  
...  
Keyword(s):  
Probiotic Strain ◽  
Uv Spectra ◽  
Organic Layer ◽  
Characteristic Peak ◽  
Antitumor Compound ◽  
Solvent Systems ◽  
Culture Supernatants ◽  
Layer Chromatography ◽  
Economical Process

Syringolin A is a non-ribosomal virulence factor secreted by few Pseudomonas strains. Syringolin A is an well known irreversible proteasome inhibitor and antitumor compound. The present study is focused on the extraction of Syringolin A through a non-tedious and economical process. Syringolin A is extracted from culture supernatants by the immiscible organic layer by mixing of dichloromethane or chloroform (trichloromethane). Syringolin A was identified by the characteristic peak at 350 nm by UV spectra. The compound was further characterized by Thin Layer Chromatography (TLC) with the retention value, Rf was found to be in the range of 0.78-0.83 run using a combination of solvent systems water and methanol.  The molecular weight of the compound was found to be 492.2614 g mol-1 identified and analyzed by UHPLC–QTOF-MS analysis. Due to its significant pharmacological importance in proliferative diseases, further studies on production and optimization of these compounds are necessary.   

scholarly journals Biochemical Characterization of a New β-Agarase from Cellulophaga Algicola

2019 ◽  
Vol 20 (9) ◽  
pp. 2143 ◽  
Author(s):  
Han ◽  
Zhang ◽  
Yang
Keyword(s):  
Structural Model ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Optimal Temperature ◽  
Salt Tolerant ◽  
Wide Ph Range ◽  
Specific Activities ◽  
Ph Range ◽  
Layer Chromatography

Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted β-agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type β-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

scholarly journals Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

Marine Drugs ◽  
2020 ◽  
Vol 18 (6) ◽  
pp. 312 ◽  
Author(s):  
Dandan Li ◽  
Shangyong Li ◽  
Yanhong Wu ◽  
Mengfei Jin ◽  
Yu Zhou ◽  
...  
Keyword(s):  
Dairy Products ◽  
Marine Bacterium ◽  
Intestinal Flora ◽  
Lactose Hydrolysis ◽  
Glycoside Hydrolase Family ◽  
Transglycosylation Activity ◽  
Ph Range ◽  
Hydrolysis Of ◽  
Hydrolase Family

As prebiotics, galacto-oligosaccharides (GOSs) can improve the intestinal flora and have important applications in medicine. β-galactosidases could promote the synthesis of GOSs in lactose and catalyze the hydrolysis of lactose. In this study, a new β-galactosidase gene (gal2A), which belongs to the glycoside hydrolase family 2, was cloned from marine bacterium Alteromonas sp. QD01 and expressed in Escherichia coli. The molecular weight of Gal2A was 117.07 kDa. The optimal pH and temperature of Gal2A were 8.0 and 40 °C, respectively. At the same time, Gal2A showed wide pH stability in the pH range of 6.0–9.5, which is suitable for lactose hydrolysis in milk. Most metal ions promoted the activity of Gal2A, especially Mn2+ and Mg2+. Importantly, Gal2A exhibited high transglycosylation activity, which can catalyze the formation of GOS from milk and lactose. These characteristics indicated that Gal2A may be ideal for producing GOSs and lactose-reducing dairy products.

scholarly journals Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7

2005 ◽  
Vol 71 (8) ◽  
pp. 4225-4232 ◽  
Author(s):  
Stephanie Shipkowski ◽  
Jean E. Brenchley
Keyword(s):  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Galactosidase Activity ◽  
Reading Frame ◽  
Substrate Preferences ◽  
Acid Protein ◽  
Paenibacillus Sp ◽  
Cold Active ◽  
Ph Range

ABSTRACT We selected for spore-forming psychrophilic bacteria able to use lactose as a carbon source and one isolate, designated Paenibacillus sp. strain C7, that was phylogenetically related to, but distinct from both Paenibacillus macquariensis and Paenibacillus antarcticus. Some Escherichia coli transformants obtained with genomic DNA from this isolate hydrolyzed X-Gal (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside) only below 30°C, an indication of cold-active β-galactosidase activity. Sequencing of the cloned insert revealed an open reading frame encoding a 756-amino acid protein that, rather than belonging to a family typically known for β-galactosidase activity, belonged to glycoside hydrolase family 3, a family of β-glucosidases. Because of this unusual placement, the recombinant enzyme (BglY) was purified and characterized. Consistent with its classification, the enzyme had seven times greater activity with the glucoside substrate ONPGlu (o-nitrophenyl-β-d-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-β-d-galactopyranoside). In addition, the enzyme had, with ONPGlu, a thermal optimum around 30 to 35°C, activity over a broad pH range (5.5 to 10.9), and an especially low Km (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-β-glucosides such as helicin, MUG (4-methylumbelliferyl-β-d-glucopyranoside), esculin, indoxyl-β-d-glucoside (a natural indigo precursor), and salicin, but had no activity with glucosidic disaccharides or lactose. These characteristics and substrate preferences make the BglY enzyme unique among the family 3 β-glucosidases. The hydrolysis of a variety of aryl-β-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-β-d-glucoside may make it useful for producing indigo.

scholarly journals Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 431
Author(s):  
Wenxin Wang ◽  
Jianxin Wang ◽  
Ruihua Yan ◽  
Runying Zeng ◽  
Yaqiang Zuo ◽  
...  
Keyword(s):  
Deep Sea ◽  
Maximum Activity ◽  
Cold Adapted ◽  
Working Temperature ◽  
Ph Values ◽  
Natural Substances ◽  
Ph Range ◽  
Layer Chromatography ◽  
Optimal Ph

The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu2+ and Zn2+, whereas Fe2+ displayed an intensification of enzymatic activity. The Km and Vmax of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Application of commercial test-systems to identify gram-negative facultatively anaerobic bacteria

2016 ◽  
Vol 3 (1) ◽  
pp. 43-48 ◽  
Author(s):  
V. Patyka ◽  
L. Butsenko ◽  
L. Pasichnyk
Keyword(s):  
Erwinia Amylovora ◽  
Anaerobic Bacteria ◽  
Biochemical Properties ◽  
Test System ◽  
Phytopathogenic Bacteria ◽  
Gram Negative ◽  
Test Systems ◽  
Facultatively Anaerobic ◽  
Microbiological Methods

Aim. To validate the suitability of commercial API 20E test-system (bioMerieux) for the identifi cation and characterization of facultative gram-negative phytopathogenic bacterial isolates. Methods. Conventional mi- crobiological methods, API 20E test-system (bioMerieux) according to the manufacturer’s instructions. Re- sults. The identifi cation results for Erwinia amylovora, Pectobacterium carotovorum and Pantoea agglome- rans isolates were derived from the conventional and API 20E test systems, which, were in line with the literature data for these species. The API 20E test-system showed high suitability for P. agglomerans isolates identifi cation. Although not all the species of facultatively anaerobic phytopathogenic bacteria may be identi- fi ed using API 20E test-system, its application will surely allow obtaining reliable data about their physiologi- cal and biochemical properties, valuable for identifi cation of bacteria, in the course of 24 h. Conclusions. The results of tests, obtained for investigated species while using API 20E test-system, and those of conventional microbiological methods coincided. The application of API 20E test-system (bioMerieux) ensures fast obtain- ing of important data, which may be used to identify phytopathogenic bacteria of Erwinia, Pectobacterium, Pantoea genera.

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