Purification of beta-acetylglucosaminase and beta-galactosidase from ram testis.
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ABSTRACT:
SUBMITTER: Caygill JC
PROVIDER: S-EPMC1264859 | biostudies-other | 1966 Feb
REPOSITORIES: biostudies-other
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Project description:Members of the human gut microbiota use glycoside hydrolase (GH) enzymes, such as β-galactosidases, to forage on host mucin glycans and dietary fibres. A human faecal metagenomic fosmid library was constructed and functionally screened to identify novel β-galactosidases. Out of the 16,000 clones screened, 30 β-galactosidase-positive clones were identified. The β-galactosidase gene found in the majority of the clones was BAD_1582 from Bifidobacterium adolescentis, subsequently named bgaC. This gene was cloned with a hexahistidine tag, expressed in Escherichia coli and His-tagged-BgaC was purified using Ni2+-NTA affinity chromatography and size filtration. The enzyme had optimal activity at pH 7.0 and 37 °C, with a wide range of pH (4-10) and temperature (0-40 °C) stability. It required a divalent metal ion co-factor; maximum activity was detected with Mg2+, while Cu2+ and Mn2+ were inhibitory. Kinetic parameters were determined using ortho-nitrophenyl-β-D-galactopyranoside (ONPG) and lactose substrates. BgaC had a Vmax of 107 μmol/min/mg and a Km of 2.5 mM for ONPG and a Vmax of 22 μmol/min/mg and a Km of 3.7 mM for lactose. It exhibited low product inhibition by galactose with a Ki of 116 mM and high tolerance for glucose (66% activity retained in presence of 700 mM glucose). In addition, BgaC possessed transglycosylation activity to produce galactooligosaccharides (GOS) from lactose, as determined by TLC and HPLC analysis. The enzymatic characteristics of B. adolescentis BgaC make it an ideal candidate for dairy industry applications and prebiotic manufacture.Key points• Bifidobacterium adolescentis BgaC β-galactosidase was selected from human faecal metagenome.• BgaC possesses sought-after properties for biotechnology, e.g. low product inhibition.• BgaC has transglycosylation activity producing prebiotic oligosaccharides. Graphical Abstract.
| S-EPMC7843569 | biostudies-literature
Project description:Plant β-galactosidases play important roles in carbohydrate-reserve mobilization, cell-wall expansion and degradation, and turnover of signalling molecules during ripening. Tomato β-galactosidase 4 (TBG4) not only has β-galactosidase activity but also has exo-β-(1,4)-galactanase activity, and prefers β-(1,4)-galactans longer than pentamers as its substrates; most other β-galactosidases only have the former activity. Recombinant TBG4 protein expressed in the yeast Pichia pastoris was crystallized by the sitting-drop vapour-diffusion method using PEG 10,000 as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-parameters a = 92.82, b = 96.30, c = 159.26 Å, and diffracted to 1.65 Å resolution. Calculation of the Matthews coefficient suggested the presence of two monomers per asymmetric unit (VM = 2.2 Å(3) Da(-1)), with a solvent content of 45%.
| S-EPMC4321468 | biostudies-literature
Project description:1. Analysis of rams testes revealed that phosphatidylcholine was the major phospholipid and accounted for about 40% of the total. Only small amounts of choline plasmalogen were present. 2. The ratio of phosphatidylcholine to choline plasmalogen in the testis was very different from that occurring in the spermatozoa. This result was in contrast with those for rat testis and rat spermatozoa (obtained from the head of the epididymis), where the ratio of the two lipids was very similar. 3. Infusions of [(32)P]orthophosphate into the testicular artery of rams resulted in incorporation of radioactivity into most phospholipids; phosphatidylinositol labelling accounted for 68% and 39% of the radioactivity after infusions lasting 3hr. and 5hr. respectively. 4. With the exception of phosphatidic acid the specific radioactivity of phosphatidylinositol was higher than that of any other lipid. 5. After the infusion of [U-(14)C]glucose, triglycerides accounted for about 60% of the radioactivity in testicular neutral lipids, whereas diglycerides had only about 15% of the radioactivity. 6. Palmitic acid (16:0) was the major component both in neutral lipids and phospholipids of ram testes. 7. The effects of gonadotrophic hormones (luteinizing hormone and follicle-stimulating hormone) on the incorporation of [(32)P]orthophosphate into total testicular phospholipids in vivo were also examined.
| S-EPMC1198653 | biostudies-other
Project description:Backgroundβ-D-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Cold-active β-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis.ResultsA genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the β-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-β-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C.ConclusionsA novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active β-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active β-D-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of Paracoccus sp. 32d β-D-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.
| S-EPMC3268748 | biostudies-literature
Project description:A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP(C)), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP(Sc)) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific--Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.
| S-EPMC3427297 | biostudies-literature
Project description:Geobacillus stearothermophilus T1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems in G. stearothermophilus T1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure-function study. The best crystals obtained for this enzyme belonged to the triclinic space group P1, with average crystallographic unit-cell parameters of a = 67.0, b = 78.1, c = 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D.
| S-EPMC3936444 | biostudies-literature
Project description:Expression and purification of β-galactosidases derived from Bifidobacterium provide a new resource for efficient lactose hydrolysis and lactose intolerance alleviation. Here, we cloned and expressed two β-galactosidases derived from Bifidobacterium. The optimal pH for BLGLB1 was 5.5, and the optimal temperature was 45 °C, at which the enzyme activity of BLGLB1 was higher than that of commercial enzyme E (300 ± 3.6 U/mg) under its optimal conditions, reaching 2200 ± 15 U/mg. The optimal pH and temperature for BPGLB1 were 6.0 and 45 °C, respectively, and the enzyme activity (0.58 ± 0.03 U/mg) under optimum conditions was significantly lower than that of BLGLB1. The structures of the two β-galactosidase were similar, with all known key sites conserved. When o-nitrophenyl-β-D-galactoside (oNPG) was used as an enzyme reaction substrate, the maximum reaction velocity (Vmax) for BLGLB1 and BPGLB1 was 3700 ± 100 U/mg and 1.1 ± 0.1 U/mg, respectively. The kinetic constant (Km) of BLGLB1 and BPGLB1 was 1.9 ± 0.1 and 1.3 ± 0.3 mmol/L, respectively. The respective catalytic constant (kcat) of BLGLB1 and BPGLB1 was 1700 ± 40 s-1 and 0.5 ± 0.02 s-1, respectively; the respective kcat/Km value of BLGLB1 and BPGLB1 was 870 L/(mmol∙s) and 0.36 L/(mmol∙s), respectively. The Km, kcat and Vmax values of BLGLB1 were superior to those of earlier reported β-galactosidase derived from Bifidobacterium. Overall, BLGLB1 has potential application in the food industry.
| S-EPMC9323360 | biostudies-literature
Project description:BACKGROUND: The development of a new cold-active beta-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production. RESULTS: In this article, we present a new beta-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this beta-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium Arthrobacter sp. 32c, sequenced, cloned, expressed in Escherichia coli and Pichia pastoris, purified and characterized. 27 mg of beta-D-galactosidase was purified from 1 L of culture with the use of an intracellular E. coli expression system. The protein was also produced extracellularly by P. pastoris in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of P. pastoris culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50 degrees C, 60% of the maximum activity of the enzyme was determined at 25 degrees C and 15% of the maximum activity was detected at 0 degrees C. CONCLUSION: The properties of Arthrobacter sp. 32cbeta-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.
| S-EPMC2723119 | biostudies-literature
Project description:Three and four N-acetylhexosaminidase components, from ram testis and epididymis respectively, have been separated by ion-exchange chromatography on DEAE-cellulose. Although they all have the same molecular weight (approx. 140000) and very similar catalytic properties towards the synthetic substrates, 4-methylumbelliferyl N-acetyl-beta-glucosaminide and N-acetyl-beta-galactosaminide, isoelectric focusing of the individual components showed that each had a distinct pI value. Isoelectric focusing has also been used to demonstrate the occurrence of multiple forms in ejaculated ram semen.
| S-EPMC1177738 | biostudies-other
Project description:?-D-Galactosidase (?-Gal) is an exoglycosidase that cleaves ?-galactosides from glycoproteins, sphingolipids and keratan sulfate. This study reports the expression, purification, crystallization and preliminary X-ray crystallographic analysis of human lysosomal ?-Gal. The sitting-drop vapour-diffusion method was used to crystallize ?-Gal in complexes with its product galactose and with the inhibitor 1-deoxygalactonojirimycin. The resulting crystals were isomorphous and belonged to space group P2(1). The crystals of the ?-Gal-galactose and the ?-Gal-inhibitor complexes had unit-cell parameters a = 94.8, b = 116.1, c = 140.3 Å, ? = 92.2° and a = 94.8, b = 116.0, c = 140.3 Å, ? = 92.2°, respectively. Diffraction data were collected to 1.8 Å resolution for both crystals.
| S-EPMC3253840 | biostudies-literature