Characterization of a glycoside hydrolase family 42 β-galactosidase from Deinococcus geothermalis (original) (raw)
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Biochimica et Biophysica Acta (BBA) - General Subjects, 2000
An extracellular K-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl K-D-galactosides but efficiently hydrolyzed K-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl K-galactopyranoside and galactosyl 1 mannotriose, followed by 1 H NMR spectroscopy, pointed out the K-anomer of D-galactose was the primary product of hydrolysis from which the L-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl K-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.
FEMS Microbiology Letters, 1999
An K-galactosidase gene from the thermophilic bacterium Bacillus stearothermophilus NUB3621 was cloned, sequenced, expressed in Escherichia coli and the recombinant protein was purified. The Bacillus enzyme, designated AgaN, is similar to Kgalactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme was estimated to be a tetramer with a molecular mass of subunits 80.3 kDa. The purified AgaN is thermostable and has a temperature optimum of activity at 75³C and a half-life of inactivation of 19 h at 70³C. AgaN displays high affinity for oligomeric substrates such as melibiose and raffinose and is able to hydrolyze raffinose in the presence of 60% sucrose with high efficiency.
Systematic and Applied Microbiology, 1998
Thermotoga maritima represents one of the few hyperthermophilic bacteria currently known. The chromosomal a-galactosidase gene of T. maritima strain MSB8 has been cloned and its nucleotide sequence was determined. The gene, designated galA, has coding capacity for a 552 residue polypeptide with a calculated molecular mass of 63,653 Da. GalA was found to be flanked by other genes probably involved in galactoside breakdown and utilization. The previously sequenced ~-galactosidase gene, lacZ, is localized immediately upstream of galA while two open reading frames that putatively encode enzymes of galactose catabolism, i.e. galactose-l-phoshpate uridylytransferase (galT) and galactokinase (gaIK), were found downstream of galA. The identified genes are extremely close together or even overlap and have the same orientation, so they could all be part of one galactoside utilization operon of T. maritima MSB8. GalA displayed low-level amino acid sequence similarity with a-galactosidases of glycosyl hydrolase family 36. However, GalA is smaller than the other members of this enzyme family. The galA gene was expressed in Escherichia coli and the recombinant a-galactosidase was purified and characterized. The molecular mass of the recombinant enzyme was estimated at about 62 kDa by denaturing gel electrophoresis. Maximal hydrolysis of the chromogenic substrate p-nitrophenyl-aD -galactopyranoside was measured at pH 5.0-5.5 and 90-95 °C (5 min assay). Divalent cations were not required for activity. The enzyme released galactose from raffinose, melibiose and the synthetic substrates p-nitrophenyland o-nitrophenyl-aD -galactopyranoside. The T. maritima a-galactosidase thus was highly specific for the galactose moiety and the a-anomeric configuration of the glycosidic linkage. Its extreme thermal stability (tll2 = 6.5 h at 85°C) makes this enzyme an interesting candidate for biotechnological applications.
European Journal of Biochemistry, 1990
A thermophilic and thermostable P-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Suljiulobus sovuturicus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 'C with o-nitrophenyl P-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus P-galactosidase was a tetramer of 240 Ifr S kDa composed of similar or identical subunits. Comparison of the amino acid composition of pgalactosidase from S. solfataricus with that from Eschevichia culi revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not crossreact with P-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for P-galactosidases from other mesopbilic and thermophilic sources. P-D-Galactosidase (P-D-gdlactoside galactohydrolase) catalyzes the hydrolysis of p-1,4-D-galactosidic linkages. This enzyme is widely distributed in nature, being found in numerous microorganisms, plant and animal tissues [1]. Of the 8-galactosidases so far investigated, that from Escherichia coli has been the most throughly studied, particularly from the genetic point of view, following the results of the work on the Lac operon [2]. The E. coli LacZ enzyme is inducible and composed of four identical protomers, each estimated to have a molecular mass of 116248 Da [3]. The enzyme exhibits both transferase and hydrolase activities [I,
Molecular cloning and characterization of alpha - galactosidase gene from Glaciozyma antarctica
Psychrophilic enzymes are proteins produced by psychrophilic organisms which recently are the limelight for industrial applications. A gene encoding α-galactosidase from a psychrophilic yeast, Glaciozyma antarctica PI12 which belongs to glycoside hydrolase family 27, was isolated and analyzed using several bioinformatic tools. The cDNA of the gene with the size of 1,404-bp encodes a protein with 467 amino acid residues. Predicted molecular weight of protein was 48.59 kDa and hence we name the gene encoding α-galactosidase as GAL48. We found that the predicted protein sequences possessed signal peptide sequence and are highly conserved among other fungal α-galactosidase.
PubMed, 2015
A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.
Characterization of β-galactosidase from an Antarctic Bacillus sp
2005
Antarctica is the coldest continent on the earth and harbours a variety of microorganisms. One of the bacterial isolates from cyanobacterial mats of Schirmacher Oasis, characterized as Bacillus sp. grew at 5-35C with an optima at 25C, and produced intracellular cold active β-galactosidase. The maximum activity was recorded at pH 6.8 and 40C during late stationary phase. At 5C, the enzyme retained 39.7% activity and at 60C, became completely inactive within 15 min. The enzyme activity was stimulated by metal ions but was inhibited by ethylene diamine tetra acetic acid. Non-denaturing polyacylamide separation followed by in situ hydrolysis of 5-bromo-4-chloro-3-indolyl-β-galactopyanoside, suggested the presence of isozymes. These properties of β-galactosidase, indicate its potential use in removal of lactose from the milk for lactose intolerant people.