scholarly journals Microbial Proline 4-Hydroxylase Screening and Gene Cloning

1999 ◽  
Vol 65 (9) ◽  
pp. 4028-4031 ◽  
Author(s):  
Takeshi Shibasaki ◽  
Hideo Mori ◽  
Shigeru Chiba ◽  
Akio Ozaki
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Original Strain ◽  
Degenerate Primers ◽  
E Coli ◽  
Dna Fragment

ABSTRACT Microbial proline 4-hydroxylases, which hydroxylate freel-proline totrans-4-hydroxy-l-proline, were screened in order to establish an industrial system for biotransformation of l-proline totrans-4-hydroxy-l-proline. Enzyme activities were detected in eight strains, including strains ofDactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library ofDactylosporangium sp. strain RH1 constructed inEscherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29,715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the β-galactosidase α-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13.6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.

scholarly journals Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

1998 ◽  
Vol 180 (2) ◽  
pp. 388-394 ◽  
Author(s):  
Masahiro Furutani ◽  
Toshii Iida ◽  
Shigeyuki Yamano ◽  
Kei Kamino ◽  
Tadashi Maruyama
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ppiase Activity ◽  
Methanococcus Thermolithotrophicus ◽  
Fk506 Binding Protein ◽  
Thermophilic Archaeon

ABSTRACT A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k cat/Km ) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.

scholarly journals The Human Cytomegalovirus UL74 Gene Encodes the Third Component of the Glycoprotein H-Glycoprotein L-Containing Envelope Complex

1998 ◽  
Vol 72 (10) ◽  
pp. 8191-8197 ◽  
Author(s):  
Mary T. Huber ◽  
Teresa Compton
Keyword(s):  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Protein Species ◽  
Gene Encoding ◽  
Peptide Backbone ◽  
Glycoprotein H ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) gCIII envelope complex is composed of glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and a third, 125-kDa protein not related to gH or gL (M. T. Huber and T. Compton, J. Virol. 71:5391–5398, 1997; L. Li, J. A. Nelson, and W. J. Britt, J. Virol. 71:3090–3097, 1997). Glycosidase digestion analysis demonstrated that the 125-kDa protein was a glycoprotein containing ca. 60 kDa of N-linked oligosaccharides on a peptide backbone of 65 kDa or less. Based on these biochemical characteristics, two HCMV open reading frames, UL74 and TRL/IRL12, were identified as candidate genes for the 125-kDa glycoprotein. To identify the gene encoding the 125-kDa glycoprotein, we purified the gCIII complex, separated the components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected gH and the 125-kDa glycoprotein to amino acid microsequence analysis. Microsequencing of an internal peptide derived from purified 125-kDa glycoprotein yielded the amino acid sequence LYVGPTK. A FASTA search revealed an exact match of this sequence to amino acids 188 to 195 of the predicted product of the candidate gene UL74, which we have designated glycoprotein O (gO). Anti-gO antibodies reacted in immunoblots with a protein species migrating at ca. 100 to 125 kDa in lysates of HCMV-infected cells and with 100- and 125-kDa protein species in purified virions. Anti-gO antibodies also immunoprecipitated the gCIII complex and recognized the 125-kDa glycoprotein component of the gCIII complex. Positional homologs of the UL74 gene were found in other betaherpesviruses, and comparisons of the predicted products of the UL74 homolog genes demonstrated a number of conserved biochemical features.

scholarly journals Studies on the subunits of human myeloperoxidase

1984 ◽  
Vol 222 (3) ◽  
pp. 701-709 ◽  
Author(s):  
R L Olsen ◽  
C Little
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Molecular Mass ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Additional Species ◽  
Main Protein

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

scholarly journals The First Archaeal ATP-Dependent Glucokinase, from the Hyperthermophilic Crenarchaeon Aeropyrum pernix, Represents a Monomeric, Extremely Thermophilic ROK Glucokinase with Broad Hexose Specificity

2002 ◽  
Vol 184 (21) ◽  
pp. 5955-5965 ◽  
Author(s):  
Thomas Hansen ◽  
Bianca Reichstein ◽  
Roland Schmid ◽  
Peter Schönheit
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Divalent Cations ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Enzyme ◽  
Start Codon ◽  
Aeropyrum Pernix

ABSTRACT An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent Km values for ATP and glucose (at 90°C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V max was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k cat/Km ), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg2+, which was most effective, could partially be replaced with Co2+, Mn2+, and Ni2+. The enzyme had a temperature optimum of at least 100°C and showed significant thermostability up to 100°C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.

Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli

1990 ◽  
Vol 68 (1) ◽  
pp. 123-137 ◽  
Author(s):  
Krishna G. Peri ◽  
Hughes Goldie ◽  
E. Bruce Waygood
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Gene Products ◽  
Reading Frame ◽  
E Coli ◽  
Palindromic Sequences ◽  
Repetitive Extragenic Palindromic

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate – polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62 000; nagA, 45 000; nagB, 29 000. In addition, another product of Mr 44 000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weights (MWs) of the gene products derived from the sequence are as follows: nagA, 40 954; nagB, 29 657; nagC, 44 664; nagE, 68 356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27 181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: [Formula: see text]Key words: N-acetylglucosamine, N-acetylglucosamine-6-P deacetylase, glucosamine-6-P isomerase, repetitive extragenic palindromic sequences, catabolite repression.

scholarly journals Characterization of an Atypical Superoxide Dismutase from Sinorhizobium meliloti

1999 ◽  
Vol 181 (15) ◽  
pp. 4509-4516 ◽  
Author(s):  
Renata Santos ◽  
Stephane Bocquet ◽  
Alain Puppo ◽  
Danièle Touati
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Genomic Library ◽  
Specific Activity ◽  
Growth Conditions ◽  
Aerobic Bacterium ◽  
Degenerate Primers ◽  
E Coli ◽  
Acid Protein

ABSTRACT Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. ThesodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M r of 22,430 and pI of 5.8.S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetictac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H2O2. The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl2 or Fe(NH4)2(SO4)2, demonstrating the cambialistic nature of the S. melilotiSOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H2O2. Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H2O2of this unusual Fe-type SOD.

scholarly journals Evidence for the Presence of an F-Type ATP Synthase Involved in Sulfate Respiration in Desulfovibrio vulgaris

2000 ◽  
Vol 182 (8) ◽  
pp. 2200-2206 ◽  
Author(s):  
Kiyoshi Ozawa ◽  
Takanori Meikari ◽  
Ken Motohashi ◽  
Masasuke Yoshida ◽  
Hideo Akutsu
Keyword(s):  
Amino Acid ◽  
Atp Synthase ◽  
Cytoplasmic Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Level ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Desulfovibrio Vulgaris ◽  
F Cells

ABSTRACT Using a library of genomic DNA from Desulfovibrio vulgaris Miyazaki F, a strict anaerobe, and two synthetic deoxyoligonucleotide probes designed for F-type ATPases, the genes for open reading frames (ORFs) 1 to 5 were cloned and sequenced. The predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and that they share considerable identity at the amino acid level with δ, α, γ, β, and ɛ subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was partially purified from the cytoplasmic membrane fraction of theD. vulgaris Miyazaki F cells. The N-terminal amino acid sequences of three major polypeptides separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis were identical to those of the products predicted by the sequences of ORF-2, ORF-3, and ORF-4, suggesting that an F-type ATPase is functioning in the D. vulgaris Miyazaki F cytoplasmic membrane. The amount of the F-type ATPase produced in the D. vulgaris Miyazaki F cells is similar to that in the Escherichia coli cells cultured aerobically. It indicates that the enzyme works as an ATP synthase in the D. vulgaris Miyazaki F cells in connection with sulfate respiration.

scholarly journals 2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation byRhodopseudomonas palustris

1998 ◽  
Vol 180 (9) ◽  
pp. 2330-2336 ◽  
Author(s):  
Dale A. Pelletier ◽  
Caroline S. Harwood
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ring Cleavage ◽  
Benzoate Degradation ◽  
Coa Hydrolase

ABSTRACT 2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min−1 mg of protein−1. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.

scholarly journals Isolation of the Gene for the B12-Dependent Ribonucleotide Reductase from Anabaena sp. Strain PCC 7120 and Expression in Escherichia coli

2002 ◽  
Vol 184 (23) ◽  
pp. 6544-6550 ◽  
Author(s):  
Florence K. Gleason ◽  
Neil E. Olszewski
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Ribonucleotide Reductase ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Class Ii ◽  
E Coli ◽  
Deoxyribonucleoside Triphosphate ◽  
Pcc 7120

ABSTRACT The gene for ribonucleotide reductase from Anabaena sp. strain PCC 7120 was identified and expressed in Escherichia coli. This gene codes for a 1,172-amino-acid protein that contains a 407-amino-acid intein. The intein splices itself from the protein when it is expressed in E. coli, yielding an active ribonucleotide reductase of 765 residues. The mature enzyme was purified to homogeneity from E. coli extracts. Anabaena ribonucleotide reductase is a monomer with a molecular weight of approximately 88,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Superose 12 column chromatography. The enzyme reduces ribonucleotides at the triphosphate level and requires a divalent cation and a deoxyribonucleoside triphosphate effector. The enzyme is absolutely dependent on the addition of the cofactor, 5′-adenosylcobalamin. These properties are characteristic of the class II-type reductases. The cyanobacterial enzyme has limited sequence homology to other class II reductases; the greatest similarity (38%) is to the reductase from Lactobacillus leichmannii. In contrast, the Anabaena reductase shows over 90% sequence similarity to putative reductases found in genome sequences of other cyanobacteria, such as Nostoc punctiforme, Synechococcus sp. strain WH8102, and Prochlorococcus marinus MED4, suggesting that the cyanobacterial reductases form a closely related subset of the class II enzymes.

scholarly journals Molecular and Biochemical Analysis of Two β-Galactosidases from Bifidobacterium infantisHL96

2001 ◽  
Vol 67 (9) ◽  
pp. 4256-4263 ◽  
Author(s):  
Ming-Ni Hung ◽  
Zhicheng Xia ◽  
Nien-Tai Hu ◽  
Byong H. Lee
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Yield ◽  
Amino Acid Sequences ◽  
Nucleotide Sequence Analysis ◽  
Acid Base ◽  
Calculated Molecular Mass ◽  
Transcription Terminator

ABSTRACT Two genes encoding β-galactosidase isoenzymes,β-galI and β-galIII, fromBifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. β-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5′ flanking region of β-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently fromβ-galI. The β-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of β-GalI and β-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in β-GalI, and a possible acid-base site proposed for the LacG family was located in β-GalIII, which featured a glutamate at residue 160. The coding regions of the β-galI andβ-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. β-GalI and β-GalIII were specific for β-d-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-β-d-galactopyranoside) than in response to lactose, particularly β-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by β-GalI at 37°C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with β-GalIII. The structure of the major trisaccharide produced by β-GalI catalysis was characterized asO-β-d-galactopyranosyl-(1-3)-O-β-d-galactopyranosyl-(1-4)-d-glucopyranose (3′-galactosyl-lactose).

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