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Jean E. Brenchley - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of a β galactosidase with a low temperature optimum obtained from an antarctic arthrobacter isolate
Journal of Bacteriology, 2003Co-Authors: James A Coker, K R Gutshall, Peter P Sheridan, Jennifer Lovelandcurtze, Ann J Auman, Jean E. BrenchleyAbstract:A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 Beta-Galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both Beta-Galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ Beta-Galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 Beta-Galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.
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characterization of two new glycosyl hydrolases from the lactic acid bacterium carnobacterium piscicola strain ba
Applied and Environmental Microbiology, 2001Co-Authors: Jonna M Coombs, Jean E. BrenchleyAbstract:Glycosyl hydrolases (EC 3.2.1 to 3.2.3) cleave the glycosidic bond(s) between two or more carbohydrates or the bond between a carbohydrate moiety and a noncarbohydrate moiety. Traditionally, glycosyl hydrolases were grouped together based on substrate specificity. For example, all β-galactosidases were combined into one group (EC 3.2.1.23) because of their shared ability to hydrolyze lactose. However, classification based on substrate specificity is complicated by the fact that some enzymes hydrolyze more than one substrate. Some glycosyl hydrolases have activity on both phosphorylated and nonphosphorylated substrates (3, 21) or on β-glucosides and β-galactosides (2) and some β-galactosidases have activity on β-fucosides and β-galacturonides (11, 15, 25). The increase in the number of sequenced glycosyl hydrolases and the availability of new analytical methods has permitted the reorganization of these enzymes into families based on amino acid sequence similarities and hydrophobic cluster analysis (12, 13, 14). There are presently four families containing enzymes with β-galactosidase activity, families 1, 2, 35, and 42, and three families which contain enzymes with α-galactosidase activity, families 4, 27, and 36. New glycosyl hydrolases which have been sequenced can be grouped into a specific family on the basis of DNA or deduced amino acid similarity. In many cases, however, there is no information to verify the substrate specificity of the enzymes within these groups or their possible role(s) in cellular metabolism. The glycosyl hydrolases found in lactic acid bacteria have been of special interest because of their importance to the dairy and food processing industries. In contrast to most other bacteria, nearly all lactic acid bacteria transport and utilize lactose via the phosphoenolpyruvate-dependent phosphotransferase system, which requires the concomitant activity of a phospho-β-galactosidase. β-Galactosidases belonging to a different family, and sharing sequence similarity with the well-characterized Escherichia coli lacZ-encoded enzyme, have also been detected in lactic acid bacteria such as Streptococcus thermophilus or Lactococcus lactis (7). The genus Carnobacterium is a recent taxonomic addition to the lactic acid bacteria group (4, 5). Most Carnobacterium species were isolated from meat or fish (1, 23) and are similar to those in the Lactobacillus genus but do not grow on acetate and have a higher tolerance to oxygen and high pH (24). Research on Carnobacterium species has centered on their ability to produce bacteriocins (8, 19). Recently, during our investigation of psychrophilic organisms, we isolated from soil a new Carnobacterium piscicola strain, BA, which hydrolyzed the β-galactosidase chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) at 4°C. Initial work discovered a gene, bgaB, encoding a family 42 glycosyl hydrolase that had a temperature optimum of 30°C (6). This was the first report of a gene from this family in any lactic acid bacterium. Additional sequencing of this cloned fragment suggested that the bgaB gene is centered between two regions with homology to other glycosyl hydrolases. The gene agaA is located in the region adjacent to the N-terminal end of bgaB, and shared sequence homology with a group of α-galactosidases characterized from other bacteria and some eukaryotes, including a sequence from the lactic acid bacterium Lactobacillus plantarum. Adjacent to the C-terminal end of the bgaB β-galactosidase gene was a second, unrelated β-galactosidase gene, bgaC. Genes similar to bgaC have not been reported in the lactic acid bacteria. This includes L. lactis, for which the sequence of the entire genome is known. In order to explore the functions encoded by these two new putative genes, they were subcloned and their ability to produce enzymes with α- and β-galactosidase activities was tested. The arrangement of these genes on a single fragment suggested that they might function together to degrade saccharides containing both alpha and beta linkages rather than being involved in lactose hydrolysis. We examined the regulation of these enzymes in the native C. piscicola strain BA and found that their activities decreased when the medium was supplemented with either glucose or lactose. In contrast, a phospho-galactosidase activity increased during growth with lactose. These results suggest that a phospho-galactosidase is responsible for lactose utilization and that the unusual cluster of glycosyl hydrolase genes reported here might be involved in the degradation of other polysaccharides.
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A novel Arthrobacter Beta-Galactosidase with homology to eucaryotic Beta-Galactosidases.
Journal of Bacteriology, 1997Co-Authors: K R Gutshall, K Wang, Jean E. BrenchleyAbstract:An Arthrobacter Beta-Galactosidase has homology with the lysosomal acid Beta-Galactosidases from humans and mice and with a Xanthomonas manihotis enzyme. Phylogenetic analysis of the deduced amino acid sequence showed an unusual pattern, with this procaryotic enzyme clustering within the animal clade. The gene encodes a subunit of 52 kDa, and the enzyme appears to be active as a dimer. The enzyme hydrolyzed substrates with either a beta-1,4 or a beta-1,3 linkage.
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characterization of a psychrotrophic arthrobacter gene and its cold active beta galactosidase
Applied and Environmental Microbiology, 1994Co-Authors: Donald E. Trimbur, K R Gutshall, P Prema, Jean E. BrenchleyAbstract:Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active Beta-Galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces Beta-Galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding Beta-Galactosidase activity were obtained. We have subcloned and sequenced one of these Beta-Galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The Beta-Galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ Beta-Galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.
Dietmar Haltrich - One of the best experts on this subject based on the ideXlab platform.
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high level expression of recombinant beta galactosidases in lactobacillus plantarum and lactobacillus sakei using a sakacin p based expression system
Journal of Agricultural and Food Chemistry, 2008Co-Authors: Elisabeth Halbmayr, Thomas Maischberger, Thu Ha Nguyen, Vincent G H Eijsink, Geir Mathiesen, Clemens K Peterbauer, Dietmar HaltrichAbstract:This work presents the cloning and expression of the genes encoding heterodimeric Beta-Galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sorvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of Beta-Galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of Beta-Galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and Beta-Galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant Beta-Galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native Beta-Galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of Beta-Galactosidase in a food-grade expression system, which is of high interest for applications in food industry.
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purification and characterization of two novel β galactosidases from lactobacillus reuteri
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Thu Ha Nguyen, Barbara Splechtna, Marlene Steinbock, Wolfgang Kneifel, Hans Peter Lettner, Klaus D Kulbe, Dietmar HaltrichAbstract:The intracellular Beta-Galactosidase (beta-gal) enzymes from two strains of Lactobacillus reuteri, L103 and L461, were purified by ammonium sulfate fractionation, hydrophobic interaction, and affinity chromatography. Both enzymes are heterodimers with a molecular mass of 105 kDa, consisting of a 35 kDa subunit and a 72 kDa subunit. Active staining of L. reuteri L103 and L461 beta-gal with 4-methylumbelliferyl beta-d-galactoside showed that the intact enzymes as well as the larger subunits possess Beta-Galactosidase activity. The isoelectric points of L. reuteri L461 and L103 beta-gal were found to be in the range of 3.8-4.0 and 4.6-4.8, respectively. Both enzymes are most active in the pH range of 6-8; however, they are not stable at pH 8. The L. reuteri Beta-Galactosidases are activated by various mono- and divalent cations, including Na(+), K(+), and Mn(2+), and are moderately inhibited by their reaction products d-glucose and d-galactose. Because of their origin from beneficial and potentially probiotic lactobacilli, these enzymes could be of interest for the synthesis of prebiotic galacto-oligosaccharides.
Shiyao Liu - One of the best experts on this subject based on the ideXlab platform.
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Image_1_Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004.pdf
2018Co-Authors: Huiqi Wang, Chenyi Shi, Qingbiao Xie, Yaxin Wang, Shiyao Liu, Jun TaoAbstract:Bacterial β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. β-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have Beta-Galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc.
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Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004
Frontiers Media S.A., 2018Co-Authors: Huiqi Wang, Chenyi Shi, Qingbiao Xie, Yaxin Wang, Shiyao LiuAbstract:Bacterial β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. β-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have Beta-Galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc
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Table_1_Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004.pdf
2018Co-Authors: Huiqi Wang, Chenyi Shi, Qingbiao Xie, Yaxin Wang, Shiyao Liu, Jun TaoAbstract:Bacterial β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. β-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have Beta-Galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc.
Huiqi Wang - One of the best experts on this subject based on the ideXlab platform.
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Image_1_Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004.pdf
2018Co-Authors: Huiqi Wang, Chenyi Shi, Qingbiao Xie, Yaxin Wang, Shiyao Liu, Jun TaoAbstract:Bacterial β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. β-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have Beta-Galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc.
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Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004
Frontiers Media S.A., 2018Co-Authors: Huiqi Wang, Chenyi Shi, Qingbiao Xie, Yaxin Wang, Shiyao LiuAbstract:Bacterial β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. β-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have Beta-Galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc
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Table_1_Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004.pdf
2018Co-Authors: Huiqi Wang, Chenyi Shi, Qingbiao Xie, Yaxin Wang, Shiyao Liu, Jun TaoAbstract:Bacterial β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. β-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have Beta-Galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc.
K R Gutshall - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of a β galactosidase with a low temperature optimum obtained from an antarctic arthrobacter isolate
Journal of Bacteriology, 2003Co-Authors: James A Coker, K R Gutshall, Peter P Sheridan, Jennifer Lovelandcurtze, Ann J Auman, Jean E. BrenchleyAbstract:A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 Beta-Galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both Beta-Galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ Beta-Galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 Beta-Galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.
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A novel Arthrobacter Beta-Galactosidase with homology to eucaryotic Beta-Galactosidases.
Journal of Bacteriology, 1997Co-Authors: K R Gutshall, K Wang, Jean E. BrenchleyAbstract:An Arthrobacter Beta-Galactosidase has homology with the lysosomal acid Beta-Galactosidases from humans and mice and with a Xanthomonas manihotis enzyme. Phylogenetic analysis of the deduced amino acid sequence showed an unusual pattern, with this procaryotic enzyme clustering within the animal clade. The gene encodes a subunit of 52 kDa, and the enzyme appears to be active as a dimer. The enzyme hydrolyzed substrates with either a beta-1,4 or a beta-1,3 linkage.
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characterization of a psychrotrophic arthrobacter gene and its cold active beta galactosidase
Applied and Environmental Microbiology, 1994Co-Authors: Donald E. Trimbur, K R Gutshall, P Prema, Jean E. BrenchleyAbstract:Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active Beta-Galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces Beta-Galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding Beta-Galactosidase activity were obtained. We have subcloned and sequenced one of these Beta-Galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The Beta-Galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ Beta-Galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.
