Thermoanaerobacter ethanolicus Gene Cluster Containing the α- and β-Galactosidase Genes melA and lacA and Properties of Recombinant LacA

The nucleotide sequence of a 4936-bp genomic DNA fragment from the thermophilic bacterium Thermoanaerobacter ethanolicus has been determined. The fragment contains three open reading frames (ORFs). The product of the incomplete first ORF is highly homologous to α-galactosidases (melibiases). The second ORF corresponds to the lacA gene for a thermostable β-galactosidase. The product of the third ORF is similar to α-D-mannosidases. A putative ρ-independent terminator is located immediately downstream of the lacA stop codon, suggesting a cotranscription of the α- and β-galactosidase genes. The deduced molecular weight of LacA is 86 kDa. LacA belongs to glycosylhydrolase family 2 (GH2). Native recombinant LacA is a dimer and shows the highest activity at pH 5.7–6.0 and 75–80°C. LacA is most active with lactose (480 units per mg protein, Km = 30 mM); the activities with pNP-Gal and oNP-Gal are 330 and 420 units per mg protein, respectively. Immobilized on aldehyde silochrome, LacA is even more thermostable and retains its high activity.

[1]  J. Synowiecki,et al.  Cloning and nucleotide sequence of the thermostable β-galactosidase gene fromPyrococcus woesei inEscherichia coli and some properties of the isolated enzyme , 1998, Molecular biotechnology.

[2]  B Henrissat,et al.  A classification of glycosyl hydrolases based on amino acid sequence similarities. , 1991, The Biochemical journal.

[3]  V. Zverlov,et al.  Thermotoga neapolitana bglB gene, upstream of lamA, encodes a highly thermostable beta-glucosidase that is a laminaribiase. , 1997, Microbiology.

[4]  V. Zverlov,et al.  A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Molecular Structure of the Locus , 2003, Molecular Biology.

[5]  R. E. Huber,et al.  Quaternary structure, Mg2+ interactions, and some kinetic properties of the (β-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1 , 1996, Journal of protein chemistry.

[6]  J. Pridham,et al.  Biochemistry of -galactosidases. , 2006, Advances in enzymology and related areas of molecular biology.

[7]  J. Brenchley,et al.  A novel Arthrobacter beta-galactosidase with homology to eucaryotic beta-galactosidases , 1997, Journal of bacteriology.

[8]  J. Yon,et al.  Kinetic study of the activation process of -galactosidase from Escherichia coli by Mg 2+ . , 1972, European journal of biochemistry.

[9]  R. E. Huber,et al.  Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between β‐galactosidase and other glycohydrolases , 2008, Protein science : a publication of the Protein Society.

[10]  A Bairoch,et al.  Updating the sequence-based classification of glycosyl hydrolases. , 1996, The Biochemical journal.

[11]  R. Rella,et al.  Thermostable β-galactosidase from the archaebacterium Sulfolobus solfataricus Purification and properties , 1990 .

[12]  J. Wiegel,et al.  Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum , 1979, Journal of bacteriology.

[13]  B. Matthews,et al.  A structural view of the action of Escherichia coli (lacZ) beta-galactosidase. , 2001, Biochemistry.

[14]  P. Colman,et al.  Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  G. Shaw,et al.  Cloning, Expression, and Catabolite Repression of a Gene Encoding β-Galactosidase of Bacillus megateriumATCC 14581 , 1998 .

[16]  V. Zverlov,et al.  Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5 , 1999, Applied Microbiology and Biotechnology.

[17]  T. Nakayama,et al.  Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula. , 1994, The Journal of biological chemistry.

[18]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[19]  B. Matthews,et al.  Three-dimensional structure of β-galactosidase from E. coli. , 1994, Nature.

[20]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[21]  C. Robert,et al.  Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences. , 1991, Journal of general microbiology.

[22]  Y. B. Tewari.  Thermodynamics of industrially-important, enzyme-catalyzed reactions , 1990, Applied biochemistry and biotechnology.

[23]  J. T. Grace.  STUDIES OF EPSTEIN‐BARR VIRUS , 1970, Annals of the New York Academy of Sciences.

[24]  V. Zverlov,et al.  Two new cellulosome components encoded downstream of celI in the genome of Clostridium thermocellum: the non-processive endoglucanase CelN and the possibly structural protein CseP. , 2003, Microbiology.

[25]  O. Poch,et al.  Sequence of the Kluyveromyces lactis beta-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis. , 1992, Gene.

[26]  W. Hammes,et al.  Two genes encoding the β-galactosidase of Lactobacillus sake , 1995 .

[27]  V. Zverlov,et al.  Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-beta-1,3-glucanase bound to the outer cell surface. , 2003, Microbiology.

[28]  M. Rossi,et al.  Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties. , 1990, European journal of biochemistry.

[29]  T. Tanaka,et al.  Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119 , 1995, Applied and environmental microbiology.

[30]  V. Zverlov,et al.  Chi18A, the Endochitinase in the Cellulosome of the Thermophilic, Cellulolytic Bacterium Clostridium thermocellum , 2002, Applied and Environmental Microbiology.

[31]  B. Henrissat,et al.  Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. Withers,et al.  Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli. , 1992, The Journal of biological chemistry.

[33]  H. Bahl,et al.  Cloning and analysis of the beta-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1. , 1991, Gene.

[34]  K. Murata,et al.  Molecular Identification of Family 38 α-Mannosidase of Bacillus sp. Strain GL1, Responsible for Complete Depolymerization of Xanthan , 2002, Applied and Environmental Microbiology.

[35]  J. Vilotte.  Lowering the milk lactose content in vivo: potential interests, strategies and physiological consequences. , 2002, Reproduction, nutrition, development.

[36]  V. Zverlov,et al.  [Thermotoga neapolitana gene clusters participating in degradation of starch and maltodextins: molecular structure of the locus]. , 2003, Molekuliarnaia biologiia.