Summary
The β-glucosidase from ATCC 21400, anAgrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed phase FPLC. The partial amino acid sequence for one peptide was determined by automated Edman degradation. The sequence was used to synthesize a mixture of oligodeoxyribonucleotides which was used as a hybridization probe to identify a recombinant DNA clone carrying the gene for β-glucosidase. A single clone was isolated which expressed an enzymatic activity that hydrolyzed several β-glucosides. The enzymatic activity produced by this clone could be adsorbed by rabbit antiserum raised against theAgrobacterium enzyme. The direction of transcription of the β-glucosidase gene was determined by verifying the DNA sequence 3′ to the oligodeoxyribonucleotide probe binding site. After subcloning the gene a high level of expression was obtained in the plasmid vector pUC18 using the lacZ gene promoter.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Armentrout RW, Brown RD (1981) Molecular cloning of genes for cellobiose utilization and their expression inEscherichia coli. Appl Environ Microbiol 41:1355–1362
Atkinson T, Smith M (1984) In: Oligonucleotide synthesis a practical approach. Gait NJ (ed) IRL Press, Washington
Barras F, Chambost JP, Chippaux M (1984) Cellobiose metabolism inErwinia: A genetic study. Mol Gen Genet 197:490–496
Crestfield AM, Moore S, Stein WH (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem 193:622–627
Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence ofEscherichia coli cells. Gene 6:23–28
Day AG, Withers SG (1986) The purification and characterization of a β-glucosidase fromAlcaligenes faecalis. Can J Biochem Cell Biol (in press)
Gilkes NR, Kilburn DG, Langsford ML, Miller RC, Jr, Wakarchuk WW, Warren RAJ, Whittle DJ, Wong WKR (1984) Isolation and characterization ofEscherichia coli clones expressing cellulase genes fromCellulomonas fimi. J Gen Microbiol 130:1377–1384
Grunstein M, Wallis J (1979) Colony hybridization. Methods Enzymol 68:379–389
Guo Li-He, Wu R (1983) Exonuclease III: Use for DNA sequence analysis and in specific deletions of nucleotides. Methods Enzymol 100:60–97
Han YW, Srinivasin VR (1968) Isolation and characterization of a cellulose utilizing bacterium. Appl Microbiol 16:1140–1145
Han YW, Srinivasin VR (1969) The purification and characterization of β-glucosidase ofAlcaligenes faecalis. J Bacteriol 100:1355–1363
Katz L, Kingsbury DT, Helinski DR (1973) Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein complex. J Bacteriol 114:577–591
Laemmli UK (1970) Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227:680–685
Lathe R, Kieny MP, Skory S, Lecocq JP (1984) Linker tailing: Unphosphorylated linker oligonucleotides for joining DNA termini. DNA 3:173–182
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193:265–276
Maniatis T, Fritsch EF, Sambrook J (1982) In: Molecular cloning. Cold Spring Harbor Laboratory, New York
Marmur J (1961) A procedure for the isolation deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218
Miller JH (1972) In: Experiments in molecular genetics. Coldspring Harbor Laboratory, New York
Penttila ME, Helena-Nevalainen KM, Raynal A, Knowles JKC (1984) Cloning ofAspergillus niger genes in yeast. Expression of the gene codingAspergillus β-glucosidase. Mol Gen Genet 194:494–499
Raynal A, Guerineau M (1984) Cloning and expression of the structural gene for β-glucosidase ofKluyveromyces fragilis inEscherichia coli andSaccharomyces cerevisiae. Mol Gen Genet 195:108–115
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sato M, Takahaski H (1967) Fermentation of C14-labelled cellobiose byCellulomonas fimi. Agri Biol Chem 31:470–474
Schimz KL, Broll B, John B (1983) Cellobiose phosphorylase (EC 2.4.1.20) ofCellulomonas: Occurrence, induction and its role in cellobiose metabolism. Arch Microbiol 135:241–249
Schleif RF, Wensink PC (1981) In: Practical methods in molecular biology Springer, Berlin Heidelberg New York
Shewale JG (1982) β-glucosidase: Its role in cellulase synthesis and hydrolysis of cellulose. Eur J Biochem 14:435–443
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Sternberg D, Vijayyakumar P, Reese ET (1977) β-glucosidase: Microbial production and effect on enzymatic hydrolysis of cellulose, Can J Microbiol 23:139–147
Wakarchuk WW, Kilburn DG, Miller RC Jr, Warren RAJ (1984) The preliminary characterization of the β-glucosidases ofCellulomonas fimi. J Gen Microbiol 130:1385–1389
Whittle DJ, Kilburn DG, Miller RC Jr, Warren RAJ (1982) Molecular cloning of aCellulomonas fimi cellulase gene inEscherichia coli. Gene 17:139–145
Workman WE, Day DF (1982) Purification and properties of β-glucosidase fromAspergillus terrus. Appl Environ Microbiol 44:1289–1295
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–119
Zoller MJ, Smith M (1983) Oligo nucleotide directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol 100:468–500
Author information
Authors and Affiliations
Additional information
Communicated by A. Böck
Rights and permissions
About this article
Cite this article
Wakarchuk, W.W., Kilburn, D.G., Miller, R.C. et al. The molecular cloning and expression of a cellobiase gene from anAgrobacterium inEscherichia coli . Molec. Gen. Genet. 205, 146–152 (1986). https://doi.org/10.1007/BF02428044
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02428044