Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli

Hancock, Kerrie R.; Rockman, E. S.; Young, Carolyn A.; Pearce, L. E.; Maddox, I. S.; Scott, David B.

doi:10.1128/jb.173.10.3084-3095.1991

Cited by 51 publications

(21 citation statements)

References 67 publications

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“…We designated this group lacG to separate it from the lacZ and lacS families. If the lacG enzymes (19), Klebsiella pneumoniae lacZ (5), Kluyveromyces lactis (25), Clostridium acetobutylicum (12), Streptococcus thermophilus (29), Lactobacillus bulgaricus (28), Leuconostoc lactis lacL and lacM (7), B. circulans bgaA (GenBank accession number L03424), B. circulans bgaB (GenBank accession number L03425), B. stearothermophilus (15), and Arthrobacter B7 isozyme 12 (this report). Consensus residues are boxed.…”

Section: Discussionmentioning

confidence: 99%

Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate

et al. 1995

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We have characterized a new psychrotrophic Arthrobacter isolate which produces ␤-galactosidase isozymes. When DNA from this isolate was transformed into an Escherichia coli host, we obtained three different fragments, designated 12, 14, and 15, each encoding a different ␤-galactosidase isozyme. The ␤-galactosidase produced from fragment 12 was of special interest because the protein subunit was smaller (about 71 versus 116 kDa) than those typically encoded by the lacZ family. The isozyme encoded by fragment 12 was purified, and its activity and thermostability were examined. Although the enzyme is highly specific towards ␤-Dgalactoside substrates, its levels in the isolate do not increase in cells grown with lactose. Nucleotide sequence determination showed that the gene encoding isozyme 12 is not similar to the other members of the lacZ family but has regions similar to ␤-galactosidase isozymes from Bacillus stearothermophilus and B. circulans. Addition of the isozyme 12 sequence to the database made it possible to examine these enzymes as possible members of a new, separate family. Our analysis of this new family showed some conserved amino acids corresponding to the lacZ acid-base catalytic region but no homology with the nucleophilic region. On the basis of these comparisons, we designated this a new lacG family.The intricate genetic and biochemical studies with the lacZencoded ␤-galactosidase (EC 3.2.1.23) of Escherichia coli provide an excellent foundation for examining and comparing the structures of other ␤-galactosidases. Because the lacZ enzyme is used so frequently, we often overlook other interesting and unusual ␤-galactosidases. The study of these new enzymes, however, can help explain the roles of various amino acids in enzyme structure, provide insight into the evolution of genes encoding ␤-galactosidases, suggest structural relationships to other glycosidases, and yield enzymes with valuable properties that differ from those of the E. coli enzyme.We had isolated psychrotrophic Arthrobacter strains which produced cold-active ␤-galactosidases and demonstrated that cells of one strain, B7, contained at least two isozymes when grown with lactose as a carbon source (22). When we used DNA from isolate B7 to transform an E. coli host, we obtained three transformants, each containing a different DNA segment encoding a ␤-galactosidase activity. One gene, designated 15, encoded the predominant isozyme found in lactose-grown cells. This enzyme has a temperature optimum about 20ЊC below that of the E. coli ␤-galactosidase and has been purified and characterized, and the gene that encodes it has been sequenced (33). This enzyme has the conserved acid-base and nucleophilic sites involved in catalysis that are typically found with the lacZ family of ␤-galactosidases. The presence of the other isozymes in this isolate raised questions about their possible functions in enabling these psychrotrophic strains to survive rapid temperature changes or in using other glycoside substrates. Our initial work with the secon...

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Section: Discussionmentioning

confidence: 99%

Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate

et al. 1995

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“…5). Asn604 is one of the amino acids forming this substrate binding pocket in the protein (14), and this residue is conserved in several other known ␤-galactosidase sequences (25,(27)(28)(29), except the evolved galactosidase gene (ebgA) of E. coli (30). In our evolved fucosidase, Asn604 is replaced by Ser.…”

Section: Kineticsmentioning

confidence: 99%

Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening

Zhang

Dawes²,

Stemmer³

1997

Proc. Natl. Acad. Sci. U.S.A.

243

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An efficient ␤-fucosidase was evolved by DNA shuff ling from the Escherichia coli lacZ ␤-galactosidase. Seven rounds of DNA shuff ling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round. Compared with native ␤-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity for onitrophenyl fucopyranoside versus o-nitrophenyl galactopyranoside and a 300-fold increased substrate specificity for p-nitrophenyl fucopyranoside versus p-nitrophenyl galactopyranoside. The evolved cell line showed a 66-fold increase in p-nitrophenyl fucosidase specific activity. The evolved fucosidase has a 10-to 20-fold increased k cat ͞K m for the fucose substrates compared with the native enzyme. The DNA sequence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme. This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuff ling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology. Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design.Proteins and enzymes with novel functions and properties can be obtained either by searching the largely unknown natural species or by improving upon currently known natural proteins or enzymes. The latter approach may be more suitable for creating properties for which natural evolutionary processes are unlikely to have been selected.One promising strategy to create such novel properties is by directed molecular evolution. Starting with known natural protein(s), multiple rounds of mutagenesis, functional screening, and amplification can be carried out. When the mutation rate, library size, and selection pressures are properly balanced, the desired phenotype of a protein generally increases with each round (1-8). The advantage of such a process is that it can be used to rapidly evolve any protein, without any knowledge of its structure.

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“…coli (25), Streptococcus thermophilus (43), Lactobacillus bulgaricus (42), and Clostridium acetobutylicum (21). A computer alignment of these sequences is given in Fig.…”

Section: R T P T P M F W R a T T D N D H G S G F S V K S A Q W Y A A mentioning

confidence: 99%

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes

et al. 1992

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A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional j-galactosidase. Deletion and complementation analysis showed that the coding region for ,-galactosidase was located on a 5. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional 1-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N-and C-terminal parts, respectively, of ,-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral 13-galactosidase gene.Two systems for lactose transport and hydrolysis among bacteria are known. The first system has only been found in gram-positive bacteria and involves a phosphoenolpyruvatedependent phosphotransferase system, by which lactose is phosphorylated during transport and subsequently hydrolyzed by a phospho-,B-galactosidase (for reviews, see references 14 and 22). In the second, more widespread system, lactose is transported across the cellular membrane by a galactoside permease, and the unmodified internalized sugar is hydrolyzed by a 13-galactosidase. Most research has focused on the lactose permease (lacY) and ,-galactosidase (lacZ) genes from Escherichia coli (for reviews, see references 3, 24, and 25), and its lacZ gene has been developed into a useful tool in molecular genetics. Similar lac genes located on chromosomal or plasmid DNA have been found in other gram-negative bacteria (20), and in one instance a lac transposon (Tn951) has been reported (11).Recently, lac genes have been characterized in lactic acid bacteria that are used as starter cultures in dairy fermentations and therefore are highly specialized lactose utilizers. Genetic studies have shown that the lactose-specific phosphotransferase system enzymes are homologous and plasmid encoded in Lactococcus lactis (14)(15)(16)30) and Lactobacillus casei (1, 2, 38). In contrast, the homologous lac genes of Streptococcus thernophilus and Lactobacillus bulgaricus are chromosomally located and have been found to encode unique lactose permeases (28, 37) and ,B-galactosidases that show high similarity to those of gram-negative bacteria (42,43). A plasmid-encoded P-galactosidase in Lactobacillus casei ATCC 393 has been reported (10). In addition, we showed recently that Leuconostoc lactis NZ6009 also contains a lactose plasmid that codes for a P-galactosidase (13). gram-positive cocci that are used for industrial milk and wine fermentations. We recently started the genetic characterization of Leuconostoc spp. (13) and focused on the plasmidlocated lac genes in L. lactis NZ6009 (12). Here we describe the molecular characterization of a DNA fragment from the lactose plasmid pNZ63 that ...

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Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli

Cited by 51 publications

References 67 publications

Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate

Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate

Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes

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