Cloning and expression of ?-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Lee, Byong H.; Robert, Normand; Jacques, Chantal; Ricard, Line

doi:10.1007/bf01086342

Cited by 7 publications

(3 citation statements)

References 15 publications

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“…Investigators have screened microorganisms capable of producing high levels of Bgalactosidase, and in recent years different research groups (Hirata et al, 1985;Herman and McKay, 1986;Saito et al, 1989;Lee et al, 1990) have reported the cloning of the B-galactosidase gene from different thermophilic bacteria. Although a number of sources for B-galactosidase now exist, most of them are from thermophilie bacteria.…”

Section: Discussionmentioning

confidence: 99%

Cloning and expression of ?-galactosidase from psychotrophic Bacillus subtilis KL88 into Escherichia coli

Torres

Lee

1995

Biotechnol Lett

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The B-galactosidase gene from Bacillus subtilis KL88 was cloned into Escherichia coli and the gene product characterized for its potential use in the dairy industry. The two recombinant plasmids that we obtanied encoded a B-galactosidase with the same catalytic and thermal characteristics as the native B-galactosidase from B. subtilis. The recombinant B-galactosidases exhibited high activity at low temperature (10°C), with maximum activity at 50°C and an optimum pH of 6.0. Its molecular weight was estimated to be 90 Kd. The restriction maps of the recombinant plasmids were constructed. The 13-galactosidase gene was located in a 2.3 Kb fragment.

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Section: Discussionmentioning

confidence: 99%

Cloning and expression of ?-galactosidase from psychotrophic Bacillus subtilis KL88 into Escherichia coli

Torres

Lee

1995

Biotechnol Lett

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“…Despite its widespread and longtime use in fermentation processes, progress in the genetics of this organism has been made only recently. Some genes have been isolated and analyzed (2,16,19,32,33,41,50). Among these are the lacS (lactose permease) and lacZ (3-galactosidase) genes, which are separated by only three nucleotides and are probably translationally coupled (32).…”

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confidence: 99%

Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus

et al. 1993

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Several pGEM5-and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilw at the loci of the lactose-metabolizing genes.Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food.

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“…Although there is abundant information on B-galactosidase, most studies have been done with thennophilic (Hirata et al, 1985;Herman and Mckay, 1986;Saito et al, 1989;Lee et al, 1990) and mesophilic bacteria, specially from lactic acid bacteria (de Vos and Vaugham, 1994). Very little information is available on new g-galactosidases with high activity in hydrolysing lactose at the low temperature at which milk and milk products am usually stored.…”

Section: Discussionmentioning

confidence: 99%

Expression of a ?-galactosidase gene from Lactobacillus sake in Escherichia coli

Torres¹,

Lee²

1995

Biotechnol Lett

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A B-galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. 'sa& was partially digested with the restriction enzyme SaDAI, and the 3-6 Kb fragment was ligated to the cloning vector pSP72 digested with BarnHI. One E. coli transformant expressing B-galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant B-galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 5O"C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.

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Cloning and expression of ?-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Cited by 7 publications

References 15 publications

Cloning and expression of ?-galactosidase from psychotrophic Bacillus subtilis KL88 into Escherichia coli

Cloning and expression of ?-galactosidase from psychotrophic Bacillus subtilis KL88 into Escherichia coli

Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus

Expression of a ?-galactosidase gene from Lactobacillus sake in Escherichia coli

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