Dataset Information

The melREDCA Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in Bacillus subtilis.

ABSTRACT: Bacillus subtilis is a heterotrophic soil bacterium that hydrolyzes different polysaccharides mainly found in the decomposed plants. These carbohydrates are mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides (RFOs). RFOs are soluble ?-galactosides, such as raffinose, stachyose, and verbascose, that rank second only after sucrose in abundance. Genome sequencing and transcriptome analysis of B. subtilis indicated the presence of a putative ?-galactosidase-encoding gene (melA) located in the msmRE-amyDC-melA operon. Characterization of the MelA protein showed that it is a strictly Mn2+- and NAD+-dependent ?-galactosidase able to hydrolyze melibiose, raffinose, and stachyose. Transcription of the msmER-amyDC-melA operon is under control of a ?A-type promoter located upstream of msmR (P msmR ), which is negatively regulated by MsmR. The activity of P msmR was induced in the presence of melibiose and raffinose. MsmR is a transcriptional repressor that binds to two binding sites at P msmR located upstream of the -35 box and downstream of the transcriptional start site. MsmEX-AmyCD forms an ATP-binding cassette (ABC) transporter that probably transports melibiose into the cell. Since msmRE-amyDC-melA is a melibiose utilization system, we renamed the operon melREDCA IMPORTANCE Bacillus subtilis utilizes different polysaccharides produced by plants. These carbohydrates are primarily degraded by extracellular hydrolases, and the resulting oligo-, di-, and monosaccharides are transported into the cytosol via phosphoenolpyruvate-dependent phosphotransferase systems (PTS), major facilitator superfamily, and ATP-binding cassette (ABC) transporters. In this study, a new carbohydrate utilization system of B. subtilis responsible for the utilization of ?-galactosides of the raffinose family of oligosaccharides (RFOs) was investigated. RFOs are synthesized from sucrose in plants and are mainly found in the storage organs of plant leaves. Our results revealed the modus operandi of a new carbohydrate utilization system in B. subtilis.

SUBMITTER: Morabbi Heravi K

PROVIDER: S-EPMC6620409 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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The melREDCA Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in Bacillus subtilis.

Morabbi Heravi Kambiz K Watzlawick Hildegard H Altenbuchner Josef J

Journal of bacteriology 20190710 15

Bacillus subtilis is a heterotrophic soil bacterium that hydrolyzes different polysaccharides mainly found in the decomposed plants. These carbohydrates are mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides (RFOs). RFOs are soluble α-galactosides, such as raffinose, stachyose, and verbascose, that rank second only after sucrose in abundance. Genome sequencing and transcriptome analysis of B. subtilis indicated the presence of a putative α-galactosidase-e ...[more]

PMID: 31138628

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Project description:BACKGROUND:?-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of ?-galactosidase of Saccharomyces cerevisiae. RESULTS:The MEL1 gene coding for the ?-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-?-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with ?-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of ?-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS:ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

Dataset Information

The melREDCA Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in Bacillus subtilis.

Publications

The <i>melREDCA</i> Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in <i>Bacillus subtilis</i>.

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