( 2010 )
Sporolactobacillus putidus sp. nov., an endospore-forming lactic acid bacterium isolated from spoiled orange juice.
PMID : 19684317 : DOI : 10.1099/ijs.0.002048-0
A Gram-positive, endospore-forming, lactic acid bacterium was isolated from spoiled orange juice. The organism, strain QC81-06(T), grew microaerobically or anaerobically at 30-45 degrees C (optimum 35 degrees C) and pH 3.5-5.5 (optimum pH 4.5), and produced acid from various sugars. D-Lactic acid was produced. It contained menaquinone-7 as the major isoprenoid quinone. The G+C content of the genomic DNA was 47.5 mol%. The predominant cellular fatty acids of the strain were iso-C(16 : 0), anteiso-C(15 : 0) and anteiso-C(17 : 0). Phylogenetic analyses based on the 16S rRNA gene and gyrB gene (DNA gyrase B subunit gene) revealed that strain QC81-06(T) clustered with Sporolactobacillus species but the strain was clearly distinct from other Sporolactobacillus species with significant bootstrap values. The levels of 16S rRNA gene and gyrB gene sequence similarities between strain QC81-06(T) and the other strains of the cluster were 96.0-97.0 % and 75.1-77.2 %, respectively. On the basis of these results, strain QC81-06(T) should be classified as a novel Sporolactobacillus species for which the name Sporolactobacillus putidus is proposed. The type strain is strain QC81-06(T) (=DSM 21265(T)=JCM 15325(T)).
( 2014 )
Mn??/Mg ??-dependent pyruvate kinase from a D-lactic acid-producing bacterium Sporolactobacillus inulinus: characterization of a novel Mn??-mediated allosterically regulated enzyme.
PMID : 23695776 : DOI : 10.1007/s00253-013-4907-9
Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in D-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve D-lactic acid production. A novel Mn(2+)/Mg(2+)-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn(2+) compared to those of the common PYK cofactor Mg(2+), and the circular dichroism spectra of the S. inulinus PYK suggested a Mn(2+)-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO? and KH?PO? significantly affected glycolysis flux and D-lactic acid production, which is consistent with the allosteric regulation of Mn(2+) and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus D-lactic acid production.
( 2012 )
Glucokinase contributes to glucose phosphorylation in D-lactic acid production by Sporolactobacillus inulinus Y2-8.
PMID : 22892885 : DOI : 10.1007/s10295-012-1176-z
Sporolactobacillus inulinus, a homofermentative lactic acid bacterium, is a species capable of efficient industrial D-lactic acid production from glucose. Glucose phosphorylation is the key step of glucose metabolism, and fine-tuned expression of which can improve D-lactic acid production. During growth on high-concentration glucose, a fast induction of high glucokinase (GLK) activity was observed, and paralleled the patterns of glucose consumption and D-lactic acid accumulation, while phosphoenolpyruvate phosphotransferase system (PTS) activity was completely repressed. The transmembrane proton gradient of 1.3-1.5 units was expected to generate a large proton motive force to the uptake of glucose. This suggests that the GLK pathway is the major route for glucose utilization, with the uptake of glucose through PTS-independent transport systems and phosphorylation of glucose by GLK in S. inulinus D-lactic acid production. The gene encoding GLK was cloned from S. inulinus and expressed in Escherichia coli. The amino acid sequence revealed significant similarity to GLK sequences from Bacillaceae. The recombinant GLK was purified and shown to be a homodimer with a subunit molecular mass of 34.5 kDa. Strikingly, it demonstrated an unusual broad substrate specificity, catalyzing phosphorylation of 2-deoxyglucose, mannitol, maltose, galactose and glucosamine, in addition to glucose. This report documented the key step concerning glucose phosphorylation of S. inulinus, which will help to understand the regulation of glucose metabolism and D-lactic acid production.