Complete genome sequence analysis of a strain Lactobacillus pentosus ZFM94 and its probiotic characteristics.

Lactic acid bacteria have been attracting increased attentions recent years because of harboring probiotic properties. In present study, a Lactobacillus pentosus strain ZFM94 was screened from healthy infant feces and its probiotic characteristics were investigated. We found that ZFM94 was resistant to environmental stresses (temperature, pH and NaCl), tolerant to gastrointestinal juice and bile salts, with inhibitory action against pathogens and capacity of folate production etc. Additionally, complete genome sequence of the strain was analyzed to highlight the probiotic features at genetic level. Genomic characteristics along with the experimental studies is critically important for building an appropriate probiotic profile of novel strains. Genes that correspond to phenotypes mentioned above were identified. Moreover, genes potentially related to its adaptation, such as carbon metabolism and carbohydrate transporter, carbohydrate-active enzymes, and a novel gene cluster RaS-RiPPs, were also revealed. Together, ZFM94 could be considered as a potential probiotic candidate.

Expression and biochemical characterization of a new alkaline tannase from Lactobacillus pentosus.

Lactobacillus pentosus BA-7 and L. pentosus QA1-5 are tannin-tolerant lactic acid bacteria that were isolated from Miang, a traditional fermented tea-leaf found in northern Thailand and a tannin-rich substrate. Tannase encoding genes were isolated, cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant tannase was produced with production yields of 40 and 39 KU/L for LpTanBA-7 and LpTanQA1-5, respectively. Both revealed the same molecular weight of 50 kDa as estimated by SDS-PAGE and were optimally active under alkaline pH conditions LpTanQA1-5 revealed optimal temperatures in a range of 37-40 °C as is typically found in lactic acid bacteria, while LpTanBA-7 was active at higher temperatures with an optimum temperature range of 45-55 °C. LpTanBA-7 was found to be more stable within the same range of temperatures than LpTanQA1-5. Furthermore, it was active and stable toward various organic solvents and produced 50 mg/mL of gallic acid from 100 mg/mL tannic acid. Based on the results, LpTanBA-7 is considered a new alkali-moderately thermophilic tannase obtained from lactic acid bacterium that may be capable of a feasible production capacity of gallic acid and its esters. Furthermore, tannase that is active at high temperatures could also be used in tea products in order to develop a sweet aftertaste, as well as to improve levels of antioxidant activity.

Expression of genes involved in metabolism of phenolic compounds by Lactobacillus pentosus and its relevance for table-olive fermentations.

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-ß Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.

Characterization of Biomimetic Cofactors According to Stability, Redox Potentials, and Enzymatic Conversion by NADH Oxidase from Lactobacillus pentosus.

Oxidoreductases are attractive biocatalysts that convert achiral substrates into products of higher value, but they are also for the most part dependent on nicotinamide cofactors. Recently, biomimetic nicotinamide derivatives have received attention as less costly alternatives to natural cofactors. However, recycling of biomimetics is still challenging because there are only limited opportunities. Here, we have characterized various biomimetic cofactors with regard to stability and redox potentials to find the best alternative to natural cofactors. Further, the cofactor spectrum of NADH oxidase from Lactobacillus pentosus (LpNox) could be expanded, and the enzymatic activity was also compared to activities with different small-molecule catalysts. As a result, we succeeded in identifying several strategies for regeneration of oxidized biomimetics.

Cloning and characterization of two distinct water-forming NADH oxidases from Lactobacillus pentosus for the regeneration of NAD.

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K M of 99.0 µM (LpNox1) and 27.6 µM (LpNox2), and yielding catalytic efficiency k cat/K M of 1.0 and 0.2 µM(-1) s(-1), respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD(+) was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD(+) regeneration in dehydrogenase-catalyzed oxidations.

Bioinformatic analysis of dihydrofolate reductase predicted in the genome sequence of Lactobacillus pentosus KCA1.

Physiologic studies of Lactobacillus species show that some species cannot synthesize folate de novo, which is required for growth. Folate plays a critical role in regulating the amount of tetrahydrofolate in the cell that is utilized for DNA replication, and proliferation of the erythropoietic system. We recently sequenced the genome of Lactobacillus pentosus KCA1, isolated from a Nigerian subject. The genome has open reading frames coding for the complete genes required for folate biosynthesis. Our previous study shows that rats fed with L. pentosus KCA1 led to enhancement of haematological parameters. Bioinformatic tool such as ClustalW algorithm was used to analyze dihydrofolate reductase (folA/dfrA) encoded in the genome sequence of L. pentosus KCA1 for comparative multiple sequence alignments. I-TASSER was used to predict the 3-D model structure of the protein and potential active binding site residues. Result show that two unique amino acid substitutions were found in KCA1_1610 sequence at position 85 with alanine (A-Ala85), while other strains have aspartic acid (D-Asp) for other L. pentosus and threonine (T-Thr) for L. plantarum strains at the same position. The result suggests that dihydrofolate reductase can be used as a distinguishing marker between L. pentosus KCA1 and other pentosus including L. plantarum strains. The secondary structure prediction with I-TASSER revealed 5 alpha helices and 8 beta-strands. Twelve binding site residues were predicted in KCA1_1610 relative to the template protein 2zzaA in protein database (PDB). The predicted structure of KCA1_1610 dihydrofolate reductase can serve as a new template as an addition to structural genomics and generation of models for use in drug screening and physiological function inference.