Strain S1 Research Articles

A novel biotechnology for enhanced ciprofloxacin removal via bioaugmentation of Paraclostridium sp.

To enhance the removal of ciprofloxacin (CIP, 1000–10,000 μg/L) in pharmaceutical wastewater, an efficient CIP-degrading bacterium, Paraclostridium sp. (strain S2), was immobilized and added into a sulfate-reducing up-flow sludge bed (SRUSB) bioreactor during long-term operation (over 80 days). CIP removal was increased by 18.7 % in the bioaugmented bioreactor with immobilized strain S2 compared to that in the control bioreactor under high CIP concentration (i.e., 10,000 μg/L), and the acute toxicity of the effluent in the bioaugmented bioreactor was significantly decreased by 81.1 % compared to that of the control. These results were attributed to the specific adaptability of strain S2 to environments via the alteration of its microbial physiology (e.g., extracellular polymeric substance (EPS) secretion, membrane permeability, cell surface charge, and hydrophobicity), which ensured its sustainability in the bioreactor, thus enhancing CIP removal. This study presents a novel biotechnology for CIP-contaminated wastewater treatment via the bioaugmentation by strain S2.

English

Both alternative sigma factor RpoS and ubiquitous secondary messenger c-di-GMP participate in the biofilm forming of Salmonella Pullorum; however, the relationship between RpoS and c-di-GMP-regulated genes during biofilm forming remains unclear. In this study, nine genes related with c-di-GMP regulation were found to be differentially expressed (P<0.01) by RNA-seq analysis when compared with S. Pullorum strain S9 and its rpoS deletion strain S9S. Specifically, the rpoS deletion strain S9S had higher transcription level of gene STM1703 and lower c-di-GMP concentration and biofilm-forming ability than S9 (P<0.01). The STM1703 gene deletion in strains S9 and S9S significantly enhanced the c-di-GMP concentration and the biofilm-forming ability (P<0.01). qRT–PCR analysis showed that rpoS deletion or P193L substitution in RpoS increased the transcription level of the STM1703 gene by decreasing the transcription levels of the csrA and STM1344 genes (P<0.01). Overall, RpoS-mediated STM1703 negatively regulates the biofilm formation of S. Pullorum by degrading c-di-GMP

Prospecting bacterial volatile organic compounds antifungal activities against postharvest diseases

Chemical pesticides have a plenty of negative impacts on human health and on the environment. Thus, modern agriculture cropping systems are moving towards more eco-friendly alternatives. This study aims to investigate the bioprotective effect of three volatile organic compounds (VOCs: N-Ethylaniline, 2-Heptanone and 3-Methylbutan-1-ol) produced by endophytic bacteria against 21 phytopathogenic fungal strains and their bioprotective effect on horticulture products i.e. tomato and lemon fruits and Potato tubers. The results showed that N-Ethylaniline and 3-methylbutan-1-ol had better antagonistic activity against the fungal strains by inhibiting the mycelia growth of the studied fungal strains at different concentrations. The N-Ethylaniline showed the lowest effective concentration (EC50) against B. cinerea strain S5 (0,258 mL/L headspace), Fusarium solani strain SB4.15.1 (0,496 mL/L headspace) and Colletotrichum gloeosporioides strain ManS3Fr02 (0,206 mL/L headspace). At EC50 this compound significantly reduced B. cinerea and C. gloeosporioides infections on tomato and lemon fruits, respectively. However, N-Ethylaniline didn’t showed significant effect on F. solani infection on Potato tubers. This study showed the broad spectrum of in vitro antifungal activity of N-Ethylaniline and its effect to reduce postharvest infections of some fungal diseases suggesting its potential use as a biofumigant.

Successive mycelial subculturing decreased lignocellulase activity and increased ROS accumulation in Volvariella volvacea.

Strain degradation is a common problem in many artificially-cultivated edible mushrooms. As a fungus with poor tolerance to low-temperature, Volvariella volvacea cannot delay its degradation by long-term low temperature storage like other fungi, so its degradation is particularly severe, which hinders industrial applications. Periodic mycelial subculture is a common storage method for V. volvacea, but excessive subculturing can also lead to strain degeneration. After 20 months of continuous subculturing every 3 days, V. volvacea strains S1–S20 were obtained, and their characteristics throughout the subculture process were analyzed. With increasing number of subculture, the growth rate, mycelial biomass, the number of fruiting bodies and biological efficiency gradually decreased while the production cycle and the time to primordium formation was lengthened. Strains S13–S20, obtained after 13–20 months of mycelial subculturing, also lacked the ability to produce fruiting bodies during cultivation experiments. Determination of reactive oxygen species (ROS) content as well as enzyme activity showed that decreased lignocellulase activity, along with excessive accumulation of ROS, was concomitant with the subculture-associated degeneration of V. volvacea. Reverse transcription polymerase chain reaction (RT-PCR) was eventually used to analyze the gene expression for lignocellulase and antioxidant enzymes in subcultured V. volvacea strains, with the results found to be consistent with prior observations regarding enzyme activities. These findings could form the basis of further studies on the degeneration mechanism of V. volvacea and other fungi.

Screening of new Paenibacillus polymyxa S3 and its disease resistance of grass carp (Ctenopharyngodon idellus).

A new strain of Paenibacillus polymyxa S3 with antagonistic effects on 11 major fish pathogens (especially Aeromonas hydrophila), but had no toxicity to grass carp, was screened from the sediment of fishponds. In vivo colonization studies showed that strain S3 could be colonized and distributed in the gill and abdomen of the grass carp. Bioassay results showed that the weight growth rate of grass carp in the strain S3 oral group (16.01%) and strain S3 immersion group (16.44%) was significantly higher than those of the control group (8.61%). At the same time, the activities of ACP, AKP, CAT and GSH-Px in the serum of grass carp in oral and immersion groups were significantly higher than those of the control group. In addition, the treatment with strain S3 could significantly upregulate the expression of the antioxidant-related genes and immune-related genes Keap1, Nrf2, C3, LZM, IgM, TLR-4 and MyD-88 in grass carp tissues. The challenge test showed that strain S3 treatment significantly increased the survival rate of grass carp infected with Aeromonas hydrophila. Whole genome sequencing analysis showed that strain S3 had 16 active metabolite gene clusters, indicating that it had abundant gene resources, which provided important support for its development for fish microecological preparations. In summary, a new strain of Paenibacillus polymyxa S3 with antibacterial activity against a variety of fish pathogens was screened in this study and its probiotic function was evaluated, proving its potential value in fisheries.

Bacterial synthesis of magnetic Fe3O4 nanoparticles: Decolorization Acid Red 88 using FeNPs/Ca-Alg beads

• A new route to biosynthesis of FeNPs using a novel hyperthermophile bacterial strain isolated from Choghart iron mine. • FeNPs/Ca-Alg beads had a high ability to decolorize Acid Red 88 dye. • Metabolites decolorization of Acid Red 88 dye by FeNPs/Ca-Alg beads was evaluated using GC/MS analysis. • FeNPs indicated great anti-cancer activity. • FeNPs/Ca-Alg beads effectively kill pathogenic bacteria. The research studied the bacterial synthesis of magnetic iron oxide nanoparticles (FeNPs), their biological activity, and the biodegradation efficiency of Acid Red 88. An iron-resistant bacterial strain S2 was isolated from Choghart iron mine soil and selected to synthesize FeNPs. The phylogenetic analysis depended on 16S rDNA sequence comparison resolution and showed that strain S2 was 99.68% similar to Bacillus zhangzhouensis . FeNPs were identified by UV–Vis, FTIR, and XRD spectroscopy. SEM-EDX conclusions corroborated the size of FeNPs in the range of 20–70 nm in the rhombus form. The finding indicated that FeNPs had antimicrobial activity on tested bacteria. According to the brine shrimp lethality (BSL) test, FeNPs showed great anticancer activity with LC 50 value of 0.5 μg/ml. The biodegradation of Acid Red 88 was investigated separately by FeNPs/Ca-Alg beads and FeNPs powder. FeNPs/Ca-Alg beads decolorized Acid Red 88 with 100% efficiency after 24 h, while FeNPs powder decolorized Acid Red 88 with 100% efficiency after 72 h. Moreover, Ca-Alg beads were applied as control and could not decolorize the dye. Decolorization of Acid Red 88 using FeNPs/Ca-Alg beads was investigated by GC/MS analysis. In this analysis, no mass was found related to the initial mass of Acid Red 88. According to these conclusions, it can be inferred that the dye is completely decomposed. The toxicity of Acid Red 88 dye, and the decolorization products, was assessed using BSL and phytotoxicity test. The conclusions determined that Acid Red 88 dye had toxicity effect. Further, the decolorization products could not kill half of the shrimp due to their low toxicity and did not inhibit the germination of radish seeds.

Potential probiotic properties and molecular identification of lactic acid Bacteria isolated from fermented millet porridge or ragi koozh and jalebi batter

The consumer market has significantly increased in the search for nutritional and probiotic formulated products. In the present study, probiotic characterization parameters have been evaluated for the bacteria isolated from fermented millet porridge or ragi koozh and jalebi batter. Initially, 30 colonies were randomly picked and screened for lactic acid production. Lactic acid-producing 13 isolates showing gram-positive cell wall, non-spore-forming capacity and catalase-negative strains were subjected to probiotic characterization. Among 13 isolates, 2 isolates S9 and S13 exhibited good tolerance towards in-vitro characterization parameters such as acid and bile tolerance, adhesion to hydrocarbons, autoaggregation, co-aggregation, antibacterial activity, and safety measures. The potential probiotic strains S9 and S13 were identified by molecular methods, and evolutionary imprints and submitted to GenBank as Weisella cibaria and Enterococcus lactis. Results showed the efficacy of probiotic characteristics of isolates S9 and S13 which can be used for the formulation of probiotic products.

Biodegradation and optimization of bilge water in a sequencing batch reactor using response surface methodology

Bilge water is a significant source of pollution in the marine environment and has captured widespread international attention. In this study, a sequencing batch reactor (SBR) combined with strain S2 identified as Bacillus licheniformis was employed to assess the biodegradation of Chemical Oxygen Demand (COD) from bilge water. The influencing variables such as temperature, pH level and inoculum concentration on the performance SBR system were optimized by utilizing response surface methodology (RSM). The experimental results showed that the maximum COD removal of 77.81% was reached at the optimal SBR operation conditions of temperature 35.44 °C pH 8.13, and inoculum concentration 31.47 mL. In the practical application of SBR, it was found that the decrease in hydraulic retention time (HRT) was accompanied by a decrease in COD degradation rate. The biodegradation kinetics of COD in bilge water were well fitted with the first–order equation with a higher R2 value of 0.98106. In conclusion, COD in bilge water can be efficiently biodegraded by SBR under the optimization of RSM.

An intranasally administrated SARS-CoV-2 beta variant subunit booster vaccine prevents beta variant replication in rhesus macaques.

Emergence of SARS-CoV-2 variants and waning of vaccine/infection-induced immunity pose threats to curbing the COVID-19 pandemic. Effective, safe, and convenient booster vaccines are in need. We hypothesized that a variant-modified mucosal booster vaccine might induce local immunity to prevent SARS-CoV-2 infection at the port of entry. The beta-variant is one of the hardest to cross-neutralize. Herein, we assessed the protective efficacy of an intranasal booster composed of beta variant-spike protein S1 with IL-15 and TLR agonists in previously immunized macaques. The macaques were first vaccinated with Wuhan strain S1 with the same adjuvant. A total of 1 year later, negligibly detectable SARS-CoV-2-specific antibody remained. Nevertheless, the booster induced vigorous humoral immunity including serum- and bronchoalveolar lavage (BAL)-IgG, secretory nasal- and BAL-IgA, and neutralizing antibody against the original strain and/or beta variant. Beta-variant S1-specific CD4+ and CD8+ T cell responses were also elicited in PBMC and BAL. Following SARS-CoV-2 beta variant challenge, the vaccinated group demonstrated significant protection against viral replication in the upper and lower respiratory tracts, with almost full protection in the nasal cavity. The fact that one intranasal beta-variant booster administrated 1 year after the first vaccination provoked protective immunity against beta variant infections may inform future SARS-CoV-2 booster design and administration timing.

A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System.

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25–35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.

Complete Genome Analysis of Rhodococcus opacus S8 Capable of Degrading Alkanes and Producing Biosurfactant Reveals Its Genetic Adaptation for Crude Oil Decomposition.

Microorganisms capable of decomposing hydrophobic substrates in cold climates are of considerable interest both in terms of studying adaptive reactions to low temperatures and in terms of their application in biotechnologies for cleaning up oil spills in a crude-oil polluted soil. The aim of this work was to investigate the genome of Rhodococcus opacus S8 and explore behavior traits of this strain grown in the presence of hexadecane. The genome size of strain S8 is 8.78 Mb, of which the chromosome size is 7.75 Mb. The S8 strain contains 2 circular plasmids of 135 kb and 105 kb and a linear plasmid with a size of 788 kb. The analysis of the genome revealed the presence of genes responsible for the degradation of alkanes and synthesis of biosurfactants. The peculiarities of morphology of microbial cells when interacting with a hydrophobic substrate were revealed. An adaptive mechanism responsible in the absence of oxygen for maintaining the process of degradation of hexadecane is discussed. The data obtained show that the strain S8 has great potential to be used in biotechnologies.

Regulatory mechanism of Haa1p and Tye7p in Saccharomyces cerevisiae when fermenting mixed glucose and xylose with or without inhibitors

BackgroundVarious inhibitors coexist in the hydrolysate derived from lignocellulosic biomass. They inhibit the performance of Saccharomyces cerevisiae and further restrict the development of industrial bioethanol production. Transcription factors are regarded as targets for constructing robust S. cerevisiae by genetic engineering. The tolerance-related transcription factors have been successively reported, while their regulatory mechanisms are not clear. In this study, we revealed the regulation mechanisms of Haa1p and Tye7p that had outstanding contributions to the improvement of the fermentation performance and multiple inhibitor tolerance of S. cerevisiae.ResultsComparative transcriptomic analyses were applied to reveal the regulatory mechanisms of Haa1p and Tye7p under mixed sugar fermentation conditions with mixed inhibitors [acetic acid and furfural (AFur)] or without inhibitor (C) using the original strain s6 (S), the HAA1-overexpressing strain s6H3 (H), and the TYE7-overexpressing strain s6T3 (T). The expression of the pathways related to carbohydrate, amino acid, transcription, translation, cofactors, and vitamins metabolism was enhanced in the strains s6H3 and s6T3. Compared to C_H vs. C_S group, the unique DEGs in AFur_H vs. AFur_S group were further involved in oxidative phosphorylation, purine metabolism, vitamin B6 metabolism, and spliceosome under the regulation of Haa1p. A similar pattern appeared under the regulation of Tye7p, and the unique DEGs in AFur_T vs. AFur_S group were also involved in riboflavin metabolism and spliceosome. The most significant difference between the regulations of Haa1p and Tye7p was the intracellular energy supply. Haa1p preferred to enhance oxidative phosphorylation, while Tye7p tended to upregulate glycolysis/gluconeogenesis.ConclusionsGlobal gene expressions could be rewired with the overexpression of HAA1 or TYE7. The positive perturbations of energy and amino acid metabolism were beneficial to the improvement of the fermentation performance of the strain. Furthermore, strengthening of key cofactor metabolism, and transcriptional and translational regulation were helpful in improving the strain tolerance. This work provides a novel and comprehensive understanding of the regulation mechanisms of Haa1p and Tye7p in S. cerevisiae.

Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum

Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants. A guide RNA plasmid, pKM-CRISPR, was constructed, with spacers targeting the ksgA and mnuA genes transcribed under the control of the vlhA1.1 promoter in a backbone plasmid carrying the oriC of M. imitans, and this plasmid was introduced into electrocompetent M. gallisepticum strain S6 cells. PCR assays targeting the ksgA gene, followed by Sanger sequence analyses of the phenotypically resistant transformants, detected polymorphisms within the targeted region of ksgA, confirming the activity of the endogenous CRISPR/Cas system. The nuclease activity of the kasugamycin resistant colonies was then assessed using zymogram assays. The complete or partial loss of nuclease activity in the majority of kasugamycin resistant isolates transformed with the CRISPR plasmid confirmed that the endogenous CRISPR/Cas system had effectively interfered with the function of both ksgA and mnuA genes. Sanger sequencing and RT-qPCR analyses of the mnuA gene suggested that the M. gallisepticum CRISPR/Cas system can be programmed to cleave both DNA and RNA.

A dual-function phage regulator controls the response of cohabiting phage elements via regulation of the bacterial SOS response.

SummaryListeria monocytogenes strain 10403S harbors two phage elements in its chromosome; one produces infective virions and the other tailocins. It was previously demonstrated that induction of the two elements is coordinated, as they are regulated by the same anti-repressor. In this study, we identified AriS as another phage regulator that controls the two elements, bearing the capacity to inhibit their lytic induction under SOS conditions. AriS is a two-domain protein that possesses two distinct activities, one regulating the genes of its encoding phage and the other downregulating the bacterial SOS response. While the first activity associates with the AriS N-terminal AntA/AntB domain, the second associates with its C-terminal ANT/KilAC domain. The ANT/KilAC domain is conserved in many AriS-like proteins of listerial and non-listerial prophages, suggesting that temperate phages acquired such dual-function regulators to align their response with the other phage elements that cohabit the genome.

Biological characteristics and whole-genome analysis of the potential probiotic, Lactobacillus reuteri S5

Lactic acid bacteria are micro-organisms used for probiotic purposes and form major parts of human and mammalian intestinal microbiota, exerting important health-promoting effects on the host. Here, we evaluated Lactobacillus reuteri strain S5 isolated from the intestines of healthy white feather broilers. Lactobacillus reuteri S5 grew best after 20 h of incubation in MRS medium. Lactic acid production was 1·42 mmol l-1 at 24 h, which was well tolerated. Activities of T-AOC, GSH-Px and T-SOD in the cell-free fermentation supernatant of L. reuteri S5 were higher than those in the bacteria, and the strain showed good hydrophobicity in vitro. The dominant carbon and nitrogen sources of L. reuteri S5 were glucose and soybean meal. A high-quality complete genome map of L. reuteri S5 was obtained using a Pacbio nanopore third-generation sequencing platform. The results showed that L. reuteri S5 possesses a complete primary metabolic pathway, encoding the main functional enzymes of the glycolysis pathway and pentose phosphate pathway. The genome contains genes encoding antioxidants and conferring tolerance to inorganic salt ions, acids and bile salts. This study shows that L.reuteri S5 is a probiotic strain with excellent probiotic characteristics and has great potential for the development of feed additives to promote animal health.

SCREENING OF PECTINASE PRODUCING BACTERIA FROM SOIL AND PRODUCTION, PARTIAL PURIFICATION OF ENZYME

Bacterial pectinase enzyme is very much important at industrial level for different processes This present study focus on bacterial pectinase. Pectinase producing bacterial spp.is screen out from soil. About seven cultures of bacterial spp. were isolated from farmland soil sample using pectin agar plates. The selected isolates showed the zone of hydrolysis to confirmed the production of pectinase from the isolates. Among them the culture S4 showed prominent zone of hydrolysis surrounding the colony. Pectinase activity of each isolate were checked at an interval of 24 hrsusing DNSA method to access the enzyme efficiency produced by respective isolates. Keeping the ideal growth conditions the highest pectinase activity obtained among isolates was 11.43 U/ ml, by culture S4.Optimum condition for pectinase production was found at 72hrs. incubation period, 37˚C temperature and alkaline pH of 10 could be the optimum cultural conditions for bacterial strain S4. Bacterial strain S4 gave 40.85 U/ml partially purified enzyme.

Biodegradation of used motor oil by Streptomyces ginkgonis KM-1–2, isolated from soil polluted by waste oils in the region of Azzaba (Skikda-Algeria)

Actinobacteria have many properties that make them good candidates for the bioremediation of sites contaminated by several organic and inorganic pollutants. However, studies on the biodegradation of used motor oils by Actinobacteria, compared to other bacteria, remain little studied. Actinobacteria were isolated from soil contaminated with used motor oils and sewage sludge in order to select a species that can effectively degrade such pollutants. This study aims to assess their degradation capacity of the various hydrocarbon fractions contained in the oil by gas chromatography-mass spectrometry (GC-MS). Five Actinobacteria isolates were isolated by the enrichment method. The S1.1. A strain was considered the best biosurfactant producing strain. It presents the highest emulsification index compared to other isolated Actinobacteria (82.6%). Phenotypic and molecular identification by sequencing the 16 S rDNA gene makes it possible to assign this isolate to the species Streptomyces ginkgonis KM-1–2. Gravimetric analysis results of biodegraded used motor oil indicated that this strain is capable of degrading 76.4% of an initial 50 ml/l concentration of used motor oil after 4 weeks of incubation. The results of GC-MS analysis of the residual motor oil showed that strain S1.1. A degraded some long and intermediate chain alkanes completely or to shorter fractions. The Streptomyces ginkgonis strain KM-1–2 was also able to degrade certain alkylated mono-aromatic hydrocarbons linked to benzene, as well as certain alkylated and non-alkylated polycyclic aromatic hydrocarbons linked to anthracene, naphthalene, phenanthrene, fluorene, and azulene. This strain exhibited the highest emulsification index at 82.6%. This bacterium shows a significant biodegradation capacity and could, consequently, be used in the processes of bioaugmentation of sites contaminated by these oils.

Draft Genome Sequence of Bacteriocin-Encoding Enterococcus faecium Strain S6, Isolated from Camel Milk.

ABSTRACTEnterococcus faecium strain S6 is a newly identified bacteriocin producer isolated from raw camel milk. The draft genome sequence is composed of 2,617,971 bp, with 2,407 coding genes and a G+C content of 37.99%. The genome sequence analysis provided details into the antimicrobial properties of strain S6.

Seleção in vitro de bactérias probióticas do peixe ornamental Amazônico Nannostomus beckfordi

ABSTRACT This study aimed to isolate and select in vitro bacteria with probiotic potential for the Amazon ornamental fish Nannostomus beckfordi. For isolate, twelve fish underwent surgery procedure to remove their intestinal tract, macerate and then inoculate in the plate petri containing de Man Rugosa Sharped Agar (MRS). After bacterial growth (48 hours at 35ºC), selected strains were inoculated in MRS broth and submitted to resistance test with NaCl (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%), pH (4, 5, 6, 8 and 9) and bile salts (5% w/v). Inhibition test against pathogenic bacteria Aeromonas hydrophila, Pseudomonas aeroginosa, Streptococcus agalactiae and Aeromonas Jandaei was also performed. Within the isolated strains group (23 strains), only six (S1, S2, S3, S4, S5 and S6) showed probiotic potential. Strains S1 and S6 showed the greater resistance for NaCl (0.5% and 1%) and pH (5 and 6), but only S1 obtained better results to resist the bile salts. Even against pathogenic bacteria, the S1 showed the best results with inhibition halos greater than 9 mm. In the end, this bacterial strain (S1) was identified as Enterococcus faecium 11037CHB. Thus, this is the first report regarding isolated autochthonous bacterium E. faecium with probiotic potential of N. beckfordi.

Improving the kinetic parameters of nicotine oxidizing enzymes by homologous structure comparison and rational design

Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high kcat or low Km, but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high kcat (>1 s−1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar Km for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by ∼25% (0.011 s−1 to 0.014 s−1), and reduce the Km of the NctB protein by approximately 34% (940 μM–622 μM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications.