The applications of biocatalysts in chemical industries are characterized by activity, selectivity, and stability. One key strategy to achieve high biocatalytic activity is the identification of novel enzymes with kinetics optimized for organic synthesis by Nature. The isolation of novel cytochrome P450 monooxygenase genes from Acidovorax sp. CHX100 and their functional expression in recombinant Pseudomonas taiwanensis VLB120 enabled efficient oxidation of cyclohexane to cyclohexanol. Although initial resting cell activities of 20 U gCDW (-1) were achieved, the rapid decrease in catalytic activity due to the toxicity of cyclohexane prevented synthetic applications. Cyclohexane toxicity was reduced and cellular activities stabilized over the reaction time by delivering the toxic substrate through the vapor phase and by balancing the aqueous phase mass transfer with the cellular conversion rate. The potential of this novel CYP enzyme was exploited by transferring the shake flask reaction to an aqueous-air segmented flow biofilm membrane reactor for maximizing productivity. Cyclohexane was continuously delivered via the silicone membrane. This ensured lower reactant toxicity and continuous product formation at an average volumetric productivity of 0.4 g L tube (-1) h(-1) for several days. This highlights the potential of combining a powerful catalyst with a beneficial reactor design to overcome critical issues of cyclohexane oxidation to cyclohexanol. It opens new opportunities for biocatalytic transformations of compounds which are toxic, volatile, and have low solubility in water.

Aim:  To develop real‐time quantitative PCR methods, based on the use of probes labelled with a stable fluorescent lanthanide chelate, for the quantification of different human faecal bifidobacterial populations. Methods and Results:  The designed quantitative PCR assays were found to be specific for the corresponding Bifidobacterium species or groups (Bifidobacterium longum group, Bifidobacterium catenulatum group, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium angulatum, Bifidobacterium bifidum and Bifidobacterium dentium). The detection limits of the methodologies used ranged between 2 × 105 and 9 × 103 cells g−1 of faeces. The applicability of the developed assays was tested by analysing 20 human faecal samples. Bif. longum group was found to be the qualitatively and quantitatively predominant bifidobacterial group. Conclusions:  The real‐time PCR procedures developed here are specific, accurate, rapid and easy methods for the quantification of Bifidobacterium groups or species in human faecal samples. Significance and Impact of the Study:  The developed procedures will facilitate rapid and objective counting of large numbers of samples increasing our knowledge on the role of gut bifidobacterial microbiota in health and disease. This will contribute to the efficient use of intestinal bacterial assays in research, food and pharmaceutical development as well as in the assessment of dietary management of diseases.

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