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d-Psicose 3-epimerase is an enzyme that catalyzes the synthesis of d-psicose from d-fructose. We cloned the d-psicose 3-epimerase from Ruminococcus sp. (RDPE) and expressed it in Bacillus subtilis A311. By a two-step pH regulation of segmented fermentation, we significantly improved the RDPE production and decreased the fermentation cost. The two-step regulation consisted of the first step maintained the pH value at 7.0 for 24 H and the second step adjusted the pH value up to 7.5 slowly for another 24 H. Finally, the RDPE production was increased to 74 U/mL, which was about 2.5-fold compared with the control. Our segmented fermentation strategy provides an important experimental basis for the industrial-scale production of RDPE.

A laboratory scale experimental pipeline system is developed to investigate the leakage behavior of high pressure CO 2 . The 23‐m‐long experimental pipeline could provide an inner pressure varying from 0.1 to 10 MPa and a preset temperature within the range of 0 ∼ 50 °C to ensure a stable initial phase state for the leakage test. Experiments on CO 2 leakage with three initial inner pressures and four different nozzle orifice sizes were conducted using this system. The individual and combined effects of inner pressure and leakage orifice size on the main characteristic flow parameters of the pipeline, the structure of the leakage jet, and the temperature decrease of the plume were investigated. Based on the experimental results, limitations of the previous theoretical formula on mass loss rate were discussed. The maximum temperature decrease along the centerline of the far‐field plume by Joule‐Thomson effect was also studied. The new experimental test facility proves to be valuable in analyzing the hazardous leakage process of CO 2 from pipelines.

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (Kcat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711–720. © 2012 Wiley Periodicals, Inc.