Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae: evaluation of production kinetics and enantioselective esterification potential for pharmaceutical applications.

Downstream processing is a significant part of a production process and accounts for 50-90% of the production cost of biotechnological products. Post-fermentation localization of a microbial metabolite contributes significantly to the recovery cost of the product. Enterobacter cloacae produced naturally, acidic lipase with a 0.023:1 extracellular localization ratio. This research aimed to re-direct the localization of lipase to the extracellular milieu to reduce recovery costs using multi-objective response surface optimization (MO-RSM). The approach resulted in a 1:0.32 extracellular: intracellular lipase ratio, with product formation kinetics of Luedeking-Piret function showing a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization. The enzyme was purified by an aqueous two-phase system which extracted 95.22% lipase with 72.36 purity. Characterization of the enzyme showed a molecular weight of 55.7 kDa, kcat of 68.59 s-1, and a Km of 0.63 mmol. Lipase activity occurred optimally at pH 2.5-3.5 and 50 °C, and was stable in most organic solvents tested. The acidic lipase demonstrated pH-dependent enantioselective esterification in resolving (R, S)-ibuprofen (E = 14, pH 4.5) and (R, S)-Naproxen (E = 13, pH 2.5), with an enantioselective preference for (S)-enantiomer in both drugs thus underpinning its potential for pharmaceutical applications.

A novel strain of Stenotrophomonas acidaminiphila produces thermostable alkaline peptidase on agro-industrial wastes: process optimization, kinetic modeling and scale-up.

Bacterial alkaline peptidases, especially from Bacillus species, occupy the frontline in global enzyme market, albeit with poor production economics. Here, we report the deployment of response surface methodology approximations to optimize fermentation parameters for enhanced yield of alkaline peptidase by the non-Bacillus bacterium; Stenotrophomonas acidaminiphila. Shake flask production under optimized conditions was scaled up in a 5-L bench-scale bioreactor. Logistic and modified Gompertz models revealed significant fits for biomass formation, total protein, and substrate consumption models. Maximum specific growth rate (µmax = 0.362 h-1) of the bacterium in the optimized medium did not differ significantly from those in Luria-Bertani and trypticase soy broths. The aqueous two-phase system-purified 45.7 kDa alkaline protease retained 83% activity which improved with increasing sodium dodecyl sulfate concentration thus highlighting potential laundry application. Maximum enzyme activity occurred at 75ºC and pH 10.5 but was inhibited by 5 mM phenyl-methyl-sulfonyl fluoride suggesting a serine-protease nature.

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae through multi-objective medium optimization: aqueous two-phase purification and activity kinetics.

As physiological impairments that require replacement therapy continue to increase, so also does the need for improved production of acidic lipase from new microbial sources. Enterobacter cloacae strain UCCM 00116 produced a novel acidic lipase in kernel oil-processing waste-basal broth with 0.023:1 extracellular: intracellular localization ratio. This research re-directed enzyme localization to the extracellular milieu to reduce recovery cost using multi-objective response surface optimization of medium parameters. Results revealed a 1:0.32 extracellular:intracellular lipase ratio. Product formation kinetics, modeled by the Luedeking-Piret function, showed a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization through medium optimization. Aqueous two-phase system purification conditions extracted 95.22% lipase with 72.36 purity, a Vmax of 370.37 µmolmin-1, and a Km of 0.63 mmol. Enzyme activity was enhanced by K+ and Ca2+ ions, stable in many organic solvents, except acetone, and had pH and temperature optima at 2.5-3.5 and 50 °C, respectively.

Overproduction of a thermo-stable halo-alkaline protease on agro-waste-based optimized medium through alternate combinatorial random mutagenesis of Stenotrophomonas acidaminiphila.

A strain of Stenotrophomonas acidaminiphila, isolated from fermenting bean-processing wastewater, produced alkaline protease in pretreated cassava waste-stream, but with low yield. Strain improvement by alternate combinatorial random mutagenesis and bioprocess optimization using comparative statistical and neural network methods enhanced yield by 17.8-fold in mutant kGy-04-UV-25. Kinetics of production by selected mutant modeled by logistic and modified Gompertz functions revealed higher specific growth rate in mutant than in the parent strain, likewise volumetric and specific productivities. Purification by PEG/Na+ citrate aqueous two-phase system recovered 73.87% yield and 52.55-fold of protease. Its activity was stable at 5-35% NaCl, 45-75°C, and was significantly enhanced by 1-15 mM sodium dodecyl sulfate (SDS). The protease was inhibited by low concentrations of phenyl-methyl-sulfonyl fluoride but was activated by 1-5 mM Mn2+ suggesting a manganese-dependent serine­protease. The 45.7 kDa thermo-halo-stable alkaline protease demonstrated keratinolytic and blood-stain removal potentials showing prospects in textile and detergent industries, respectively.

A used ball of cotton wool as a source of nosocomially-acquired hepatitis C infection.

BACKGROUND AND AIMS: An error involving the reuse of the same ball of cotton wool in stopping blood flow after venous blood collection from five antenatal women prompted further investigation and follow-up studies to rule out nosocomially-acquired blood borne viruses. METHODS: The five women were screened for antibodies to the human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B surface antigen (HBsAg), using enzyme-linked immunosorbent assay (ELISA) /kits Murex HIV-1- ,2,0 (Murex Biotech, UK); ORTHO HCV 3.0 ELISA Test kit (Ortho Clinical Diagnostics, USA); and QUADRATECH CHECK 4-HBs one-step generation test kit (VEDALAB, France) respectively. The tests were repeated in 2005 on the five women, their husbands and twenty children, aged nine months to seven years borne by all the women within the period. Anti-HCV was detected in one out of the five women at the initial stage of the error (1997). No anti-HIV or HBsAg was found in any of the women. A repeat screening for anti-HIV, anti-HCV and HBsAg carried out seven years later (2005) on the five women, their husbands and twenty children aged nine months to seven years borne by all the women within the seven years revealed an HCV sero-conversion in two additional women. No anti-HCV or anti-HIV nor HBsAg was detected in any of the women, their spouses or their 20 offspring. RESULTS: Anti-HCV was detected in one out of the five women at the initial stage of the error (1997). No anti-HIV or HBsAg was detected in any of the women. A repeat re-evaluation revealed an HCV sero-conversion in two additional women. No anti-HCV or anti-HIV nor HBsAg was detected in any of the women, their spouses or any of their 20 screened offspring. CONCLUSIONS: This study provides evidence for the nosocomial transmission of HCV through the use of a contaminated ball of cotton wool. It also confirms the poor efficiency of sexual and vertical transmission of HCV and calls for improved hospital facilities and the use of skilled staff to perform essential duties.