Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens

The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of β-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD+-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0 (w/w) and ammonium sulfate 12.0 (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0 and 270.0, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery. © 2015 National Institute of Science Communication and Information Resources (NISCAIR). All rights reserved

Исследование технологии лазерного фрезерования

В роботі розглянуто технологію фрезерування виробів з різноманітних матеріалів сфокусованим лазерним випромінюванням з модульованою добротністю. Встановлено, що на продуктивність та якість процесу разом з режимами лазерної обробки насамперед впливає механізм руйнування заготовки.Technologies of laser milling of different materials using Q-switched laser systems are presented in this paper. It was established that mechanism of material removal influences significantly the productivity and quality of laser processing. Technological parameters of laser milling of the most common intractable materials were simulated numerically and verified experimentally. It was established that at laser milling of intractable materials with solid-state Q-switched lasers it is possible to achieve the maximum milling depth without the formation of liquid phase. In order to increase the productivity and quality of laser milling in the case when a significant amount of liquid phase form (intractable materials, hard alloys) it is advisable to mill in two passes. First laser pass forms crates that are positioned symmetrically and the second laser pass breaks bridges between crates thus forming the surface with required roughness.В работе рассмотрена технология фрезерования изделий из различных материалов сфокусированным лазерным излучением с модулированной добротностью. Установлено, что на производительность и качество процесса, вместе с режимами обработки прежде всего влияет механизм разрушения заготовки