{"title":"荧光假单胞菌重组d -半乳糖脱氢酶的表达及回收纯化的响应面优化。","authors":"Shadi Rokhsartalab Azar, Raika Naiebi, Ameneh Homami, Zahra Akbari, Anvarsadat Kianmehr, Rahman Mahdizadehdehosta, Faezeh Najafzadeh","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13281,"journal":{"name":"Indian journal of biochemistry & biophysics","volume":"52 1","pages":"68-74"},"PeriodicalIF":1.5000,"publicationDate":"2015-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.\",\"authors\":\"Shadi Rokhsartalab Azar, Raika Naiebi, Ameneh Homami, Zahra Akbari, Anvarsadat Kianmehr, Rahman Mahdizadehdehosta, Faezeh Najafzadeh\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. 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引用次数: 0
摘要
酶d -半乳糖脱氢酶(GalDH)已被用于诊断试剂盒筛选新生儿血清半乳糖血症。它也是测定β- d -半乳糖、α- d -半乳糖和乳糖的重要工具。本研究采用响应面法(RSM)确定两水相体系(ATPS)中荧光假单胞菌(Pseudomonas fluorescens)中重组GalDH的回收条件。鉴定的GalDH基因经PCR扩增，并进一步克隆和测序证实。利用质粒(pET28aGDH)上含有GalDH基因的大肠杆菌BL-21 (DE3)表达和纯化重组酶。以聚乙二醇(PEG)浓度、硫酸铵浓度和pH值为变量，对GalDH的纯化进行分析。为建立数学模型，以最高分离条件为基础，采用中心复合设计的RSM方法。IPTG诱导后表达重组GalDH酶。对d -半乳糖具有NAD依赖型脱氢酶活性。根据RSM模型，在pH为7.5时，最佳的ATPS由PEG-2000 14.0% (w/w)和硫酸铵12.0% (w/w)组成。在这些条件下，GalDH优先集中在顶部富含peg的相。酶活、纯化因子(PF)和回收率(R)分别为1400 U/ml、60.0%和270.0%。聚乙二醇和盐浓度对酶的回收率有显著影响。总之，我们的数据表明，RSM可以作为确定重组荧光假单胞菌GalDH回收最佳atp的合适工具。
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.
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