IOSR Journal Of Pharmacy And Biological Sciences (IOSR-JPBS) e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 14, Issue 6 Ser. II (Nov –Dec 2019), PP 52-55 www.Iosrjournals.Org DOI: 10.9790/3008-1406025255 www.iosrjournals.org 52 | Page Characterization of Crude DPEase (D-Psicose 3-Epimerase) from Recombinant Escherichia coli Deby Edyliani 1 , Budi Saksono 2 , Yurnaliza Yurnaliza 1 , Rizky Indradewi 2 , Nirwana Fazri Harahap 1 1 Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia . 2 Main Research for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl.Raya Bogor Km 46, Kec.Cibinong , Kab. Bogor, 16911. Indonesia Abstract: D-psicose is a unique monosaccharide sugar with prospective health benefits and physiologically safer than fructose. D-psicose is an enzymatic product of DPEase, converting fructose as substrate. The DPEase holds an important mechanism to produce D-psicose in laboratory and industrial scale. The DPEase is known to bind with certain cofactors in performing optimum enzymatic reaction. This study aimed to characterize the crude DPEase obtained from recombinant Escherichia coli in order to obtain the optimum condition for converting fructose to D-psicose in laboratory scale. Environmental parameters such as pH, temperature, and presence of metal ions were adjusted to observe any changes in DPEase activity. The results showed that optimum condition for DPEase activity were at 35 o C, pH 8.0, and addition of Co 2+ dan Mn 2+ enhanced the enzyme activity. Meanwhile, the presence of metal ions, e.g Ni 2+ , Cu 2+ , Zn² + , Ca² + , Mg² + and Mo 2+ were known as inhibitors to enzyme activity. --------------------------------------------------------------------------------------------------------------------------------------- Date of Submission: 22-11-2019 Date of Acceptance: 06-12-2019 --------------------------------------------------------------------------------------------------------------------------------------- I. Introduction D-psicose is a ketose sugar by having a carbon 3-epimer in D-fructosestructure [1]. This distinguishing character makes D-psicosesafe for consumption in health perspectives [2]. There are several health benefits of low-calorie D-psicose sugar, e.g it is readily absorbed in human body metabolism or in the small intestine, therebyenhances sugar consumption by diabetic patients [3], prevent the postprandial hyperglycemia in patients [4], when eating foods containing sucrose and maltose [5], [6]. Other benefits, D-psicose may be consumed for fat and weight losses [7], nerve protectors [8], antioxidant activity [9], pancreatic protector [10], with no side effects [11]. D-psicose from isalso reported to lower the hypertension [12]. D-psicoseis obtained from enzymatic reaction between the DPease enzyme and its substrate, the fructose. [13]. DPEase enzyme is the most important element in D-psicose production. In the three-dimensional structure of DPEase, the residues involved in binding of substrates or fructise are E156, W112, R215, I66 and H186, while metal binding sites are H209, D183, E150 and E244.Theresidue charactersdistinguisheDPEase from other epimerases [14]. The DPEase was originally classified as DTEase (D-tagatose 3-epimerase) [15], but recent finding on its main product, the D-psicose, has clarified its position and was grouped into the DPEase enzymes [14]. The DPEase is a metalloproteinwhich requires metal ions to increase its enzyme activity [16], especially Co 2 + andMn 2+ [17]. Due to possible nature of different metal binding properties by different source of DPEase, we need to investigate the environmental conditions which may directly improve or inhibit the enzyme activity in laboratory scale by using the recombinant E. coli as a model in this study. II. Material And Methods Recombinant E. coli containing the dpe gene from Agrobacterium tumefaciens was used as D-psicose producing strain in this study [18]. Crude enzymes were obtained from fermentation medium containing containing fructose as substrate. Enzyme assay or DPEase activity was determined based on the yield of D- psicose from D-fructose. The enzyme reaction contained a mixture of crude enzyme, fructose, Tris-HCL buffer (pH 8.0), and distilled water, reacted at 35°C for 10 minutes. The enzyme reaction was stopped in boiling water.The remaining fructose is removed or converted by adding Saccharomyces.cereviceae into the reaction mixture and analyzed by the Cysteine-carbazole method [19]. One unit of DPEase activity is determined as the amount of μmol D-psicose formed in a reaction. The protein content (mg/mL) was determined using the Lowry method [20], and bovine serum albumin was used as a standard. Characterization of DPEase based on varying environmental conditions, e.g temperature, pHand presence of metal ions.