Isolation and Purification of α-Glycerophosphate Oxidase in a Polyethylene Glycol/(NH4)2SO4 Aqueous Two-phase System_Journal of Biomedical Engineering

Authors：

MENGYao ¹ , JINJiagui ¹ , LIUShuangfeng ¹ , YANGMin ¹ , ZHANGQinglian ¹ , WANLi ¹ ,  TANGKun ²

1. School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, China;
2. Department of Respiratory, the First Affiliated Hospital of Chengdu Medical College, Chengdu 610083, China;

Corresponding author：

TANGKun, Email: Cyyfytang@yahoo.com

Keywords：

α-glycerophosphate oxidase; Enterococcus casseliflavus; aqueous two-phase system; distribution coefficient; purification fold

DOI：

10.7507/1001-5515.20140027

Video：

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Alpha-glycerophosphate oxidase (α-GPO) from Enterococcus casseliflavus was successfully isolated and purified by using polyethylene glycol (PEG)/(NH₄)₂SO₄ aqueous two-phase system (ATPS). The results showed that the chosen PEG/(NH₄)₂SO₄ ATPS could be affected by PEG molecular weight, pH, concentration of PEG and (NH₄)₂SO₄, and inorganic salt as well as additional amount of crude enzyme. After evaluating these influencing factors, the final optimum purification strategy was formed by 16.5% (m/m) PEG2000, 13.2% (m/m) (NH₄)₂SO₄, pH 7.5 and 30% (m/m) additive crude enzyme, respectively. The NaCl was a negative influencing factor which would lead to lower purification fold and activity recovery. These conditions eventually resulted in the activity recovery of 89% (m/m), distribution coefficient of 1.2 and purification fold of 7.0.

Citation： MENGYao, JINJiagui, LIUShuangfeng, YANGMin, ZHANGQinglian, WANLi, TANGKun. Isolation and Purification of α-Glycerophosphate Oxidase in a Polyethylene Glycol/(NH₄)₂SO₄ Aqueous Two-phase System. Journal of Biomedical Engineering, 2014, 31(1): 136-141. doi: 10.7507/1001-5515.20140027 Copy

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13.	龚丽芬, 龚文贤. 双水相分离提取蛋白酶和琼脂糖的初步研究[J].化学与生物工程, 2006, 23(11):36-38.
14.	谭平华, 林金清, 肖春妹. 双水相萃取技术研究进展及应用[J]. 化工生产与技术,2003, 10(1):119-123.
15.	黄淑霞, 吴晓莉, 尹卓容. PEG/(NH₄)₂SO₄双水相体系提取和纯化糖化酶[J]. 酿酒科技,2003, (2):24-25.
16.	李伟, 朱自强, 梅乐和. 双水相萃取技术在药物分离和提取中的应用[J]. 北工进展, 1998, 1:26-29.
17.	焦庆才, 刘茜陈, 陈耀祖. 双水相萃取法从发酵液中分离提取α-淀粉酶和蛋白酶的研究[J].高等学校化学学报,1998,3:391-394.
18.	杨基础, 沈忠耀, 汪家鼎. 用聚乙二醇(PEG)/磷酸盐双水相体系从枯草杆菌发酵液中提取α-淀粉酶[J]. 清华大学学报,1987, 27(3):81-88.
19.	KOPE AĆU W,JAMROZ D,WILICZKIEWICZ A,et al. Antioxidation status and histidine dipeptides content in broiler blood and muscles depending on protein sources in feed[J]. J Anim Physiol Anim Nutr (Berl),2013,97(3):586-598.

1. GRUNDY S M,CLEEMAN J I,DANIELS S R, et al. Diagnosis and management of the metabolic syndrome. An American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement. Executive Summary[J].Cardiol Rev,2005,13(6):322-327.
2. CARMON A,CHIEN J,SULLIVAN D,et al. The alpha glycerophosphate cycle in Drosophila melanogaster Ⅴ. molecular analysis of alpha glycerophosphate dehydrogenase and alpha glycerophosphate oxidase mutants[J]. J Hered,2010, 101(2):218-224.
3. CARMON A,MACINTYRE R. The alpha glycerophosphate cycle in Drosophila melanogaster Ⅵ. structure and evolution of enzyme paralogs in the genus Drosophila[J]. J Hered,2010, 101(2):225-234.
4. KIRANAS E R,KARAYANNIS M I,TZOUWARA-KARAYANNI S M. Enzymatic methods for the determination of alpha-glycerophosphate and alpha-glycerophosphate oxidase with an automated FIA system[J]. Talanta,1998, 45(5):1015-1021.
5. SULLIVAN D T,MACINTYRE R,FUDA N,et al. Analysis of glycolytic enzyme co-localization in Drosophila flight muscle[J]. J Exp Biol,2003, 206(Pt12):2031-2038.
6. ZHENG C,ZHAO Z,LI Y, et al. Effect of IPTG amount on apo- and holo-forms of glycerophosphate oxidase expressed in Escherichia coli[J]. Protein Expr Purif, 2011, 75(2):133-137.
7. COLUSSI T,PARSONAGE D,BOLES W, et al. Structure of alpha-glycerophosphate oxidase from Streptococcus sp.:a template for the mitochondrial alpha-glycerophosphate dehydrogenase[J]. Biochemistry,2008, 47(3):965-977.
8. PARSONAGE D,LUBA J,MALLETT T C,et al. The soluble alpha-glycerophosphate oxidase from Enterococcus casseliflavus. Sequence homology with the membrane-associated dehydrogenase and kinetic analysis of the recombinant enzyme[J]. J Biol Chem, 1998, 273(37):23812-23822.
9. BIZZINI A,ZHAO C,BUDIN-VERNEUIL A,et al. Glycerol is metabolized in a complex and strain-dependent manner in Enterococcus faecalis[J]. J Bacteriol, 2010, 192(3):779-785.
10. 包凌晟, 杜林方,李海林,等. 产磷酸甘油氧化酶菌株的鉴定及其酶学性质研究[J]. 中国酿造,2008,11:15-18.
11. 冯咏梅, 黎高翔. 磷酸甘油氧化酶的菌种选育及发酵条件[J]. 微生物学报, 1997, 37(1):26-31.
12. 卢戌,赵兴秀,张锐,等.链球菌产磷酸甘油氧化酶的基本特性[J].四川大学学报,2006,43(6):1379-1383.
13. 龚丽芬, 龚文贤. 双水相分离提取蛋白酶和琼脂糖的初步研究[J].化学与生物工程, 2006, 23(11):36-38.
14. 谭平华, 林金清, 肖春妹. 双水相萃取技术研究进展及应用[J]. 化工生产与技术,2003, 10(1):119-123.
15. 黄淑霞, 吴晓莉, 尹卓容. PEG/(NH₄)₂SO₄双水相体系提取和纯化糖化酶[J]. 酿酒科技,2003, (2):24-25.
16. 李伟, 朱自强, 梅乐和. 双水相萃取技术在药物分离和提取中的应用[J]. 北工进展, 1998, 1:26-29.
17. 焦庆才, 刘茜陈, 陈耀祖. 双水相萃取法从发酵液中分离提取α-淀粉酶和蛋白酶的研究[J].高等学校化学学报,1998,3:391-394.
18. 杨基础, 沈忠耀, 汪家鼎. 用聚乙二醇(PEG)/磷酸盐双水相体系从枯草杆菌发酵液中提取α-淀粉酶[J]. 清华大学学报,1987, 27(3):81-88.
19. KOPE AĆU W,JAMROZ D,WILICZKIEWICZ A,et al. Antioxidation status and histidine dipeptides content in broiler blood and muscles depending on protein sources in feed[J]. J Anim Physiol Anim Nutr (Berl),2013,97(3):586-598.

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Isolation and Purification of α-Glycerophosphate Oxidase in a Polyethylene Glycol/(NH₄)₂SO₄ Aqueous Two-phase System

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