Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032)

被引:3
作者
Lozada-Ramirez, J. D. [1 ]
Sanchez-Ferrer, A. [2 ]
Garcia-Carmona, F. [2 ]
机构
[1] Univ Americas Puebla, Sch Sci, Dept Chem & Biol Sci, Cholula 72810, Mexico
[2] Univ Murcia, Fac Biol, Dept Biochem & Mol Biol A, E-30071 Murcia, Spain
关键词
S-Adenosylhomocysteine; S-Adenosylhomocysteine hydrolase; Corynebacterium glutamicum; Immobilization; Synthesis; ADENOSYL-L-HOMOCYSTEINE; EUPERGIT-C; BINDING; ANALOGS; GENOME; ADENOSYLMETHIONINE; METHYLTRANSFERASE; PURIFICATION; METHIONINE; KINETICS;
D O I
10.1007/s00253-011-3769-2
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to EupergitA (R) C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 similar to mu M NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.
引用
收藏
页码:2317 / 2325
页数:9
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