Labrou N E, Karagouni A, Clonis Y D
Enzyme Technology Laboratory, Department of Agricultural Biology & Biotechnology, Agricultural University of Athens, lera Odos 75, 11855 Athens, Greece.
Biotechnol Bioeng. 1995 Nov 5;48(3):278-88. doi: 10.1002/bit.260480314.
Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 microM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C).
通过仿生染料亲和色谱法从博伊丁假丝酵母细胞中一步纯化甲酸脱氢酶（FDH，EC 1.2.1.2）。为此，将七种带有羧化结构作为其末端仿生部分的一氯三嗪染料Cibacron® Blue 3GA（CB3GA）的仿生类似物以及母体二氯三嗪染料Vilmafix Blue A-R（VBAR）固定在交联琼脂糖凝胶Ultrogel A6R上。评估了相应的新型仿生染料吸附剂以及带有CB3GA和VBAR的非仿生吸附剂从博伊丁假丝酵母细胞压榨破碎后获得的提取物中纯化FDH的能力。当细胞在4%甲醇上生长，压榨破碎在匀浆静置1小时（4℃）前连续进行四次时，起始提取物中FDH的比活性（1.8 U/mg）达到最佳条件。与非仿生吸附剂相比，仿生吸附剂表现出更高的纯化能力。此外，一种固定化的仿生染料，其末端仿生部分为连接在氯三嗪环上的巯基丙酮酸（BM6），表现出最高的纯化能力。在分批系统中获得的BM6吸附剂的吸附平衡数据与朗缪尔等温线吻合良好，此外，还测定了BM6吸附剂固定床中蛋白质和FDH吸附的穿透曲线。发现固定化BM6与FDH之间复合物的解离常数（K(D)）等于0.05 μM。吸附剂BM6用于从18升博伊丁假丝酵母培养物中一步纯化FDH（FDH总收率60%）。纯化的FDH在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上呈现单一条带，比活性为7.0 U/mg（30℃）。
