Braun M, Lünsdorf H, Bückmann A F
Department of Enzyme Technology, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Federal Republic of Germany.
Eur J Biochem. 1991 Mar 14;196(2):439-50. doi: 10.1111/j.1432-1033.1991.tb15835.x.
NADP(H)-dependent 12 alpha-hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48-50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900-1 scale (9000 U/l, 7 g HSDH, specific activity 1.0 U/mg crude protein, 55 U/g wet cells). By a simple two-step affinity chromatography procedure, easily adaptable to a large-scale operation, using columns of small dimensions of Sephacryl-S-400-Procion-orange-P-2R (5 cm x 28 cm) and Sephacryl-S-400-Procion-red-HE-7B (2.6 cm x 14 cm) approximately 140 mg (1.8 x 10(4) U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 U/mg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5-9.5 and a temperature optimum of approximately 55 C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5'-phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability (t 1/2 4-10 d, 25 C; 2-5 d, 37 C). Steady-state kinetic analysis for both reaction directions indicated that the reaction proceeds through an ordered bi bi mechanism with NADP(H) binding first to the free enzyme. Km, Vmax [forward (Vf) and reverse reactions (Vr)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, Km = 35 microns, Kd = 35 microns; cholic acid, Km = 72 microns, deoxycholic acid, Km = 45 microns, Vf = 160 U mg; NAPDH, Kd = 16 microns; 12-oxochenodeoxylic acid, Km = 12 microns, 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25 degrees C). N6-functionalized NADP derivatives, e.g. N6-(2-aminoethyl)NADP (Km = 4.5 mM) are poorly accepted as coenzyme by HSDH.
来自P群梭菌C 48 - 50菌株的NADP(H)依赖性12α-羟基类固醇脱氢酶（HSDH），在培养条件下仍以异常高的水平表达（约占总蛋白的1%），此时常用的昂贵脑心浸液复合培养基被廉价的工业级酵母自溶物所取代。临时开发了一种用于生产HSDH的廉价厌氧生物工艺，规模可达900 - 1（9000 U/l，7 g HSDH，比活性1.0 U/mg粗蛋白，55 U/g湿细胞）。通过简单的两步亲和层析程序，该程序易于适应大规模操作，使用尺寸较小的Sephacryl - S - 400 - Procion - orange - P - 2R（5 cm×28 cm）和Sephacryl - S - 400 - Procion - red - HE - 7B（2.6 cm×14 cm）柱，约140 mg（1.8×10⁴ U）的HSDH被纯化至表观同质，并直接从粗细胞提取物中浓缩（总收率53%，比活性128 U/mg）。经快速非变性和SDS/PAGE、等电聚焦及电子显微镜证实，HSDH的分子量约为105 kDa，由四个呈扁平四面体排列的相同亚基（26 kDa）组成。该酶的等电点相当低，为4.6，胆酸氧化的最适pH为8.5 - 9.5，最适温度约为55℃。已观察到其受SH试剂和吡哆醛5'-磷酸的抑制作用。螯合剂无抑制作用。NADP的存在显著提高了热稳定性（25℃时t₁/₂为4 - 10天；37℃时为2 - 5天）。对两个反应方向的稳态动力学分析表明，反应通过有序的双底物双产物机制进行，NADP(H)首先与游离酶结合。NADP、Km = 35 μM、Kd = 35 μM；胆酸、Km = 72 μM，脱氧胆酸、Km = 45 μM，Vf = 160 U mg；NAPDH、Kd = 16 μM；12 - 氧代鹅去氧胆酸、Km = 12 μM，66 U/mg（条件：0.1 M磷酸钾，pH 8.0，25℃）。N⁶-官能化的NADP衍生物，如N⁶-(2 - 氨基乙基)NADP（Km = 4.5 mM），作为辅酶被HSDH接受的程度很差。
