File S1 - Escherichia coli Nema Is an Efficient Chromate Reductase That Can Be Biologically Immobilized to Provide a Cell Free System for Remediation of Hexavalent Chromium

Figure S1. Representative Michaelis-Menten (panels A,C) and Lineweaver-Burk (panels B,D) plots to measure apparent kcat and apparent Km for NemA_Ec with Cr(VI) (panels A,B) and NADH (panels C,D) as substrate. Graphs are single repeats of reaction velocity measured at each substrate concentration. The apparent kcat and Km with Cr(VI) as substrate were measured at 1 mM NADH; and the apparent kcat and Km with NADH as substrate were measured at 150 µM Cr(VI). Figure S2. A. Identification of the pH optimum for NemA_Ec. 15 µg NemA_Ec were incubated at 22°C with 150 µM potassium chromate and 1 mM NADH in either 50 mM sodium phosphate buffer (pH 5.8, 6.5 or 7.0) or 50 mM Tris-Cl (pH 7.5 or 8.8). Reactions were initiated by addition of NemA_Ec and rate of Cr(VI) reduction measured by diphenyl carbazide assay. Data are the mean of three independent replicates, and error bars indicate ±1 standard deviation. B. Comparison of Cr(VI) reduction velocity at pH 7.0 in sodium phosphate buffer or Tris-Cl buffer. Reactions were established, initiated and monitored as described for A, except that either 50 mM sodium phosphate (pH 7.0) or 50 mM Tris-Cl (pH 7.0) were used as buffer. Data are the mean of three independent replicates, and error bars indicate ±1 standard deviation. Figure S3. Control reactions to ensure no spontaneous reduction of Cr(VI) by NADH and/or formic acid. Duplicate reactions of 150 µM K2CrO4, 5 mM formic acid and 1 mM NADH were incubated with (♦) or without (▪) 50 mM sodium phosphate buffer (pH 7.0). The amount of Cr(VI) remaining in each reaction at each time-point was measured by diphenyl carbazide assay. Data are the mean of three independent replicates, and error bars indicate ±1 standard deviation. Figure S4. Fluorescent micrograph of Nile Red stained E. coli producing PHA beads that display NemA_Ec. To visualize PHA beads, 1 ml of a 44 h culture of XLI-Blue cells co-expressing pMCS69 and pET-14b:PhaC-L-NemA_Ec was centrifuged (13, 000 rpm, 1 min) and the pellet resuspended in potassium phosphate buffer (pH 7.5), followed by addition of 10 μl of Nile Red stain (250 μg/ml Nile Red in DMSO). Cells were incubated in the dark for five minutes, pelleted by centrifugation, and re-suspended in potassium phosphate buffer. The micrograph was taken with an Olympus BX51 fluorescence microscope at 1000x magnification using the U-MWIG2 filter set (520–550 nm excitation wavelength and a 565 nm cut-on dichromatic mirror). (DOCX)图S1. 用于测定大肠杆菌NemA酶（NemA_Ec）以六价铬[Cr(VI)]（A、B组）和还原型烟酰胺腺嘌呤二核苷酸（NADH）（C、D组）为底物时的表观催化常数（apparent kcat）与表观米氏常数（apparent Km）的代表性米氏方程（Michaelis-Menten，A、C面板）与双倒数作图法（Lineweaver-Burk，B、D面板）结果。各图为各底物浓度下反应速率的单次重复测定值。以Cr(VI)为底物时的表观kcat与Km是在1 mM NADH条件下测定的；以NADH为底物时的表观kcat与Km则是在150 µM Cr(VI)条件下测定的。 图S2. A. 确定NemA_Ec的最适pH。将15 µg NemA_Ec与150 µM铬酸钾、1 mM NADH在22°C下孵育，缓冲液分别为50 mM磷酸钠缓冲液（pH 5.8、6.5或7.0）或50 mM Tris-Cl缓冲液（pH 7.5或8.8）。反应通过加入NemA_Ec启动，采用二苯卡巴肼测定法检测六价铬还原速率。数据为三次独立重复实验的平均值，误差棒表示±1倍标准偏差。B. 比较pH 7.0条件下，磷酸钠缓冲液与Tris-Cl缓冲液中的Cr(VI)还原速率。反应体系、启动方式与检测方法同A，仅将缓冲液替换为50 mM磷酸钠（pH 7.0）或50 mM Tris-Cl（pH 7.0）。数据为三次独立重复实验的平均值，误差棒表示±1倍标准偏差。 图S3. 对照反应，用于验证NADH和/或甲酸不会自发还原Cr(VI)。设置两组重复反应：体系包含150 µM铬酸钾、5 mM甲酸与1 mM NADH，分别加入（♦）或不加入（▪）50 mM磷酸钠缓冲液（pH 7.0）。在每个时间点采用二苯卡巴肼测定法检测各反应体系中剩余的Cr(VI)含量。数据为三次独立重复实验的平均值，误差棒表示±1倍标准偏差。 图S4. 表达展示NemA_Ec的聚羟基脂肪酸酯（PHA）微球的大肠杆菌经尼罗红（Nile Red）染色后的荧光显微照片。为可视化PHA微球，取44小时培养的共表达pMCS69与pET-14b:PhaC-L-NemA_Ec的XLI-Blue菌株1 ml，离心（13,000 rpm，1分钟）后，沉淀重悬于磷酸钾缓冲液（pH 7.5），随后加入10 μl尼罗红染液（250 μg/ml尼罗红溶于二甲基亚砜（DMSO））。将细胞于暗处孵育5分钟，离心收集后重悬于磷酸钾缓冲液。显微照片采用奥林巴斯BX51荧光显微镜拍摄，放大倍数为1000×，使用U-MWIG2滤光片组（激发波长520–550 nm，截止二向色镜波长565 nm）。(DOCX)