Dong Dalu, Pan Hangtao, Yu Ping
College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou 310035, Zhejiang Province, People's Republic of China.
Appl Microbiol Biotechnol. 2014 Jun;98(11):4995-5007. doi: 10.1007/s00253-014-5522-0. Epub 2014 Jan 21.
To improve the production of phycocyanin holo-α-subunit (CpcA) from Spirulina maxima, five genes and their spacer region sequences involved in its biosynthesis were subject to the directed evolution by error-prone PCR using the plasmid pETDuet-6 as the template. Mutants were screened by determining the CpcA yield in 96-well plates directly. A mutant strain CPCA713 with the highest CpcA yield of 17.36 mg/l in 96-well plates was obtained, and this yield was 29.7 % higher than that from the control strain ZJGSU09 containing pETDuet-6 (13.38 mg/l). Sequence alignments indicated that 10 nucleotides and 5 amino acids were mutated. Glycerol and beef extract were found to be the best carbon and nitrogen sources for accumulating CpcA in the screened CPCA713 strain, respectively. The concentrations of the key factors that affected the CpcA yield were optimized by response surface methodology with a Box-Behnken design and were as follows: glycerol, 16.0 g/l; yeast extract, 18.2 g/l; and beef extract, 4.8 g/l. Under the optimal conditions, the CpcA yield was up to 71.21 mg/l in the shake flask. Time-course of the CpcA production before and after optimization were performed and compared. After being purified by a Hi-Trap metal chelating affinity column loaded with 100 mM nickel sulfate, CpcA presented a single protein band with an estimated molecular weight of 29 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The purified CpcA had the maximal absorptive and fluorescent emission wavelengths at 623 and 650.8 nm, respectively, and was stable at temperatures of 40 °C below and pHs of 5.5-8.0, and in the dark or in the dim light. It had also a strong scavenging ability to three free radicals ·OH, ·O2 (-), and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH). The IC50 values of ·OH, ·O2 (-), and DPPH free radicals by purified CpcA were 0.08, 0.46, and 0.48 mg/ml, respectively. This study lays a good foundation for the industrial production of CpcA by engineered Escherichia coli in future.
为提高极大螺旋藻藻蓝蛋白全α亚基（CpcA）的产量，以质粒pETDuet - 6为模板，通过易错PCR对其生物合成过程中涉及的5个基因及其间隔区序列进行定向进化。通过直接测定96孔板中CpcA的产量来筛选突变体。获得了在96孔板中CpcA产量最高的突变株CPCA713，其产量为17.36 mg/L，比含有pETDuet - 6的对照菌株ZJGSU09（13.38 mg/L）高出29.7%。序列比对表明有10个核苷酸和5个氨基酸发生了突变。结果发现甘油和牛肉膏分别是筛选出的CPCA713菌株中积累CpcA的最佳碳源和氮源。采用Box - Behnken设计的响应面法对影响CpcA产量的关键因素浓度进行了优化，结果如下：甘油16.0 g/L；酵母提取物18.2 g/L；牛肉膏4.8 g/L。在最佳条件下，摇瓶中CpcA产量高达71.21 mg/L。对优化前后CpcA的生产过程进行了时间进程分析和比较。经装有100 mM硫酸镍的Hi - Trap金属螯合亲和柱纯化后，CpcA在十二烷基硫酸钠聚丙烯酰胺凝胶电泳凝胶上呈现出一条估计分子量为29 kDa的单一蛋白条带。纯化后的CpcA最大吸收波长和荧光发射波长分别为623和650.8 nm，在40℃以下、pH值为5.5 - 8.0的条件下以及黑暗或弱光环境中均稳定。它对·OH、·O2(-)和二（苯基）-（2,4,6 - 三硝基苯基）亚氨基氮鎓（DPPH）三种自由基也具有较强的清除能力。纯化后的CpcA对·OH、·O2(-)和DPPH自由基的IC50值分别为0.08、0.46和0.48 mg/ml。本研究为未来利用工程化大肠杆菌工业化生产CpcA奠定了良好基础。
