Staijen I E, Hatzimanikatis V, Witholt B
Institute of Biotechnology, Swiss Federal Institute of Technology, Zurich.
Eur J Biochem. 1997 Mar 1;244(2):462-70. doi: 10.1111/j.1432-1033.1997.00462.x.
We have studied the synthesis and stability of the monooxygenase AlkB of Pseudomonas oleovorans in its natural host and in recombinant Escherichia coli. Three strains were investigated: the prototype strain P. oleovorans and the E. coli alk+ recombinants HB101 (pGEc47) and W3110 (pGEc47). Plasmid pGEc47 allows regulated expression of alkB and synthesis of active AlkB in E. coli. The E. coli strains were selected because E. coli HB101 (pGEc47) produces similar amounts of AlkB as P. oleovorans (1.5-2% of total cell protein), whereas E. coli W3110 (pGEc47) is able to make substantially (about fivefold) more AlkB. The AlkB synthesis and degradation rates in batch cultures of the three strains were determined by means of isotopic-labeling and immunological techniques. The mean specific AlkB synthesis rates in P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47) were approximately 7, 12.5 and 45 microg x mg protein(-1) x h(-1), respectively. The half-lives of AlkB were estimated to be 80, 3 and 15 for P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47), respectively. Thus, the intracellular AlkB level in each of the three strains was the result of their AlkB synthesis and degradation rates. The AlkB level during batch growth was modelled by means of experimentally derived parameters for AlkB synthesis and degradation, and showed good agreement with AlkB levels determined by means of immunoblotting in all strains investigated.
我们研究了食油假单胞菌单加氧酶AlkB在其天然宿主及重组大肠杆菌中的合成与稳定性。研究了三株菌株：原型菌株食油假单胞菌以及大肠杆菌alk+重组体HB101(pGEc47)和W3110(pGEc47)。质粒pGEc47可调控AlkB的表达并在大肠杆菌中合成活性AlkB。选择大肠杆菌菌株是因为大肠杆菌HB101(pGEc47)产生的AlkB量与食油假单胞菌相似(占总细胞蛋白的1.5 - 2%)，而大肠杆菌W3110(pGEc47)能够产生更多(约五倍)的AlkB。通过同位素标记和免疫技术测定了这三株菌株分批培养时AlkB的合成和降解速率。食油假单胞菌、大肠杆菌HB101(pGEc47)和大肠杆菌W3110(pGEc47)中AlkB的平均比合成速率分别约为7、12.5和45μg·mg蛋白⁻¹·h⁻¹。食油假单胞菌、大肠杆菌HB101(pGEc47)和大肠杆菌W3110(pGEc47)中AlkB的半衰期估计分别为80、3和15。因此，三株菌株中细胞内AlkB水平是其AlkB合成和降解速率的结果。通过实验得出的AlkB合成和降解参数对分批生长过程中的AlkB水平进行建模，结果表明该模型与所有研究菌株中通过免疫印迹法测定的AlkB水平吻合良好。
