Ni Jinfeng, Takehara Motomi, Miyazawa Mitsuhiro, Watanabe Hirofumi
National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Protein Eng Des Sel. 2007 Nov;20(11):535-42. doi: 10.1093/protein/gzm052. Epub 2007 Oct 30.
There have been two major problems preventing applications of termite cellulases; one was difficulty for their hetelologous overexpression, and another is their low thermostability. We previously achieved adaptation of termite cellulase genes to an overexpression system of Escherichia coli by family shuffling of four orthologous cDNAs (Biosci. Biotechnol. Biochem., 2005; 69: 1711-1720). Using the adapted mutant cDNAs as parental genes combined with native-form cDNAs, we performed further family shuffling and obtained mutant cDNAs, which gave enzymes with improved thermostability. The best-evolved clone (PA68) was improved by 10 degrees C in maximum stability (retaining 90% original activity for 30 min incubation) from the parental enzymes, and kept 54% of its original activity for 150 min at 50 degrees C, whereas the most thermostable enzyme amongst the parents (A18) retained 30% of its original activity. PA68 showed 889 (micromoles of reducing sugars/min/mg of protein) in V(max) and 560 (micromoles of reducing sugars/min/mg of protein) in the specific activity against carboxymethylcellulose, which corresponds to 9.8 and 13.1 times of those of one of the ancestral enzymes rRsEG. In summary, we improved thermostability of the termite cellulase and increased the V(max) value and specific activity by combining only cDNAs encoding enzymes adapted for normal temperatures.
一直存在两个主要问题阻碍着白蚁纤维素酶的应用；一个是其异源过表达困难，另一个是其热稳定性低。我们之前通过对四个直系同源cDNA进行家族改组，实现了白蚁纤维素酶基因对大肠杆菌过表达系统的适应性（《生物科学、生物技术与生物化学》，2005年；69：1711 - 1720）。使用适应后的突变cDNA作为亲本基因与天然形式的cDNA相结合，我们进行了进一步的家族改组并获得了突变cDNA，其产生的酶具有更高的热稳定性。进化最好的克隆（PA68）与亲本酶相比，最大稳定性提高了10摄氏度（在30分钟孵育后保留90%的原始活性），并且在50摄氏度下150分钟保持其原始活性的54%，而亲本中最耐热的酶（A18）保留其原始活性的30%。PA68对羧甲基纤维素的V(max)为889（微摩尔还原糖/分钟/毫克蛋白质），比活性为560（微摩尔还原糖/分钟/毫克蛋白质），分别相当于祖先酶之一rRsEG的9.8倍和13.1倍。总之，我们通过仅组合编码适应常温的酶的cDNA，提高了白蚁纤维素酶的热稳定性，并提高了V(max)值和比活性。
