Improved activity and thermostability of Bacillus pumilus lipase by directed evolution
作者： Nagihan Akbulut , Merve Tuzlakoğlu Öztürk , Tjaard Pijning , Saliha İşsever Öztürk , Füsun Gümüşel
DOI: 10.1016/J.JBIOTEC.2012.12.016
关键词: Lipase 、 Enzyme 、 DNA shuffling 、 Biochemistry 、 Thermostability 、 Mutant 、 Bacillus pumilus 、 Specific activity 、 Biology 、 Directed evolution
摘要: To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. isolates. Using a high-throughput assay, the mutant with highest selected. This chimeric (L3-3), carrying crossover positions and three point mutations, has specific 6.4 8.2 times higher than parent enzymes. The also is more tolerant various detergents organic solvents, 9 longer half-life at 50 °C. Homology modeling L3-3, based on highly homologous subtilis A, shows that increased thermostability likely due structural rigidification reduced surface hydrophobicity. Increased may result location mutations close active site. Together, our results show it possible evolve, by shuffling, variants improved applicability as biocatalysts, even if enzymes are similar.
core.ac.uk 
sci-hub.se 参考文章(35)
in chief George M. Garrity, Bergey's Manual of Systematic Bacteriology ,(1986)
C. Chothia, A.M. Lesk, The relation between the divergence of sequence and structure in proteins. The EMBO Journal. ,vol. 5, pp. 823- 826 ,(1986) , 10.1002/J.1460-2075.1986.TB04288.X
Andreas Crameri, Sun-Ai Raillard, Ericka Bermudez, Willem P. C. Stemmer, DNA shuffling of a family of genes from diverse species accelerates directed evolution Nature. ,vol. 391, pp. 288- 291 ,(1998) , 10.1038/34663
Ashok Pandey, Sailas Benjamin, Carlos R Soccol, Poonam Nigam, Nadia Krieger, Vanete T Soccol, None, The realm of microbial lipases in biotechnology Biotechnology and Applied Biochemistry. ,vol. 29, pp. 119- 131 ,(1999) , 10.1111/J.1470-8744.1999.TB00541.X
U K Winkler, M Stuckmann, Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology. ,vol. 138, pp. 663- 670 ,(1979) , 10.1128/JB.138.3.663-670.1979
M. T. Reetz, Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications Proceedings of the National Academy of Sciences of the United States of America. ,vol. 101, pp. 5716- 5722 ,(2004) , 10.1073/PNAS.0306866101
Gyeong Tae Eom, Jae Kwang Song, Jung Hoon Ahn, Yeon Soo Seo, Joon Shick Rhee, Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System Applied and Environmental Microbiology. ,vol. 71, pp. 3468- 3474 ,(2005) , 10.1128/AEM.71.7.3468-3474.2005
Chin John Hun, Raja Noor Zaliha Abd. Rahman, Abu Bakar Salleh, Mahiran Basri, A newly isolated organic solvent tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase Biochemical Engineering Journal. ,vol. 15, pp. 147- 151 ,(2003) , 10.1016/S1369-703X(02)00185-7
Claudia Schmidt-Dannert, Frances H Arnold, Directed evolution of industrial enzymes. Trends in Biotechnology. ,vol. 17, pp. 135- 136 ,(1999) , 10.1016/S0167-7799(98)01283-9
Wolfgang Kabsch, Christian Sander, None, Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features Biopolymers. ,vol. 22, pp. 2577- 2637 ,(1983) , 10.1002/BIP.360221211