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| Directed Evolution in vitro of Armillariella tabescens MAN47 β-Mannanase with Higher Thermalstability and Acid Tolerance |
| WU Xiu-xiu, LV Xiao-hui, HU Ya-dong, XIE Chun-fang, LIU Da-ling, YAO Dong-sheng |
| Institute of Microbial Biotechnology, Jinan University, Guangzhou 510632, China |
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Abstract A mutant database has been built and mutants with acid tolerance or thermostability or high activity which would be taken as parent gene of DNA shuffling was also screened. Then mutants with higher thermalstablility and acid tolerance from DNA Shuffling database was searched by facilitation and 96 deep wall plate culture screen. Futhermore, some information about bioinformatics of β-mannanase by sequence comparison and homologous model was also been got. Through two cycles of DNA shuffling, a mutant database was built. Then one optimum 1108 was screened from about 104 mutants. The evoluted β-mannanase displayed both higher thermalstability and acid tolerance than wide type. The evoluted enzyme 1108 retained high activity after treatment at 90℃ for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of 1108 under 80℃ and acid treatment was 10 times and 5 times as wide type respectively. The sequence comparison illustrated that there were three nucleotide substitutions T289A, A535T, T1085C which carried corresponding amino acid changes Ser97Thr, Val362Ala, Ile179Leu. According to homologous modeling by SWISS-MODEL Repository and amino acid analysis, There is a foundation mutations locate near the catylyze core, So there is a speculation Ser97Thr, Val362Ala, Ile179Leu have something with acid tolerance activity and thermalstablility respectively.
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Received: 31 October 2011
Published: 25 March 2012
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