Degradation of Polycaprolactone by Streptomyces thermoviolaceus subsp. thermoviolaceus 76T-2

• Main Authors: Ting-Kuang Chueh, 闕廷光
• Other Authors: Mei-Kwei Yang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/01615657924677677915

碩士 === 輔仁大學 === 生命科學系碩士班 === 100 === Abstract A thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus 76T-2 that can degrade Polycaprolactone (PCL) was isolated from soil in Taiwan. The optimum medium for strain 76T-2 is UF medium. It can grow at 30℃ to 55℃ and the optimum temperature is 45℃. When incubated in the liquid cultures with 0.3％ emulsified PCL, strain 76T-2 could degrade emulsified PCL completely in 1 hour at 40℃. For producing the PCL depolymerase, the 76T-2 strain was incubated in Basal medium with 0.5% PCL powder at 40℃ for 9 hours. The proteins in the medium were precipitated by the 50-80% concentration of ammonium sulfate. There were two proteins exhibited PCL degrading activity after transferred into PCL gel, those two proteins were named protein I and protein II, and the molecular weight of protein I is 55 kDa and protein II is 25 kDa. The protein N-terminal amino acids sequence of protein II is Ala-Asn-Phe-Val-Val-Ser-Glu-Ala, it’s similar to a family 19 chitinase Chi25 of S. thermoviolaceus OPC-520 strain. For check the relation between protein II and Chi25, we designed a pair of primers Fc1 and Rc1 to clone the gene of Chi25. Then we produced a 810 bp DNA sequence by polymerase chain reaction with Fc1, Rc1 according the chromosome of 76T-2 strain. The 810 bp DNA sequence was expressed by E.coli ER2566 for producing Chi25. The Chi25 produced by E.coli ER2566 could express chitinase activity.