Functional Expression of a Laccase PEL3 from Pleurotus eryngii and a Pectin Methylesterase PMEII from Ficus pumila var. awkeotsang

• Main Authors: Yo-Zong Chang, 張祐宗
• Other Authors: Douglas J. H. Shyu
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/01084198080316716610

碩士 === 國立屏東科技大學 === 生物科技系所 === 101 === In this work, Pichia pastoris is used as host for heterologous expression of P. eryngii laccase 3 (PEL3) cDNA and jelly fig Pectin methylesteraseⅡ (PMEII) cDNA for the production of a large amounts of recombinant proteins for two purposes. One is for the investigation of the protein function, and the other is for the utilization in the industry. Laccases (EC 1.10.3.2) are useful industrial enzymes ubiquitously present in various organisms, especially in fungi. Laccases are blue multi-copper oxidases, with redox potential, capable of oxidizing phenols and aromatic amines and reducing molecular oxygen to water by one-electron transfer mechanism. The laccases have been found mainly in white-rot fungi, and Pleurotus eryngii is an edible white rot fungus with high economic values. It is known that two laccase genes PEL3 and PEL4 are identified in P. eryngii genome. The results indicate that the optimum conditions for the expression of recombinant proteins is to culture the recombinant strain in medium added with 0.4 mM copper at 30C for 3 days. Laccase activity is determined using ABTS as substrate, and it is found that enzyme activity is stable at a pH range of 4.0-6.0 and it exhibits thermostability below 60C. Compared with our previous study, P. eryngii laccase 4 (PEL4) displayed higher enzyme activity in all test. PEL3 and PEL4 are tested for decolorization of six dyes classified as azo and triphenylmethane dyes. Malachite green is more easily decolorized than the others. The laccases from P. eryngii appear to be a good candidate for application in the industrial effluent treatment. Pectin methylesteraseⅡ is the key enzyme responsible for the gelation of jelly curd in the water extract of Ficus pumila var. awkeotsang. The major function is to de-esterify the methoxylated pectin into a pectic acid. The PMEII cDNA was previously cloned from jelly fig cDNA library. Heterologous expression of PMEII in P. pastoris for the investigation of the protein function is conducted in this study.