Wang Bo, Yan Ying, Tian Yongsheng, Zhao Wei, Li Zhengjun, Gao Jianjie, Peng Rihe, Yao Quanhong
Biotechnological Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.
Crop Breeding and Cultivation Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China.
World J Microbiol Biotechnol. 2016 Mar;32(3):40. doi: 10.1007/s11274-015-1999-7. Epub 2016 Feb 11.
Laccases have received considerable attention in recent decades because of their ability to oxidise a large spectrum of phenolic and non-phenolic organic substrates and highly recalcitrant environmental pollutants. In this research, a laccase gene from Colletotrichum lagenarium was chemically synthesised using yeast bias codons and expressed in Pichia pastoris. The molecular mass of the recombinant laccase was estimated to be 64.6 kDa by SDS-PAGE, and the enzyme exhibited maximum activity at pH 3.6-4.0 but more stability in buffer with higher pH (>pH 3.6). The optimal reaction temperature of the enzyme was 40 °C, beyond which stability significantly decreased. By using 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as a substrate, K m and V max values of 0.34 mM and 7.11 mM min(-1) mg(-1), respectively, were obtained. Using ABTS as a mediator, the laccase could oxidise hydroquinone to p-benzoquinone and decolourise the synthetic dyes malachite green, crystal violet and orange G. These results indicated that the laccase could be used to treat industrial effluents containing artificial dyes.
近几十年来，漆酶因其能够氧化多种酚类和非酚类有机底物以及高度难降解的环境污染物而受到广泛关注。在本研究中，利用酵母偏好密码子化学合成了来自葫芦科炭疽菌的漆酶基因，并在毕赤酵母中表达。通过SDS-PAGE估计重组漆酶的分子量为64.6 kDa，该酶在pH 3.6-4.0时表现出最大活性，但在较高pH值（>pH 3.6）的缓冲液中更稳定。该酶的最佳反应温度为40°C，超过此温度稳定性显著下降。以2,2'-联氮-双-(3-乙基苯并噻唑啉-6-磺酸)（ABTS）为底物，分别得到K m和V max值为0.34 mM和7.11 mM min(-1) mg(-1)。以ABTS为介质，漆酶可将对苯二酚氧化为对苯醌，并使合成染料孔雀石绿、结晶紫和橙G脱色。这些结果表明，该漆酶可用于处理含有人造染料的工业废水。
