Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, P.O. Box 1334, Durban, 4000, South Africa.
Biocatalysis and Technical Biology Research Group, Institute of Biomedical and Microbial Biotechnology, Cape Peninsula University of Technology, Bellville Campus, Symphony Way, PO Box 1906, Bellville, 7535, South Africa.
Int J Biol Macromol. 2018 Mar;108:642-649. doi: 10.1016/j.ijbiomac.2017.11.169. Epub 2017 Dec 2.
This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500 ± 10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. K values for ABTS, SGZ and 2,6-DMP were 142.85 μM, 10 μM and 54.55 μM and the corresponding k values were 60.6 s, 25.36 s and 27.84 s, respectively. The t values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (E) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications.
本工作首次报道了来自草分枝杆菌 A3(2) 的小漆酶 (SLAC) 在巴斯德毕赤酵母中的分泌表达。利用 AOX1 启动子和α因子作为分泌信号，携带漆酶基因 (rSLAC) 的重组巴斯德毕赤酵母产生了高浓度的细胞外漆酶（500±10 U/l），通过 80°C 预孵育 30 min 可进一步提高漆酶产量 7 倍。该酶（∼38 kDa）在 80°C 时具有最佳活性，但最佳 pH 值随所用底物而异。ABTS、SGZ 和 2,6-DMP 的 K 值分别为 142.85 μM、10 μM 和 54.55 μM，相应的 k 值分别为 60.6 s、25.36 s 和 27.84 s。rSLAC 在 60°C、70°C 和 80°C 的 t 值分别为 60 h、32 h 和 10 h。该酶的失活动力学能量 (E) 为 117.275 kJ/mol，而 rSLAC 热失活的 ΔG、ΔH 和 ΔS 均为正值。rSLAC 在 6 h 内无需添加介体即可将超过 90%的亮蓝 G 和锥虫蓝染料脱色。在巴斯德毕赤酵母中表达的高浓度 SLAC 提高了其在各种工业应用中的潜力。
