Co-culture is an effective way to improve laccases fermentation level. The previous study showed that the transcription levels of lacc2 and lacc6 changed significantly during co-culture. In this study, lacc2 and lacc6 were successfully cloned and expressed in Pichia pastoris GS115. The crude enzyme solution of LACC2 and LACC6 were purified to investigate the enzymatic properties. When ABTS was used as the substrate, the optimal temperatures and pH of LACC2 and LACC6 were 50°C and 3.0, respectively. Moreover, low concentrations of K+, Cu2+, Co2+ and Mn2+ enhanced the activities of LACC2 more than LACC6. In addition, LACC2 showed better tolerance to organic reagents and surfactants than LACC6, while LACC2 was more vulnerable to the inhibitors than LACC6. The enzyme kinetics results showed that ABTS was the optimal substrate of LACC2 and LACC6, and the Km of LACC2 and LACC6 were 0.13 mmol/L and 0.24 mmol/L, respectively. This study provided important basic data on the properties of isoenzymes for the mechanism analysis of R. mucilaginosa enhanced P. eryngii var. ferulae laccases production during co-culture, which was beneficial to the comprehensive analysis of the promotion mechanism.
YUAN Chang
,
ZHAO Liting
,
GU Zhenghua
,
LI Youran
,
SHI Guiyang
,
DING Zhongyang
. Heterologous expression and enzymatic properties of laccase isozymes ofPleurotus eryngii var. ferulae[J]. Food and Fermentation Industries, 2020
, 46(13)
: 10
-17
.
DOI: 10.13995/j.cnki.11-1802/ts.023642
[1] THURSTON C F.The structure and function of fungal laccases[J].Microbiology,1994,140(1):19-26.
[2] 吴怡,马鸿飞,曹永佳,等.真菌漆酶的性质、生产、纯化及固定化研究进展[J].生物技术通报,2019(9):1-10.
[3] 周雪,朱启忠.白腐真菌漆酶的纯化及性质[J].生物加工过程,2011,9(2):53-57.
[4] KEUM Y S,LI Q X.Fungal laccase-catalyzed degradation of hydroxy polychlorinated biphenyls[J].Chemosphere,2004,56(1):23-30.
[5] 张昱,赵倩,李冠华.漆酶在食品领域的应用研究进展[J].食品工业科技,2016,37(17):385-389.
[6] ABADULLA E,TZANOW T,COSTA S,et al.Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta[J].Cellular and Molecular Life Sciences,2000,66(8):3 357-3 362.
[7] SODEN D M,DOBSONO A D.Differential regulation of laccase gene expression in Pleurotus sajor-caju[J].Microbiology,2001,147(7):1 755-1 763.
[8] KUMAR A,ARORA S,JAIN K,et al.Metabolic coupling in the co-cultured fungal-yeast suite of Trametes ljubarskyi and Rhodotorula mucilaginosa leads to hypersecretion of laccase isozymes [J].Fungal Biology,2019,123(12):913-926.
[9] WANG Han,PENG Lin, DING Zhongyang,et al.Stimulated laccase production of Pleurotus ferulae JM301 fungus by Rhodotorula mucilaginosa yeast in co-culture[J].Process Biochemistry,2015,50(6):901-905.
[10] ZHANG Qi,ZHAO Liting,LI Youran,Comparative transcriptomics and transcriptional regulation analysis of enhanced laccase production induced by co-culture of Pleurotus eryngii var. ferulae with Rhodotorula mucilaginosa[J].Applied Microbiology and Biotechnology,2020,104(1):241-255.
[11] 陈友枝.Pleurotus ferulae发酵产漆酶的研究[D].无锡:江南大学,2013.
[12] 马俊,安启坤,唐文竹.菊粉外切酶的异源表达、纯化及酶学性质[J].食品发酵与工业,2019,45(4):25-30.
[13] BALDRIAN P.Fungal laccases-occurrence and properties[J].FEMS Microbiology Reviews,2006,30(2):215-242.
[14] ZHU Changwei,BAO Guangwen,HUANG Shun.Optimization of laccase production in the white-rot fungus Pleurotus ostreatus (ACCC 52857) induced through yeast extract and copper[J].Biotechnology & Biotechnological Equipment,2016,30(2):270-276.
[15] BAO Wenhua,PENG Rihe,ZHANG Zhen,et al.Expression, characterization and 2,4,6-trichlorophenol degradation of laccase from Monilinia fructigena[J].Molecular Biology Reports,2012,39(4):3 871-3 877.
[16] HU Xia,WANG Chunyan,WANG Le,et al.Influence of temperature, pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis[J].World Journal of Microbiology and Biotechnology,2014,30(4):1 285-1 290.
[17] 马倩倩.白腐真菌Pleurotus ostreatus漆酶纯化和酶学性质研究[D].锦州:辽宁工业大学,2019.
[18] 林俊,刘海,张晓晴,等.不同离子对漆酶酶活的影响[J].化学试剂,2019,41(8):830-835.
[19] 朱显峰,丁涛.常见金属离子对漆酶酶活的影响[J].化学研究,2003,14(3):50-51;54.
[20] 赵丽婷.共培养胶红酵母对阿魏蘑漆酶基因表达及其相关蛋白的影响[D].无锡:江南大学,2017.
[21] DOU Xin,TIAN Qiaopeng,WANG Qi,et al.Characterization of laccase from Ganoderma sp. SYBC L48 and its performance on acid red one decolorization[J].Chinese Journal of Environmental Engineering,2019,13(4):856-864.
[22] 廖海君.平菇漆酶基因生物信息学分析和杂优-2平菇漆酶分离纯化及酶学性质、功能基团研究[D].重庆:西南大学,2017.
[23] ZHANG Jiwei,QU Yinbo,XIAO Peng,et al.Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2[J].Journal of Bioscience & Bioengineering,2012,113(6):697-703.
[24] LI Q,PEI L J,ZHAO L G,et al.Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris[J]. Applied Biochemistry & Microbiology,2014,50(2):163-170.
[25] MURUGESAN K,ARULMANI M,NAM I,et al.Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes[J].Applied Microbiology and Biotechnology,2006,72(5):939-946.
[26] LIU Lihua,LIN Zhiwei,ZHENG Teng,et al.Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969[J].Enzyme and Microbial Technology,2009,44(6-7):426-433.
[27] 李成名,王佳懿,卢磊.短小芽孢杆菌LC01漆酶基因在毕赤酵母中的胞外表达及重组漆酶酶学性质研究[J].生物技术通报,2018,34(3):185-193.
[28] WANG Jiayi,LU Lei,FENG Fujuan.Improving indigo carmine decolorization ability of a Bacillus amyloliquefaciens laccase by site-directed mutagenesis[J].Catalysts,2017,7(9):275.
[29] 赵秀艳,常飞,方泽民,等.黏细菌漆酶序列筛选及其重组酶酶学性质[J].生物工程学报,2017,33(4):609-619.
[30] 王建玲,桂爽,付禹,等.肺炎克雷伯氏菌漆酶基因的克隆及其在毕赤酵母中的高效表达[J].天津科技大学学报,2019,34(1):14-19.
[31] DITTMER N T,GORMAN M J,KANOST M R.Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm,Manduca sexta[J].Insect Biochemistry and Molecular Biology,2009,39(9):596-606.
[32] 刘振刚.小菜蛾漆酶Ⅱ分子鉴定及其酶学特性[D].泰安:山东农业大学,2017.