Identification of the Thermostable Laccase Gene ba4 and Characterization of Its Enzymatic Properties

doi:10.13560/j.cnki.biotech.bull.1985.2021-1422

Abstract

Abstract:

Laccase（EC 1.10.3.2）is an oxidoreductase,and it has application value in the oxidative degradation of toxic and carcinogenic compounds. Through sequence analysis,thermostable laccase gene ba4 was screened from the UniParc database. The gene full length was 1,860 bp,encoding 620 amino acids. By optimal reaction temperature regression prediction model（PMT）,it was predicted that BA4 laccase was a thermostable enzyme. Comparison analysis in the NCBI protein database showed 58.75% similarity with the multicopper oxidase of copper-resistance system from Klebsiella michiganensis（STW26195.1）,proving that laccase ba4 was new laccase gene in the Copper_res_A superfamily. The entire sequence was synthesized and heterologously expressed in Escherichia coli BL21（DE3）and purified. The results of characterization demonstrated that the enzyme had high activity at a temperature of 45-65℃,the optimal temperature and pH were 50℃ and 5.5. The Michaelis constant K_m measured with ABTS as the substrate was（2 144.5±358.5）μmol/L,the k_catwas（44.06±3.14）min^-1,the maximum reaction rate V_max was 623.2 μmol/（min·g）,and the k_cat/K_mwas（0.020 9±0.002）L/（μmol·min）. The degradation rate reached > 90% while laccase BA4（60-70 U/L）reacted with zearalenone（0.1 mg/mL）at 50℃ for 2 h. The degradation rate was 30% when laccase BA4（70-80 U/L）reacted with gossypol（1 mg/mL）at 40 and 50℃ for 1 h. The promising enzymatic properties of laccase BA4 and the effective degradation of zearalenone and gossypol have laid a good foundation for the application of the enzyme.

Key words: laccase, enzymatic property, zearalenone, gossypol

WANG Yu-chen, DING Zun-dan, GUAN Fei-fei, TIAN Jian, LIU Guo-an, WU Ning-feng. Identification of the Thermostable Laccase Gene ba4 and Characterization of Its Enzymatic Properties[J]. Biotechnology Bulletin, 2022, 38(8): 252-260.

Figures/Tables 9

Table 1 Determination system for laccase enzyme activity

溶液 Solution	加入量 Added amount/μL
A液：0.2 mol/L Na₂HPO₄-0.1 mol/L 柠檬酸（pH 6.0）	750
B液：5 mmol/L ABTS	200
BA4蛋白	50

Table 2 Concentrations of gossypol in marking the standa-rd curve

No.	1	2	3	4	5
棉酚Gossypol/（mg·mL^-1）	0.00	0.05	0.1	0.15	0.2

Fig. 1 Gossypol standard curve

Table 3 Concentrations of ZEN in marking the standard curve

No.	1	2	3	4	5	6
玉米赤霉烯酮浓度 ZEN/（mg·mL^-1）	0.0000	0.0625	0.1250	0.2500	0.5000	1.0000

Fig. 2 ZEN standard curve

Fig. 3 Expression of BA4 after micro aerobic fermentation for 20 h M：Protein marker;1：supernatant of crushed IPTG-induced laccase BA4;2：precipitation of crushed IPTG-induced laccase BA4;3：NTA-200 eluant

Fig. 4 Effects of temperarure and pH on BA4

Fig. 5 Michaelis-menten equation curve of laccase BA4

Fig. 6 Degradation of ZEN and gossypol by laccase BA4 A：Chromatogram. CK：ZEN reference substance;treatment group：laccase BA4 treating ZEN at 40℃ and 50℃,respectively. B：Relative degradation rate of ZEN. Treating ZEN with laccase BA4 at 40℃ and 50℃,respectively,and the relative degradation rate was calculated based on the peak area of the chromatogram. C：Chromatogram. CK：Gossypol reference substance. Treatment group：Laccase BA4 treated gossypol at 40℃ and 50℃,respectively. D：Relative degradation rate of gossypol. Laccase BA4 was treated with gossypol at 40℃ and 50℃,respectively,and the relative degradation rate was calculated based on the peak area of the chromatogram

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