漆酶BmLac的异源表达、酶学特性及棉酚降解的研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-1469

摘要/Abstract

摘要：

为开发能高效降解游离棉酚的酶蛋白。从土壤中筛选得到一株Bacillus amyloliquefaciens XP-13，从其基因组扩增获得了基因BmLac，进而构建Ppic9k-BmLac重组质粒，将其导入毕赤酵母GS115实现了异源表达，对漆酶BmLac的酶学特性、棉酚降解效果进行研究。结果表明，以ABTS为底物时，该酶的最适反应条件为pH 4，55℃，最适条件下酶活是101.56 U/mL；在90℃下孵育1 h后，相对酶活仍保留70%以上；在pH 3-7孵育1 h，其相对酶活均保留96%以上。1 mmol/L的Cu²⁺能使该酶酶活提高1.93倍，但高浓度（10 mmol/L）的Fe²⁺、Fe³⁺和Al³⁺完全抑制该酶活性。在55℃下反应2 h后，该酶对棉酚的降解率高达94.57%（无介体）和98.73%（有ABTS）。综上，漆酶BmLac具有良好的热稳定性和广泛的pH适应性，并能有效降解棉酚，该酶为有效降解棉籽粕中的游离棉酚提供基础支撑。

关键词: 游离棉酚, 解淀粉芽孢杆菌, 漆酶, 酶学性质, 棉籽粕

Abstract:

To develop enzymatic proteins for the efficient degradation of gossypol, a strain of B. amyloliquefaciens XP-13 was screened from soil, the gene BmLac was amplified from its genome, then the recombinant plasmid Ppic9k-BmLac was constructed and introduced into Pichia pastoris GS115 for heterologous expression. The characteristics of the laccase BmLac and the degradation effect of gossypol were studied. The results showed that the enzyme activity was 101.56 U/mL at pH 4 and 55℃ under the optimal reaction conditions with ABTS as the substrate; the relative enzyme activity was retained over 70% after incubation at 90℃ for 1 h; the relative enzyme activity was retained over 96% after incubation at pH 3-7 for 1 h. The enzyme activity increased 1.93 times by adding 1 mmol/L Cu²⁺, but high concentrations（10 mmol/L）of Fe²⁺, Fe³⁺ and Al³⁺ completely inhibited the enzyme activity. After reaction at 55℃ for 2 h, the degradation rate of gossyrol by this enzyme reached 94.57%（without mediator）and 98.73%（with ABTS）. In conclusion, the laccase BmLac has good thermal stability and wide pH adaptability and can effectively degrade gossypol, and this enzyme provides the basic support for the effective degradation of free gossypol in the cottonseed meal.

Key words: free gossypol, Bacillus amyloliquefaciens, laccase, enzymatic characterization, cottonseed meal

魏婷柳, 苗华彪, 吴倩, 黄遵锡. 漆酶BmLac的异源表达、酶学特性及棉酚降解的研究[J]. 生物技术通报, 2023, 39(12): 320-328.

WEI Ting-liu, MIAO Hua-biao, WU Qian, HUANG Zun-xi. Heterologous Expression, Enzymatic Characterization of Laccase BmLac and Degradation of Gossypol by It[J]. Biotechnology Bulletin, 2023, 39(12): 320-328.

图/表 9

图1 XP-13 菌株鉴定 A：16S rDNA凝胶电泳图；B：菌株XP-13芽孢光学显微镜图；C：菌株XP-13基于16S rDNA构建系统发育树

Fig. 1 Identification of XP-13 strain A: Gel electrophoresis of 16S rDNA. B: Light micrograph of strain XP-13 bacteriop-hage. C: Phylogenetic tree construction of strain XP-13 based on 16S rDNA

图2 漆酶BmLac的分析 A：BmLac二级结构；B：BmLac以1GSK同源建模三级结构（紫色表示β-sheet，蓝色表示α-helix）

Fig. 2 Analysis of laccase BmLac A : The secondary structure of BmLac. B: The 3D structure of BmLac with 1GSK homology (purple indicates β-sheet. Blue indicates α-helix)

图3 BmLac和pPic9k凝胶电泳图 A：BmLac基因凝胶电泳图；B：pPic9k双酶切凝胶电泳图；1：目的条带；M：DNA maker

Fig. 3 BmLac and pPic9k gel electrophoresis A: Gel electrophoresis map of BmLac gene. B: Gel electrophoresis map of pPic9k double digestion. 1 : Target band; M: DNA maker

图4 BmLac的SDS-PAGE M：蛋白Maker；1：失活的BmLac粗酶液；2：0.5 mmol/L ABTS染色BmLac粗酶液1 h

Fig. 4 SDS-PAGE of BmLac M: Protein maker. 1: Inactivated BmLac crude enzyme solution. 2: 0.5 mmol/L ABTS staining BmLac crude enzyme solution for 1 h

图5 BmLac最适pH及pH稳定性 A：最适pH；B：pH稳定性

Fig. 5 Optimal pH and pH stability of the BmLac A: Optimal pH. B : pH stability

图6 BmLac最适温度及温度稳定性 A：最适温度；B：温度稳定性

Fig. 6 Optimal temperature and temperature stability of the BmLac A : Optimal temperature. B : Temperature stability

表1 金属离子和化学试剂对BmLac的影响

Table 1 Effects of metal ions and chemical reagents on BmLac

试剂 Reagent	相对酶活1 Relative activity 1/%	相对酶活2 Relative activity 2/%
None	100±1.43	100±4.20
CuSO₄	193.09±5.05	98.71±2.44
MnSO₄	102.05±1.99	96.56±3.25
Pb（CH₃CO₂）₂	99.69±2.78	69.35±6.03
MgSO₄	98.36±2.15	97.15±4.36
ZnSO₄	98.26±1.13	86.13±4.69
NiSO₄	94.32±1.63	61.77±0.43
KCl	93.15±3.11	58.60±5.83
NaCl	90.74±0.49	59.52±1.77
CaCl₂	90.33±2.07	45.32±4.09
FeCl₃	90.43±1.83	0.00
LiCl	88.34±1.78	65.11±3.04
CoCl₂	84.30±3.28	19.84±1.26
AlCl₃	71.92±2.70	0.00
Fe SO₄	2.92±0.80	0.00
Guanidine hydrochloride	95.40±3.40	69.68±2.12
Urea	92.53±2.93	103.49±3.87
EDTA	86.75±1.29	42.37±1.25

图7 BmLac的动力学曲线

Fig. 7 Enzyme kinetic curves of BmLac

图8 BmLac降解棉酚的HPLC分析 A：不加介体ABTS；B：加介体1.25 mmol/L ABTS；C：棉酚降解率

Fig. 8 HPLC analysis of BmLac degrading gossypol A：Without mediator ABTS. B：With 1.25 mmol/L ABTS. C：Gossypol degradation rate

参考文献 23

[24]	Givaudan A, Effosse A, Faure D, et al. Polyphenol oxidase in Azos-pirillum lipoferum isolated from rice rhizosphere: evidence for laccase activity in non-motile strains of Azospirillum lipoferum[J]. FEMS Microbiol Lett, 1993, 108(2): 205-210. doi: 10.1111/fml.1993.108.issue-2 URL
[25]	Yang S, Long Y, et al. Gene cloning, identification, and characterization of the multicopper oxidase CumA from Pseudomonas sp. 593[J]. Biotechnol Appl Biochem, 2017, 64(3): 347-355. doi: 10.1002/bab.2017.64.issue-3 URL
[26]	Zeng J, Lin XG, Zhang J, et al. Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli[J]. Appl Microbiol Biotechnol, 2011, 89(6): 1841-1849. doi: 10.1007/s00253-010-3009-1 pmid: 21120471
[27]	Qiao WC, Chu JP, Ding SJ, et al. Characterization of a thermo-alkali-stable laccase from Bacillus subtilis cjp3 and its application in dyes decolorization[J]. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2017, 52(8): 710-717. doi: 10.1080/10934529.2017.1301747 URL
[28]	Brander S, Mikkelsen JD, Kepp KP. Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacil-lus clausii[J]. PLoS One, 2014, 9(6): e99402. doi: 10.1371/journal.pone.0099402 URL
[29]	Wang HB, Huang L, Li YZ, et al. Characterization and application of a novel laccase derived from Bacillus amyloliquefaciens[J]. Int J Biol Macromol, 2020, 150: 982-990. doi: 10.1016/j.ijbiomac.2019.11.117 URL
[30]	Lu L, Wang TN, Xu TF, et al. Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization[J]. Bioresour Technol, 2013, 134: 81-86. doi: 10.1016/j.biortech.2013.02.015 URL
[31]	Reiss R, Ihssen J, Thöny-Meyer L. Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum[J]. BMC Biotechnol, 2011, 11: 9. doi: 10.1186/1472-6750-11-9
[32]	Wan J, Sun XW, Liu C, et al. Decolorization of textile dye RB19 using volcanic rock matrix immobilized Bacillus thuringiensis cells with surface displayed laccase[J]. World J Microbiol Biotechnol, 2017, 33(6): 123. doi: 10.1007/s11274-017-2290-x URL
[33]	Wang L, Chen M, Luo XC, et al. Intramolecular annulation of gossypol by laccase to produce safe cottonseed protein[J]. Front Chem, 2020, 8: 583176. doi: 10.3389/fchem.2020.583176 URL
[34]	张乐珊. 重组漆酶OhLac的分子改造及其降解性能研究[D]. 杨凌: 西北农林科技大学, 2022.
	Zhang LS. Molecular modification and degradation of recombinant laccase OhLac[D]. Yangling: Northwest A & F University, 2022.
[35]	周梦宇, 古丽斯坦·阿不来提, 姚军, 等. 高效液相色谱法同时测定棉籽油中游离棉酚及其降解产物四甲氧基棉酚含量[J]. 食品科学, 2019, 40(16): 261-266. doi: 10.7506/spkx1002-6630-20180926-282
	Zhou MY, Abulaiti G, Yao J, et al. Simultaneous determination of free gossypol and its degradation product tetramethoxy gossypol in commercially available cottonseed oil by high performance liquid chromatography[J]. Food Sci, 2019, 40(16): 261-266.
[36]	Gadelha ICN, Fonseca NBS, Oloris SCS, et al. Gossypol toxicity from cottonseed products[J]. Sci World J, 2014, 231635.
[37]	王雨辰, 丁尊丹, 关菲菲, 等. 耐热漆酶ba4基因鉴定与酶学性质分析[J]. 生物技术通报, 2022, 38(8): 252-260. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1422
	Wang YC, Ding ZD, Guan FF, et al. Identification and characterization of heat-resistant laccase Ba4 gene[J]. Biotechnol Bull, 2022, 38(8): 252-260.
[38]	Sharma P, Goel R, Capalash N. Bacterial laccases[J]. World J Microbiol Biotechnol, 2007, 23(6): 823-832. doi: 10.1007/s11274-006-9305-3 URL
[39]	El-Bendary MA, Ezzat SM, et al. Optimization of spore laccase production by Bacillus amyloliquefaciens isolated from wastewater and its potential in green biodecolorization of synthetic textile dyes[J]. Prep Biochem Biotechnol, 2021, 51(1): 16-27. doi: 10.1080/10826068.2020.1786698 URL
[40]	You LF, Liu ZM, Lin JF, et al. Molecular cloning of a laccase gene from Ganoderma lucidum and heterologous expression in Pichia pastoris[J]. J Basic Microbiol, 2014, 54(Suppl 1): S134-S141.
[41]	Radveikienė I, Vidžiūnaitė R, Meškienė R, et al. Characterization of a yellow laccase from Botrytis cinerea 241[J]. J Fungi(Basel), 2021, 7(2): 143.
[42]	Li T, Chu XX, Yuan ZT, et al. Biochemical and structural properties of a high-temperature-active laccase from Bacillus pumilus and its application in the decolorization of food dyes[J]. Foods, 2022, 11(10): 1387. doi: 10.3390/foods11101387 URL
[43]	Zhang WR, Wang WW, Wang JH, et al. Isolation and characterization of a novel laccase for lignin degradation, LacZ1[J]. Appl Environ Microbiol, 2021, 87(23): e0135521. doi: 10.1128/AEM.01355-21 URL
[44]	Lin JH, Liu YJ, Chen S, et al. Reversible immobilization of laccase onto metal-ion-chelated magnetic microspheres for bisphenol A removal[J]. Int J Biol Macromol, 2016, 84: 189-199. doi: 10.1016/j.ijbiomac.2015.12.013 pmid: 26691384
[45]	Irshad M. Production and optimization of ligninolytic enzymes by white rot fungus Schizophyllum commune IBL-06 in solid state medium banana stalks[J]. Afr J Biotechnol, 2011, 10(79): 18234-18242.
[1]	Rehemujiang H, Yimamu A, Wang YL. Effect of dietary cotton stalk on nitrogen and free gossypol metabolism in sheep[J]. Asian-Australas J Anim Sci, 2019, 32(2): 233-240. doi: 10.5713/ajas.18.0057 URL
[2]	Zhang YH, Zhang ZY, et al. Isolation and characterization of a novel gossypol-degrading bacteria Bacillus subtilis strain Rumen Bacillus Subtilis[J]. Asian-Australas J Anim Sci, 2018, 31(1): 63-70. doi: 10.5713/ajas.17.0018 URL
[3]	Wang WK, Wang YL, Li WJ, et al. In situ rumen degradation characteristics and bacterial colonization of whole cottonseed, cottonseed hull and cottonseed meal with different gossypol content[J]. AMB Expr, 2021, 11(1): 91. doi: 10.1186/s13568-021-01244-2
[4]	宣秋希, 乔琳, 侯晓林, 等. 固态生料发酵棉籽粕菌种筛选及发酵工艺的研究[J]. 动物营养学报, 2022, 34(5): 3376-3391. doi: 10.3969/j.issn.1006-267x.2022.05.062
	Xuan QX, Qiao L, Hou XL, et al. Screening of strains for solid-state fermentation of raw cottonseed meal and study on fermentation technology[J]. Chin J Animal Nutr, 2022, 34(5): 3376-3391.
[5]	Tian X, Ruan JX, Huang JQ, et al. Gossypol: phytoalexin of cotton[J]. Sci China Life Sci, 2016, 59(2): 122-129. doi: 10.1007/s11427-016-5003-z pmid: 26803304
[6]	Santana AT, Guelfi M, Medeiros HCD, et al. Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E[J]. Biol Res, 2015, 48(1): 43. doi: 10.1186/s40659-015-0026-7 URL
[7]	Mena H, Santos JE, Huber JT, et al. The effects of feeding varying amounts of gossypol from whole cottonseed and cottonseed meal in lactating dairy cows[J]. J Dairy Sci, 2001, 84(10): 2231-2239. pmid: 11699455
[8]	Tang JW, Sun H, et al. Effects of replacement of soybean meal by fermented cottonseed meal on growth performance, serum biochemical parameters and immune function of yellow-feathered broilers[J]. Asian-Australas J Anim Sci, 2012, 25(3): 393-400. doi: 10.5713/ajas.2011.11381 URL
[9]	Liu YX, Wang LL, Zhao L, et al. Structure, properties of gossypol and its derivatives—from physiological activities to drug discovery and drug design[J]. Nat Prod Rep, 2022, 39(6): 1282-1304. doi: 10.1039/D1NP00080B URL
[10]	Wang X, Howell CP, Chen F, et al. Gossypol—a polyphenolic compound from cotton plant[J]. Adv Food Nutr Res, 2009, 58: 215-263. doi: 10.1016/S1043-4526(09)58006-0 pmid: 19878861
[11]	胡雷雨, 徐方旭, 高岩, 等. 棉籽饼粕综合利用及发展趋势研究[J]. 园艺与种苗, 2017, 37(11): 74-76.
	Hu LY, Xu FX, Gao Y, et al. Study on the comprehensive utilization and development trend of cottonseed cake[J]. Hortic Seed, 2017, 37(11): 74-76.
[12]	Zhang WJ, Xu ZR, Sun JY, et al. Effect of selected fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal[J]. J Zhejiang Univ - Sci B, 2006, 7(9): 690-695. doi: 10.1631/jzus.2006.B0690 URL
[13]	Nikolaivits E, Siaperas R, Agrafiotis A, et al. Functional and transcriptomic investigation of laccase activity in the presence of PCB29 identifies two novel enzymes and the multicopper oxidase repertoire of a marine-derived fungus[J]. Sci Total Environ, 2021, 775: 145818. doi: 10.1016/j.scitotenv.2021.145818 URL
[14]	Janusz G, Pawlik A, Świderska-Burek U, et al. Laccase properties, physiological functions, and evolution[J]. Int J Mol Sci, 2020, 21(3): 966. doi: 10.3390/ijms21030966 URL
[15]	Malhotra M, Suman SK. Laccase-mediated delignification and detoxification of lignocellulosic biomass: removing obstacles in energy generation[J]. Environ Sci Pollut Res, 2021, 28(42): 58929-58944. doi: 10.1007/s11356-021-13283-0
[16]	Mao GT, Wang K, Wang FY, et al. An engineered thermostable laccase with great ability to decolorize and detoxify malachite green[J]. Int J Mol Sci, 2021, 22(21): 11755. doi: 10.3390/ijms222111755 URL
[17]	Gupta V, Balda S, Gupta N, et al. Functional substitution of domain 3(T1 copper center)of a novel laccase with Cu ions[J]. Int J Biol Macromol, 2019, 123: 1052-1061. doi: 10.1016/j.ijbiomac.2018.11.174 URL
[18]	Karittapattawan P, Benchawattananon R. Evaluation of laccase production by monokaryotic strains of edible mushrooms[J]. Pak J Biol Sci, 2021, 24(4): 454-460. doi: 10.3923/pjbs.2021.454.460 pmid: 34486304
[19]	Le TT, Murugesan K, Lee CS, et al. Degradation of synthetic pollutants in real wastewater using laccase encapsulated in core-shell magnetic copper alginate beads[J]. Bioresour Technol, 2016, 216: 203-210. doi: 10.1016/j.biortech.2016.05.077 URL
[20]	Martins LO, Durão P, Brissos V, et al. Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology[J]. Cell Mol Life Sci, 2015, 72(5): 911-922. doi: 10.1007/s00018-014-1822-x pmid: 25572294
[21]	Cheng CM, Patel AK, Singhania RR, et al. Heterologous expression of bacterial CotA-laccase, characterization and its application for biodegradation of malachite green[J]. Bioresour Technol, 2021, 340: 125708. doi: 10.1016/j.biortech.2021.125708 URL
[22]	Chauhan PS, Goradia B, Saxena A. Bacterial laccase: recent update on production, properties and industrial applications[J]. 3 Biotech, 2017, 7(5): 323. doi: 10.1007/s13205-017-0955-7 pmid: 28955620
[23]	Guan ZB, Luo Q, Wang HR, et al. Bacterial laccases: promising biological green tools for industrial applications[J]. Cell Mol Life Sci, 2018, 75(19): 3569-3592. doi: 10.1007/s00018-018-2883-z