Fermentation Technology of G. resinaceum LZ02 Producing Laccase and Partial Enzymatic Properties

doi:10.6048/j.issn.1001-4330.2022.06.020

Abstract

Abstract:

【Objective】 A strain G. resinaceum LZ02 with high-yield laccase was screened from Tianshan Mountains in Xinjiang. This project aims to optimize its fermentation technology and study its partial enzymatic properties.【Method】 The single factor experiment was used to optimize the enzymatic production conditions and study the enzymatic characteristics. In the meanwhile, the protein was detected by two dimensional electrophoresis.【Result】 The optimal carbon was 2.5% wheat bran and 1% glucose, nitrogen sources was 0.5% casein. Adding Cu²⁺, Mg²⁺, Fe²⁺, Zn²⁺and Ca²⁺ could promote the yield of laccase, the effect of Co²⁺ was adverse. Veratryl and Guaiacol delayed enzyme production peak from the second day to the ninth day, Tannic acidhad inhibiting effect to produce enzyme. The study of enzymatic properties showed optimal reaction temperature of laccase was 60℃ and there was still activity at 80℃, optimal reaction pH was 3.0 and enzyme activity was stable when pH was 4.0-5.0 and temperature was under 40℃. In addition, Co²⁺, Cd²⁺, Cr²⁺and Pb²⁺ could damage enzyme stability. Bidirectional electrophoresis was performed on the bacteria and fermentation liquid in which isoelectric point of laccase was determined to be 4.1. A pure enzyme protein sequence was obtained by mass spectrometry that showed the protein to be Laccase (Ganoderma Lucidum).【Conclusion】 The synthesis and secretion of laccase from G. sessinaceum are strictly regulated by nutrient level, culture condition, growth stage and inducers in the medium. Lignin or lignin-related aromatic compounds, N and C sources can also regulate the synthesis of laccase. The response of laccase activity to different kinds and concentrations metal ions and inducers is different, indicating that laccase activity has a more complex physiological function and regulatory mechanism.

Key words: G. resinaceum LZ02; laccase; optimizing fermentation technology; enzymatic properties; mass spectrometry sequencing

摘要：

【目的】 从采自新疆天山的无柄灵芝菌(G.resinaceum)中筛选获得1株高产漆酶的LZ02菌株,对其产酶条件进行优化,研究部分酶学性质。【方法】 采用单因素实验,对其产酶条件优化并研究其酶学特性,采用双向电泳检测酶蛋白。【结果】 LZ02菌株产漆酶的最佳碳源为2.5%麦麸+1%葡萄糖,最佳氮源为干酪素; Cu²⁺、Mg²⁺、Fe²⁺、 Zn²⁺和Ca²⁺的添加对LZ02产漆酶有一定的促进作用,而Co²⁺具有明显的抑制作用;藜芦醇和愈创木酚抑制产酶并使产酶高峰由第3 d推后至第9 d,添加没食子酸和ABTS对产酶影响不大,单宁酸对产酶有抑制作用。LZ02菌漆酶最适反应温度为60℃,且在80℃仍具有漆酶活性;在40℃,pH 4.0~5.0,酶活性最稳定;最适反应pH为3.0,Mn²⁺ 、Zn²⁺、 Cu²⁺ 、Ba²⁺和K⁺对酶稳定性有一定的促进作用,Co²⁺、Cd²⁺、Cr²⁺和Pb²⁺对酶的稳定性具有明显的破坏作用;对菌体及发酵液进行了双向电泳检测,测定其等电点为4.1;获得了1个纯化酶蛋白质谱序列,该蛋白与Ganoderma Lucidum来源的Laccase高度同源。【结论】 无柄灵芝菌(G.resinaceum)漆酶的合成和分泌受到营养水平、培养条件、生长阶段以及培养基中诱导剂的严格调控,木质素或木质素相关的芳香类化合物、N源和C源也能调节漆酶的合成;漆酶活性对不同种类、不同浓度的金属离子以及诱导剂的响应不尽相同,其具有较复杂的生理功能及调控机制。

关键词: 无柄灵芝菌, 漆酶, 产酶条件, 酶学特性, 质谱测序

CLC Number:

Q939.97
S188

YANG Xinping, XIE Yuqing, ZHOU Liuyang, DAI Jinping, WANG Xiaowu, ZHANG Huitao, WANG Zhifang, FENG Lei. Fermentation Technology of G. resinaceum LZ02 Producing Laccase and Partial Enzymatic Properties[J]. Xinjiang Agricultural Sciences, 2022, 59(6): 1482-1490.

杨新平, 谢玉清, 周留艳, 代金平, 王小武, 张慧涛, 王志方, 冯蕾. 无柄灵芝菌LZ02高产漆酶条件及部分酶学性质分析[J]. 新疆农业科学, 2022, 59(6): 1482-1490.

Figures/Tables 12

Fig.1 Effect of different carbon sources on the production of LZ02 laccase Note: 1. maltose; 2. sucrose; 3. glycerin; 4. glucose;5. sodium ; 6. 7. bran; 8.CK

Fig.2 Effect of different nitrogen sources on the production of LZ02 laccase Note: 1.casein; 2.yeast extract powder.pepton; 4.ammonium nitrate; 5.ammonium sulfate; 6.ammonium dihydrogen phosphate; 7.CK

Fig.3 Effect of different metallic ion on the production of LZ02 laccase

Fig.4 Effect of different inducer on the production of LZ02 laccase

Fig.5 The optimum reaction temperature of laccase

Fig.6 Thermal stability of laccase

Fig.7 Optimal pH of laccase

Fig.8 pH on laccase stability

Fig.9 Effect of different metal ion on laccase stability

Fig.10 SDS-PAGE of purification steps of F. fomentarius Note: M.protein markers; 1. ammonium sulfate salting-out(60%); 2. ion- chromatography; 3. ammonium sulfate salting-out(80%); 4. gel filtration chromatography

Fig.11 Protein two-dimensional electrophoresis Note: a. ultrasonic disintegration sample cell; b. upernatant of fermentation liquor

Table 1 The basic information and match peptide sequences in mass spectrometry analysis of the laccase of G.resinaceum LZ02

起始 Start	终止 End	观测值 Observed	分子量实验值 Mr(expt)	分子量计算值 Mr(cale)	得分 Score	氨基酸同源序列 The correspondence sequence of amino acid
36	126	773.92	1 545.82	1 545.82	96	R.FPLGSDSTLINGLGR.S
15	144	864.9	1 727.79	1 727.79	97	R.SAGSTVYNYDNPVWR.D
37	198	1 039.01	2 076.01	2 076.04	104	R.ANPNFGNVGFTDGINSAILR.Y
16	198	1 039.02	2 076.03	2 076.04	64	R.ANPNFGNVGFTDGINSAILR.Y
27	198	693.02	2 076.03	2 076.04	98	R.ANPNFGNVGFTDGINSAILR.Y

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