新疆农业科学 ›› 2022, Vol. 59 ›› Issue (6): 1482-1490.DOI: 10.6048/j.issn.1001-4330.2022.06.020
杨新平(), 谢玉清, 周留艳, 代金平, 王小武, 张慧涛, 王志方, 冯蕾()
收稿日期:
2021-10-17
出版日期:
2022-06-20
发布日期:
2022-07-07
通信作者:
冯蕾
作者简介:
杨新平(1968-),男,新疆乌鲁木齐市人,副研究员,研究方向为生物肥料研发,(E-mial) 920553133@qq.com
基金资助:
YANG Xinping(), XIE Yuqing, ZHOU Liuyang, DAI Jinping, WANG Xiaowu, ZHANG Huitao, WANG Zhifang, FENG Lei()
Received:
2021-10-17
Online:
2022-06-20
Published:
2022-07-07
Correspondence author:
FENG Lei
Supported by:
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。
中图分类号:
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.
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.
图1 不同C源下LZ02产漆酶变化 注:1.麦芽糖;2.蔗糖;3.甘油; 4.葡萄糖;5.羧甲基纤维素钠;6.淀粉;7.麦麸;8.对照
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
图2 不同N源下LZ02产漆酶变化 注:1.干酪素;2.酵母粉;3.蛋白胨;4.硝酸铵;5.硫酸铵;6.磷酸二氢铵;7.对照
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
图10 菌株不同纯化步骤的SDS-PAGE 注:M.蛋白相对分子量标样;1.硫酸铵盐析(60%);2.离子交换层析;3.硫酸铵盐析(80%);4.凝胶过滤层析
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
图11 木蹄层孔菌(F. fomentarius)LZ02菌体及发酵液的蛋白质双向电泳 注:a.菌体;b.发酵液
Fig.11 Protein two-dimensional electrophoresis Note: a. ultrasonic disintegration sample cell; b. upernatant of fermentation liquor
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 |
表1 无柄灵芝菌(G.resinaceum)LZ02漆酶质谱分析中基本信息和匹配的肽段序列
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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