端梗霉Z45产漆酶培养基的优化及其对染料的脱色

doi:10.13560/j.cnki.biotech.bull.1985.2021-1221

摘要/Abstract

摘要：

菌株Acrophialophora sp. Z45是一株产漆酶的端梗霉属真菌,本文依据不同培养基中菌株Z45对愈创木酚的氧化圈大小探究了影响该菌株产漆酶的因素并设计正交试验优化其产漆酶培养基,进而比较了菌株Z45在土豆葡萄糖培养基和优化后的产漆酶培养基中的漆酶活力差异,最后基于漆酶与染料脱色的相关性评价了菌株Z45对8种合成染料的脱色能力。结果表明,单因素及正交试验确定了菌株Z45优化后的产漆酶培养基为：基础产酶培养基中以蔗糖为碳源、硝酸钠为氮源、C/N比为45∶1、pH为5.0;在优化后的产漆酶培养基中菌株Z45的漆酶活性显著提高;菌株Z45对溴酚蓝、中性红、亚甲基蓝、甲基蓝和结晶紫等5种染料均有一定的脱色能力,其中对三苯甲烷染料甲基蓝的脱色能力最强,菌株Z45的粗酶液能使甲基蓝快速脱色。在较低甲基蓝浓度的固体培养基上,甲基蓝完全脱色的时间不受染料浓度的影响;而较高甲基蓝浓度下,甲基蓝完全脱色的时间随染料浓度升高逐渐延长。

关键词: 端梗霉, 漆酶, 正交试验, 染料脱色

Abstract:

The strain Acrophialophora sp. Z45 is a laccase-producing fungus,which was discovered in the previous study of our laboratory. In this study,based on the size of guaiacol-oxidation zone on different medium,the factors influencing the laccase production by strain Z45 were studied. The orthogonal experiments were designed to optimize the basic medium for laccase production and the difference in laccase activity before and after medium optimization was then compared. Finally,the ability of strain Z45 to decolorize eight synthetic dyes was evaluated based on the correlation between laccase and dye decolorization. The results based on the single factor experiment and orthogonal test showed that the optimized medium for laccase production was as follows：sucrose as carbon source,sodium nitrate as nitrogen source,the carbon to nitrogen ratio was 45∶1,with a pH value of 5.0. The laccase activity of strain Z45 was significantly improved after medium optimization. Strain Z45 had certain decolorization ability to bromophenol blue,neutral red,methylene blue,methyl blue and crystalline violet,among which the decolorization ability to triphenylmethane dye methyl blue was the strongest. The crude enzyme solution of strain Z45 decolorized methyl blue rapidly. On the solid medium with lower concentrations of methyl blue,the time for complete decolorization of methyl blue was not affected by the dye concentration,but at higher concentrations of methyl blue,the time for complete decolorization was gradually extended with the increase of dye concentration.

Key words: Acrophialophora sp., laccase, orthogonal test, dye decolorization

贾晨波, 苏一黄, 马秀梅, 王春利, 徐春燕. 端梗霉Z45产漆酶培养基的优化及其对染料的脱色[J]. 生物技术通报, 2022, 38(6): 252-260.

JIA Chen-bo, SU Yi-huang, MA Xiu-mei, WANG Chun-li, XU Chun-yan. Medium Optimization for Laccase Production by Acrophialophora sp. Z45 and Its Decolorization of Dyes[J]. Biotechnology Bulletin, 2022, 38(6): 252-260.

图/表 10

图1 G-PDA与PDA平板上菌株Z45不同培养时期的基本情况 1d-FS、2d-FS、4d-FS和6d-FS分别代表菌株培养1 d、2 d、4 d和6 d时正面的图片;1d-BS、2d-BS、4d-BS和6d-BS分别代表菌株培养1 d、2 d、4 d和6 d时背面的图片

Fig. 1 Basic performance of strain Z45 on the G-PDA and PDA medium 1d-FS,2d-FS,4d-FS and 6d-FS indicate the growth and discoloration circles on the front side of 1,2,4 and 6 d,respectively;1d-BS,2d-BS,4d-BS and 6d-BS indicate the growth and discoloration circles on the back side of 1,2,4 and 6 d,respectively

图2 不同条件下的变色圈直径 A中横坐标依次表示碳源为葡萄糖、蔗糖、乳糖、麦芽糖、淀粉、羧甲基纤维素钠;B中横坐标依次表示氮源为硝酸钠、硝酸铵、硫酸铵、蛋白胨、酵母粉、尿素;不同字母表示差异显著（P<0.05）

Fig.2 Diameter of discoloration circle under different condition A：horizontal ordinate indicates the carbon source is glucose,sucrose,lactose,maltose,starch,sodium carboxymethyl cellulose,respectively;B：horizontal ordinate indicates the nitrogen source is sodium nitrate,ammonium nitrate,ammonium sulfate,peptone,yeast powder,and urea from left to right,respectively. Different letters indicates significant difference（P<0.05）

表1 正交试验的结果与直观分析

Table 1 Results of orthogonal test and the visual analysis

编号 No.	A（碳源） A（Carbon source）	B（氮源） B（Nitrogen source）	C（碳氮比） C（C/N）	D（pH） D（pH）	变色圈直径 Diameter of discoloration circle/mm
1	淀粉Starch	硝酸钠Sodium nitrate	35∶1	4.5	13.33
2	淀粉Starch	硝酸铵Ammonium nitrate	40∶1	5.0	10.00
3	淀粉Starch	硫酸铵Ammonium sulfate	45∶1	5.5	10.67
4	乳糖Lactose	硝酸钠Sodium nitrate	40∶1	5.5	14.00
5	乳糖Lactose	硝酸铵Ammonium nitrate	45∶1	4.5	12.00
6	乳糖Lactose	硫酸铵Ammonium sulfate	35∶1	5.0	9.33
7	蔗糖Sucrose	硝酸钠Sodium nitrate	45∶1	5.0	23.33
8	蔗糖Sucrose	硝酸铵Ammonium nitrate	35∶1	5.5	14.67
9	蔗糖Sucrose	硫酸铵Ammonium sulfate	40∶1	4.5	12.00
k1	11.33	16.89	12.44	12.44
k2	11.78	12.22	12.00	14.22
k3	16.67	10.67	15.33	13.11
MAX	16.67	16.89	15.33	14.22
极差R	5.34	6.22	3.33	1.78

表2 正交试验的方差分析

Table 2 ANOVA of the orthogonal test

方差来源 Source of variance	III 型平方和 Type III sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	显著性 Significance
A	144.296	2	72.148	20.292	**
B	199.407	2	99.704	28.042	**
C	64.296	2	32.148	9.042	**
D	18.074	2	9.037	2.542
误差Error	64.000	18	3.556

图3 不同培养基中的漆酶活性

Fig. 3 Laccase activities in different media

图4 不同粗酶液加入量下甲基蓝的脱色速率

Fig.4 Decolorization rate of methyl blue with different amount of crude laccase

图5 菌株Z45对甲基蓝染料的脱色

Fig.5 Decolorization of methyl blue by strain Z45

图6 PDA培养基上不同浓度甲基蓝染料的脱色情况

Fig.6 Decolorization of methyl blue under different concentrations on PDA medium

图7 优化后的产漆酶培养基中不同浓度甲基蓝的脱色情况

Fig. 7 Decolorization of methyl blue under different conc-entrations on the optimized medium for laccase pro-duction

图8 粗酶液对不同浓度甲基蓝溶液的脱色情况

Fig. 8 Decolorization of methyl blue under different conc-entrations by crude enzyme solution

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