烟曲霉A-16产纤维素酶工艺优化及酶学特性

doi:10.13560/j.cnki.biotech.bull.1985.2021-1507

摘要/Abstract

摘要：

分离筛选高效降解稻草的菌株,研究菌株产纤维素酶工艺条件及酶学性质。采用刚果红染色法从腐败木质下的土壤中分离筛选到一株产纤维素酶菌株,结合菌株的形态特征和18S rDNA序列同源性比较进行鉴定;通过单因素试验和响应面分析法确定菌株最适产酶条件,并对纤维素酶的稳定性进行研究。分离纯化得到的菌株命名为烟曲霉（Aspergillus fumigatus A-16）;响应面实验结果表明,最优产纤维素酶工艺参数为：稻草粉添加量7 g/100 mL,pH 6.0,温度65℃,发酵时间5 d;在此最优条件下,该菌产生的羧甲基纤维素酶（CMCase）和滤纸酶（FPA）活力分别为2 954.76 U/mL和1 086.37 U/mL;其总活力较优化前提高了26.4%。该纤维素酶的适宜反应温度为70℃,适宜pH 6.0。在80℃热处理90 min条件下酶活力可保持在80%以上,说明该酶热稳定性较好。同时,在pH 5.0-7.0范围内比较稳定,放置1 d后可保持70%以上的酶活力。该研究可为利用富含纤维素的生物质原料开发洁净能源及食品级葡萄糖资源提供了基础支撑。

关键词: 烟曲霉, 纤维素酶, 产酶条件, 酶学特性

Abstract:

The aim of this work is to screen efficient straw degradable strains and study their process of cellulase and characteristics. A cellulase-producing strain was isolated from soil under decayed wood using the Congo red staining test,and then was identified through morphological characteristics and 18S rDNA sequence comparisons. The condition of enzyme production was determined by single factor experiment and response surface experiments,and the stability of cellulase was investigated. As results,the isolated strain was named Aspergillus fumigatus（Aspergillus fumigatus A-16）. The response surface test showed that the optimal process parameters of producing cellulase were as follows：straw powder 7 g/100 mL,pH 6.0,fermentation temperature 65℃,and fermentation time 5 d. Under this optimal fermentation conditions,the carboxymethyl cellulase（CACase）and filter paper activities（FPA）was 2 954.76 U/mL and 1 086.37 U/mL,and the total enzyme activity was 26.4% higher than that before optimization. The suitable reaction temperature was 70℃ and pH 6.0 of CMCase and FPA. The relative enzyme activity of the enzyme retained at over 80% after being heated for 90 min at 80℃,indicating that it had a good thermal stability. Meanwhile,the relative enzyme activity of the enzyme was stable in the pH 5.0-7.0 and could be remained > 70% after 1 d. This study may provide basic support for exploiting clean energy and food-grade glucose resource by using cellulose-rich biomass as a raw material.

Key words: Aspergillus fumigatus, cellulase, condition of producing enzyme, enzymatic properties

张开平, 刘燕丽, 涂绵亮, 李继伟, 吴文标. 烟曲霉A-16产纤维素酶工艺优化及酶学特性[J]. 生物技术通报, 2022, 38(9): 215-225.

ZHANG Kai-ping, LIU Yan-li, TU Mian-liang, LI Ji-wei, WU Wen-biao. Optimization of Producing Cellulase by Aspergillus fumigatus A-16 and Its Enzymatic Properties[J]. Biotechnology Bulletin, 2022, 38(9): 215-225.

图/表 12

表1 产纤维素酶菌株酶活性

Table 1 Enzymatic activity of the cellulase-producing strains

菌株编号 Number of strain	D/d值 D/d value	羧甲基纤维素酶活力 CMCase /（U·mL^-1）	滤纸酶活力 FPA /（U·mL^-1）
A-02	5.83±0.66	926.09±65.43	289.41±23.28
A-07	5.65±0.71	757.26±17.55	116.52±16.65
A-10	7.12±0.48	1487.39±85.23	418.14±43.12
A-11	6.64±0.50	1004.58±36.52	396.41±40.33
A-16	9.87±0.42	2516.24±117.41	686.48±43.20
A-19	8.15±0.17	1986.61±123.28	768.87±27.40
A-22	8.41±0.24	2387.40±67.20	718.41±33.11
A-26	7.57±0.30	2060.24±25.64	671.92±40.29

图1 菌株A-16菌落形态

Fig.1 Colony morphology of the strain A-16

图2 菌株A-16基于18S rDNA的系统发育树

Fig.2 Phylogenetic tree of the strain A-16 based on 18S rDNA

图3 稻草粉添加量对A. fumigatus A-16菌株纤维素酶活力的影响不同小写字母表示差异显著（P<0.05）;下同

Fig.3 Effects of straw powder on the cellulase activity of strain A. fumigatus A-16 Different lowercase letters indicate significant differences（P<0.05）. The same below

图4 初始pH对A. fumigatus A-16菌株纤维素酶活力的影响

Fig.4 Effects of initial pH on the cellulase activity of strain A. fumigatus A-16

图5 温度对A. fumigatus A-16菌株纤维素酶活力的影响

Fig.5 Effects of temperature on the cellulase activity of strain A. fumigatus A-16

图6 发酵时间对A. fumigatus A-16菌株纤维素酶活力的影响

Fig.6 Effects of fermentation time on the cellulase activity of strain A. fumigatus A-16

图7 响应曲面图 A：稻草粉添加量和pH交互作用;B：稻草粉添加量和发酵温度交互作用;C：稻草粉添加量和发酵时间交互作用;D：pH和发酵温度交互作用;E：pH和发酵时间交互作用;F：发酵温度和发酵时间交互作用

Fig.7 Response surface diagram A：Interaction of straw powder and initial pH. B：Interaction of straw powder and fermentation temperature. C：Interaction of straw powder and fermentation time. D：Interaction of pH and fermentation temperature. E：Interaction of pH and fermentation time. F：Interaction of fermentation temperature and fermentation time

表2 响应面设计及试验结果

Table 2 Response surface design and test results

试验号 Test No.	稻草粉添加量 Straw powder/ （g·100 mL^-1）	pH	发酵温度 Fermentation temperature/℃	发酵时间 Fermentation time/d	羧甲基纤维素酶活力 CMCase / （U·mL^-1）
1	10	6.0	75	5	2 063.71±3.15
2	6	5.0	65	5	2 531.72±4.86
3	6	6.0	65	6	2 657.27±5.22
4	6	7.0	65	5	2 513.37±1.82
5	8	7.0	65	6	2 086.14±5.44
6	8	5.0	65	6	2 447.22±2.18
7	10	5.0	65	5	2 073.50±2.97
8	8	7.0	65	4	2 413.23±4.92
9	8	6.0	65	5	2 888.50±2.83
10	6	6.0	65	4	2 481.15±2.24
11	8	6.0	55	6	2 370.83±3.01
12	8	6.0	65	5	2 954.07±2.16
13	10	7.0	65	5	2 018.95±3.68
14	8	7.0	55	5	2 004.27±6.20
15	8	5.0	55	5	2 274.48±4.97
16	10	6.0	65	6	2 168.28±2.94
17	10	6.0	55	5	2 075.56±4.99
18	8	7.0	75	5	2 635.90±3.13
19	8	6.0	65	5	2 951.56±3.51
20	10	6.0	65	4	2 217.78±4.23
21	6	6.0	75	5	2 406.44±7.46
22	8	6.0	65	5	2 898.67±4.35
23	8	5.0	65	4	2 536.43±3.79
24	6	6.0	55	5	2 618.73±4.34
25	8	6.0	75	6	2 267.33±5.75
26	8	5.0	75	5	2 288.70±5.14
27	8	6.0	75	4	2 298.64±2.95
28	8	6.0	65	5	2 820.48±3.77
29	8	6.0	55	4	2 258.87±3.49

表3 响应面模型及回归方程的方差分析

Table 3 Analysis of variance of the response surface model and the regression equations

方差来源 Source	总偏差平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F	P	显著性 Significance
Model	2.224E+006	14	1.589E+005	9.61	<0.0001	**
A	5.593E+005	1	5.593E+005	33.84	<0.0001	**
B	19200.00	1	19200.00	1.16	0.2994
C	10680.33	1	10680.33	0.65	0.4349
D	3640.08	1	3640.08	0.22	0.6461
AB	334.89	1	3640.08	0.02	0.8888
AC	10040.04	1	10040.04	0.61	0.4487
AD	12746.41	1	12746.41	0.77	0.3947
BC	95326.56	1	95326.56	5.77	0.0308	*
BD	14149.10	1	14149.10	0.31	0.5861
CD	5133.72	1	5133.72	0.86	0.3705
^A2	5.886E+005	1	5.886E+005	35.61	< 0.0001	**
B²	5.899E+005	1	5.899E+005	35.69	< 0.0001	**
C²	7.240E+005	1	7.240E+005	43.80	< 0.0001	**
D²	4.190E+005	1	4.190E+005	25.35	0.0002	**
残差	2.314E+005	14	16529.14
失拟项	2.108E+005	10	21083.15	4.10	0.0932
纯误差	20576.45	4	5144.11
所有项	2.455E+006	28

图8 菌株A-16产纤维素酶作用的最适温度及其热稳定性 A：CMCase和FPA酶作用最适反应温度;B：CMCase和FPA酶的热稳定性

Fig.8 Optimal temperature of cellulase activity of strain A-16 and and its thermo-stabilities A：Optimal temperature for CMCase and FPA enzymatic activity. B：Thermo-stabilities of the CMCase and FPA

图9 菌株A-16产纤维素酶作用的最适pH及其pH稳定性 A：CMCase和FPA酶作用最适反应pH;B：CMCase和FPA酶的pH稳定性

Fig. 9 Optimal pH of cellulase activity of strain A-16 and its pH stabilities A：Optimal pH for CMCase and FPA enzymatic activity. B：pH stabilities of the CMCase and FPA

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