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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2022, Vol. 48 Issue (5): 614-624    DOI: 10.3785/j.issn.1008-9209.2021.10.211
资源利用与环境保护     
纤维素降解菌长枝木霉菌（Trichoderma longibrachiatum）ZJ-10的筛选及产酶条件优化
李子婧(),刘帆,汤胜,马庆旭,韩科峰(),吴良欢()
浙江大学环境与资源学院，浙江省农业资源与环境重点实验室，杭州 310058
Screening of cellulose-degrading fungus Trichoderma longibrachiatum ZJ-10 and optimization of enzyme production conditions
Zijing LI(),Fan LIU,Sheng TANG,Qingxu MA,Kefeng HAN(),Lianghuan WU()
Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
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摘要：

为解决畜禽废弃物及园林废弃物中纤维素降解难的问题，本试验采用刚果红染色法、滤纸崩解试验和测定内切葡聚糖酶（羧甲基纤维素酶）活力法从竹屑、枯枝烂叶、羊粪等废弃物中筛选高效的纤维素降解真菌，并对该真菌进行分子生物学鉴定。结果表明，本研究筛选到1株高效纤维素降解真菌，经形态学观察和菌种鉴定确定其为长枝木霉菌（Trichoderma longibrachiatum），并命名为T. longibrachiatum ZJ-10。单因素试验得出菌株ZJ-10最适产酶培养条件为接种量3%，初始pH 6.5，转速160 r/min，温度40 ℃，培养5 d，此时达到最大产酶活力；通过Plackett-Burman试验设计、Box-Benhnken最陡爬坡路径方法及响应面法得出菌株ZJ-10最适产酶培养基配方为NaCl 5 g/L、蛋白胨7 g/L、羧甲基纤维素钠12 g/L。在最适条件下测定的菌株ZJ-10的酶活力可达到80.32 U/mL，比优化前提高了26.45%。综上所述，本研究筛选出1株产内切葡聚糖酶活力较强的木霉真菌T. longibrachiatum ZJ-10，为畜禽及园林废弃物资源化利用提供了良好的菌种资源。
关键词： 木霉菌属;  纤维素;  真菌;  条件优化;  响应面法    
Abstract:

To improve the degradation efficiency of cellulose distributed in livestock, poultry wastes and garden wastes, a high-efficiency cellulose-degrading fungus which was from bamboo shavings, dead branches, and rotted leaves, and sheep dung was screened with Congo red staining, filter paper disintegration test and the endoglucanase [carboxyl methyl cellulose (CMC)] activity was tested. The physiological and molecular identification of the strain was carried out. The results showed that a high-efficiency cellulose-degrading fungus was screened in this study, which was identified as Trichoderma longibrachiatum by morphological observation and fungus species identification, and named as T. longibrachiatum ZJ-10. Single factor test showed that the conditions for achieving the maximum enzyme production activity showed as follows: 3% inoculation, initial pH 6.5, rotation speed of 160 r/min, 40 ℃, and cultured for 5 d. According to Plackett-Burman experimental design, Box-Benhnken steepest climbing path method and response surface methodology, the optimal enzyme production medium formula was 5 g/L NaCl, 7 g/L peptone, and 12 g/L CMC-Na. Under the optimal conditions, the CMC enzyme activity of strain ZJ-10 could reach 80.32 U/mL, which was 26.45% higher than that of the former optimization. In conclusion, strain of T. longibrachiatum ZJ-10 with strong CMC enzyme activity was screened in this study, which provides a good strain resource for the utilization of livestock, poultry and garden waste resources.
Key words: Trichoderma    cellulose    fungi    optimization of conditions    response surface methodology
收稿日期: 2021-10-21 出版日期: 2022-11-02
CLC:  X 713  
基金资助: 浙江省宁波市科技计划项目(202002N3106);国家重点研发计划项目(2020YFD1100402)
通讯作者: 韩科峰,吴良欢     E-mail: lizijing@zju.edu.cn;hkf1982@163.com;finm@zju.edu.cn
作者简介: 李子婧（https://orcid.org/0000-0002-2215-0128），E-mail：lizijing@zju.edu.cn
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引用本文:

李子婧,刘帆,汤胜,马庆旭,韩科峰,吴良欢. 纤维素降解菌长枝木霉菌（Trichoderma longibrachiatum）ZJ-10的筛选及产酶条件优化[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(5): 614-624.

Zijing LI,Fan LIU,Sheng TANG,Qingxu MA,Kefeng HAN,Lianghuan WU. Screening of cellulose-degrading fungus Trichoderma longibrachiatum ZJ-10 and optimization of enzyme production conditions. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(5): 614-624.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2021.10.211    或    https://www.zjujournals.com/agr/CN/Y2022/V48/I5/614

菌株名称

Strain name

水解能力（H/C）

Hydrolysis capacity (H/C)

滤纸崩解情况Disintegration of filter paper

滤纸分解率

Decomposition rate of filter paper/%

ZJ-11.71±0.01+++4.44±0.31
ZJ-21.26±0.03++1.63±0.08
ZJ-31.71±0.05+++5.49±0.30
ZJ-41.34±0.02+1.05±0.16
ZJ-51.33±0.04++1.75±0.10
ZJ-61.33±0.12++1.71±0.15
ZJ-71.85±0.02++++14.82±0.52
ZJ-81.55±0.03+1.28±0.06
ZJ-91.63±0.06+++6.42±0.13
ZJ-101.95±0.03+++++23.58±0.80
ZJ-111.37±0.03+1.22±0.12
ZJ-121.32±0.02+1.30±0.20
ZJ-131.56±0.03+++6.88±0.20
ZJ-141.36±0.03+1.44±0.18
ZJ-151.89±0.02+++++19.77±0.63
ZJ-161.36±0.05+1.24±0.35
ZJ-171.82±0.02++++11.98±0.43
表1  不同菌株的水解能力、滤纸崩解情况及分解率
图1  不同培养时间对纤维素降解真菌产酶的影响
图2  基于菌株ZJ-10及相关rDNA ITS序列的系统进化树
图3  接种量（A）、初始pH（B）、转速（C）、温度（D）对菌株ZJ-10产酶的影响

处理

Treatment

X1X2X3X4X5X6X7

CMC酶活力

CMC enzyme activity/(U/mL)

11－1－1－111－164.60
2111－1－11158.85
3－11－11－1－1151.63
41－11－1－1－1169.15
511111－1－163.60
611－11－11－158.94
7－111－11－1－169.05
8－1－11111167.96
9－1－111－11－175.43
101－1－111－1154.90
11－11－1－111156.53
12－1－1－1－1－1－1－165.18
效应 Effect－2.62－6.448.71－1.82－0.421.47－6.30
系数 Coefficient－1.31－3.224.36－0.91－0.210.73－3.15
T－1.87－4.606.23－1.30－0.301.04－4.50
P0.132 80.009 9**0.003 3**0.262 20.776 40.351 80.010 7*
R2 (R2Adj)0.956 2 (0.879 5)
表2  Plackett-Burman试验设计及各因素响应评价

试验号

Test No.

NaCl/(g/L)(A)

蛋白胨

Peptone/(g/L) (B)

CMC-Na/(g/L)(C)

CMC酶活力

CMC enzyme activity/(U/mL)

123861.16
234967.36
3451071.94
4561174.17
5671272.03
6781369.19
表3  NaCl、蛋白胨和CMC-Na最适质量浓度范围确定
试验号Test No.ABC

CMC酶活力

CMC enzyme activity/(U/mL)

方差分析 Analysis of variation

参量

Parameter

平方和

Sum of squares

F值

F value

P值

P value

1461062.31A53.8710.100.015 5*
2661066.31B137.6125.810.001 4**
3561172.20C220.9241.430.000 4**
4551057.29AB8.701.630.242 1
5571278.54AC4.280.800.399 8
6661272.85BC1.490.280.613 6
7561175.31A240.927.680.027 7*
8471164.21B282.5415.480.005 6**
9561174.12C214.372.700.144 6
10451159.41模型 Model578.5812.060.001 7**
11671175.61误差 Error37.32
12561174.29失拟项 Lack of fit29.074.700.084 6
13571064.91纯误差 Pure error8.25
14551268.48总和 Sum615.90
15461272.99R20.939 4
16651164.91R2Adj0.861 5
17561171.98
表4  Box-Benhnken试验结果与分析
图4  NaCl、蛋白胨和CMC-Na对CMC酶活力影响的响应面图
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