香菇菌渣高效纤维素降解菌的筛选及产酶优化

doi:10.13560/j.cnki.biotech.bull.1985.2021-1120

摘要/Abstract

摘要：

利用香菇菌渣开发生物有机肥目前是解决废弃菌渣资源化的主要趋势,为了更好地腐熟香菇菌渣,从废弃香菇菌渣中筛选出1株高效纤维素降解菌株DGW1,并优化产纤维素酶条件及评估降解菌渣能力,以期为后续利用香菇菌渣进行堆肥发酵生产生物有机肥奠定基础。采用CMC-Na平板法和刚果红染色法初步筛选,结合滤纸条崩解试验和纤维素酶活性测定进行复筛,并通过形态学观察和18S rDNA分子生物学鉴定菌株,优化菌株DGW1的产纤维素降解酶条件。结果表明:获得的高效纤维素降解菌株DGW1鉴定为里氏木霉,7 d内可将滤纸条降解为糊状。菌株DGW1最佳产酶条件为温度30℃、时间4 d,滤纸酶的酶活（FPase）为112.71 U/g,并在pH 4-7都能达到较高酶活且不低于96 U/g。菌株DGW1对香菇菌渣的降解率为31.14%（14 d）。因此,获得的菌株DGW1能够高效降解香菇菌渣,其纤维素酶活较高,对香菇菌渣发酵堆肥,制备生物有机肥具有较大应用潜力。

关键词: 香菇菌渣, 里氏木霉DGW1, 酶活力, 产酶条件

Abstract:

Utilizing spent Lentinula edodes substrate to develop organic fertilizer is major trend of converting waste bacterial residue into resources. In order to compost the spent L. edodes substrate,a high efficient cellulose-degrading strain DGW1 was isolated from the spent L. edodes substrate,the conditions of cellulase production were optimized,and the degradation capacity of the spent L. edodes substrate was evaluated,which lays a foundation for the subsequent composting fermentation of spent L. edodes substrate to produce bio-organic fertilizer. The strains were preliminarily screened by CMC-Na culturing method and Congo red dyeing and re-screened in combination with filter paper disintegration test and cellulase activity test. The strain was identified by morphological observation and 18S rDNA molecular biology,and the conditions of producing cellulose-degrading enzyme by the strain DGW1 were optimized. The results showed that the highly cellulolytic strain DGW1 was identified as Trichoderma reesei. The filter paper strips were degraded to paste in the 7 d. The optimal conditions of the strain DGW1 were temperature 30℃,time 4 d,the filter paper activity（FPase）was 112.71 U/g,and the high enzyme activity at pH 4-7 was not lower than 96 U/g. The degradation rate of the strain DGW1 on spent L. edodes substrate was 31.14%（14 d）. Therefore,the strain DGW1 can effectively degrade spent L. edodes substrate,and its cellulase activity was high,which has great application potential for composting of spent L. edodes substrate and preparation of bio-organic fertilizer.

Key words: spent Lentinula edodes substrate, Trichoderma reesei DGW1, enzyme activity, enzyme production condition

陶治东, 何艳慧, 邓子禾, 孙琳琳, 武占省. 香菇菌渣高效纤维素降解菌的筛选及产酶优化[J]. 生物技术通报, 2021, 37(11): 158-165.

TAO Zhi-dong, HE Yan-hui, DENG Zi-he, SUN Lin-lin, WU Zhan-sheng. Screening of High-efficiency Cellulose-degrading Microorganism from Spent Lentinula edodes Substrate and Optimization of Its Enzyme Production[J]. Biotechnology Bulletin, 2021, 37(11): 158-165.

图/表 8

图1 刚果红染色定性筛选 A:透明圈大小;B:部分菌株染色图

Fig.1 Qualitative screening results of Congo red staining A:Transparent circle size. B:Staining of some strains

表1 22株分离菌株的滤纸条降解

Table 1 Filter strip degradation by 22 isolated strains

菌株编号 Strain No.	滤纸条崩解程度 Degradation degree of filter paper	筛选温度 Screening temperature/℃	菌株编号 Strain No.	滤纸条崩解程度 Degradation degree of filter paper	筛选温度 Screening temperature/℃
DF1	+	28	YD3	+++	28
GF1	++	45	YG4	++	45
GF2	+	45	YD2	++	28
YG1	+	45	DGW1	++++	28
DG3	+	28	FYG2	+	45
GF3	+++	45	FYG3	++	45
YG3	+	45	FFD1	+	28
YD4	++++	28	FYG1	++	45
GG1	+	45	FDY2	+	28
YG2	+++	45	FGF1	++	45
YD1	++	28	FFD2	+	28

图2 纤维素降解菌滤纸酶活

Fig.2 FPase of cellulose-degrading microorganism

图3 18S rDNA琼脂糖凝胶电泳

Fig.3 Agarose-gel electrophoresis picture of 18S rDNA

图4 菌株DGW1基于18S rDNA序列同源性构建的系统发育树

Fig.4 Phylogenetic tree of strain DGW1 based on 18S rDNA sequence homology

图5 菌株DGW1形态 A:菌株DGW1形态;B:菌株DGW1孢子形态;C:菌株DGW1菌丝形态;D:扫描电镜菌株DGW1菌丝形态

Fig.5 Morphology of the strain DGW1 A:Morphology of the strain DGW1;B:spore morphology of strain DGW1;C:mycelial morphology of strain DGW1;D:mycelial morphology of strain DGW1 under SCM

图6 不同条件下菌株DGW1的酶活 A:不同pH下菌株DGW1酶活;B:不同温度下菌株DGW1酶活;C:不同时间下菌株DGW1酶活

Fig.6 Enzyme activity of strain DGW1 under different conditions A:Enzyme activity of strain DGW1 at different pH. B:Enzyme activity of strain DGW1 at different temperature. C:Enzyme activity of strain DGW1 at different times

图7 不同菌株菌渣杆降解率

Fig.7 Rates of different strains degrading spent mushroom substrate

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