高产阿魏酸酯酶菌株的筛选鉴定及其酶活测定条件优化

doi:10.13560/j.cnki.biotech.bull.1985.2020-0304

摘要/Abstract

摘要：

通过透明圈初筛和固体发酵复筛从野外采集的和实验室保藏的共106株大型真菌菌株中筛选得到两株高产阿魏酸酯酶的菌株,经ITS序列测定结合子实体形态鉴定为一色齿毛菌（Cerrena unicolor）和血红密孔菌（Pycnoporus sanguineus）;以米糠为诱导培养基测定了两个菌株产阿魏酸酯酶的酶活曲线,并选取活性最高的粗酶液进行了酶活测定条件优化。结果表明：以米糠为诱导培养基时,一色齿毛菌和血红密孔菌的阿魏酸酯酶活性分别在第4天和第6天达到最高;经优化得出一色齿毛菌粗酶液的阿魏酸酯酶最适测定条件为40℃、反应时间25 min、添加30 μL粗酶液和400 μL阿魏酸甲酯溶液,血红密孔菌粗酶液的最适测条件为30℃、反应时间35 min、添加20 μL粗酶液和400 μL阿魏酸甲酯溶液。

关键词: 大型真菌, 阿魏酸酯酶, 筛选鉴定, 酶活测定

Abstract:

Using transparent zone and solid fermentation,two strains with high feruloyl esterase（FAE）yield were selected from 106 macrofungi that were collected from field and deposited in the lab an. They were identified as Cerrena unicolor and Pycnoporus sanguineus by ITS sequence analysis and morphological identification. Then the FAE activity of two screened strains were determined using rice bran as induction medium and the crude enzyme with the highest activity was selected separately to optimize the enzyme activity determination conditions. The results demonstrated that the activity of FAE by C. unicolor and P. sanguineus reached the peak at 4 d and 6 d respectively. The optimal determination conditions for FAE activity in the crude enzyme solution of C. unicolor was 40℃,25 min reaction time,30 μL crude enzyme and 400 μL the methyl ferulate. The optimal determination conditions for FAE activity in the crude enzyme solution of P. sanguineus was 30℃,35 min reaction time,20 μL crude enzyme solution and 400 μL methyl ferulate.

Key words: macrofungi, feruloyl esterase, screening and identification, determination of enzyme activity

王祥锋, 汪乔, 袁慧君, 王丽. 高产阿魏酸酯酶菌株的筛选鉴定及其酶活测定条件优化[J]. 生物技术通报, 2020, 36(10): 135-141.

WANG Xiang-feng, WANG Qiao, YUAN Hui-jun, WANG Li. Screening and Identification of High-yield Feruloyl Esterase Strains and Optimizing of the Enzyme Activity Assay Conditions[J]. Biotechnology Bulletin, 2020, 36(10): 135-141.

图/表 9

图1 16株菌株的透明圈直径大小

图2 发酵麦麸培养基中16个菌株的阿魏酸酯酶活性

图3 发酵米糠培养基中16个菌株的阿魏酸酯酶活性

图4 菌株D、M的ITS序列系统发育树分析

图5 两个菌株的野外子实体图片左侧为菌株D,右侧为菌株M

图6 两个菌株的酶活力随发酵时间变化曲线

图7 两个菌株粗酶液中酶活最适反应温度测定

图8 两个菌株粗酶液中酶活最佳反应时间测定

图9 粗酶液添加量对酶活力的影响

参考文献 26

[1]	Bonzom C, Schild L, Gustafsson H, et al. Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification[J]. BMC Biochemistry, 2018,19(1):1-12. URL pmid: 29390959
[2]	孙晓明, 辛嘉英, 王艳, 等. 微生物发酵产阿魏酸酯酶及释放阿魏酸研究概述[J]. 食品研究与开发, 2019,40(1):201-206.
	Sun XM, Xin JY, Wang Y, et al. Summary on the conditions of ferulic acid esterase and ferulic acid production by microorganism fermentation[J]. Food Research And Development, 2019,40(1):201-206.
[3]	陈静, 朱汇源, 侯运华. 阿魏酸酯酶基因工程菌研究进展[J]. 生物技术通报, 2012(9):35-40.
	Chen J, Zhu HY, Hou YH. Review on gene engineering strains of feruloyl esterases[J]. Biotechnology Bulletin, 2012(9):35-40.
[4]	Oliveira DM, Mota TR, Oliva B, et al. Feruloyl esterases:Biocatalysts to overcome biomass recalcitrance and for the production of bioactive compounds[J]. Bioresource Technology, 2019,278:408-423. URL pmid: 30704902
[5]	王丽, 孙钦栋, 王贺祥. 阿魏酸酯酶的研究与应用进展[J]. 山东农业大学学报:自然科学版, 2016,47(4):628-635.
	Wang L, Sun QD, Wang HX. A review on research and applications of feruloyl esterase[J]. Journal of Shandong Agricultural University:Natural Science Edition, 2016,47(4):628-635.
[6]	Terrett OM, Dupree P. Covalent interactions between lignin and hemicelluloses in plant secondary cell walls[J]. Current Opinion in Biotechnology, 2019,56:97-104.
[7]	Bartolome B, Faulds CB, Kroon PA. An Aspergillus niger esterase(ferulic acid esterase III)and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase(XylD)release a 5-5'-ferulic dehydrodimer(diferulic acid)from barley and wheat cell walls[J]. Appl Environ Microbiol, 1997,63(1):208-212. URL pmid: 8979352
[8]	de Vries RP, van Kuyk PA, Kester HCM. The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds[J]. J Biochem, 2002,363:377-386.
[9]	Romero BE, Grajales HD, Armendáriz RM, et al. Type C feruloyl esterase from Aspergillus ochraceus:A butanol specific biocatalyst for the synjournal of hydroxycinnamates in a ternary solvent system[J]. Electronic Journal of Biotechnology, 2018,35:1-9. doi: 10.1016/j.ejbt.2018.06.004 URL
[10]	Borneman WS, Ljungdahl LG, Hartley RD. Isolation and partial characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2[J]. Applied and Environmental Microbiology, 1991,57(8):2337-2344.
[11]	Borneman WS, Ljungdahl LG, Hartley RD. Purification and partial characterization of two feruloyl esterases from the anaerobic Neocallimastix strain MC-2[J]. Applied and Environmental Microbiology, 1992,58(11):3762-3766. URL pmid: 1482195
[12]	Abeijón Mukdsi MC, Haro C, González SN. Functional goat milk cheese with feruloyl esterase activity[J]. Journal of Functional Foods, 2013,5(2):801-809.
[13]	Kirk TK, Tien M. Lignin-degrading enzyme from Phanerochaete chrysosporium[J]. Applied Biochemistry and Biotechnology, 1984,9(4):317-318.
[14]	Hyde KD, Xu JC, Rapior S, et al. The amazing potential of fungi:50 ways we can exploit fungi industrially[J]. Fungal Diversity, 2019,97(1):1-136.
[15]	孙雪言, 朱涵予, 王媛媛, 等. 大型真菌菌核生物活性研究进展[J]. 食品工业科技, 2018,39(21):328-332.
	Sun XY, Zhu HY, Wang YY, et al. Research progress in the biological activities of macrofungal sclerotia[J]. Science and Technology of Food Industry, 2018,39(21):328-332.
[16]	胡泓宇, 周燕燕, 陈静. 一株产阿魏酸酯酶丝状真菌的分离鉴定及其粗酶性质研究[J]. 生物技术通报, 2014(7):162-167.
	Hu HY, Zhou YY, Chen J. Isolation, identification and enzyme properties of a strain producing feruloyl esterases[J]. Biotechnology Bulletin, 2014(7):162-167.
[17]	刘元元, 徐泽平. 阿魏酸酯酶活性的分光光度法测定及影响因素研究[J]. 食品工业科技, 2013,34(13):246-250.
	Liu YY, Xu ZP. Study on the determination of ferulic acid esterase enzyme activity with spectrophotometer method and its impact factors[J]. Science and Technology of Food Industry, 2013,34(13):246-250.
[18]	Mateos-Deo5 JC, Asaff-Torres A, Müller-Santos Marcelo, et al. From classical to high throughput screening methods for feruloyl esterases:A review[J]. Combinatorial Chemistry & High Throughput Screening, 2016,19(8):616-628. doi: 10.2174/1386207319666151110154722 URL pmid: 26552434
[19]	桑姝丽. 1株海洋来源产阿魏酸酯酶菌株的筛选及其对木质纤维材料的降解能力[J]. 江苏农业科学, 2017,45(20):275-278.
	Sang SL. Screening of a marine strain producing ferulic acid esterase and its effect on lignocellulosic acid degradability of dimensional materials[J]. Jiangsu Agricultural Sciences, 2017,45(20):275-278.
[20]	Haase-Aschoff P, Linke D, Berger RG. Detection of feruloyl- and cinnamoyl esterases from basidiomycetes in the presence of interfering laccase[J]. Bioresource Technology, 2013,130:231-238. doi: 10.1016/j.biortech.2012.12.039 URL pmid: 23306132
[21]	MacKenzie CR, Bilous D. Ferulic acid esterase activity from Schizophyllum commune[J]. Applied and Environmental Microbiology, 1988,54:1170-1173. URL pmid: 16347629
[22]	Mathew S, Abraham TE. Ferulic acid:An antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and its application[J]. Critical Reviews in Biotechnology, 2004,24(2-3):59-83. doi: 10.1080/07388550490491467 URL pmid: 15493526
[23]	Jaszek M, Miłek J, Żuchowski J, et al. Effective and complex stimulation of the biodegradation system of fungus Cerrena unicolor by rapeseed meal fermentation[J]. Acta Biochimica Polonica, 2016,63(3):549-554. URL pmid: 27253496
[24]	宋自力, 张伟, 廖头根. 血红密孔菌高产漆酶菌株的筛选及其对烟梗的生物降解[J]. 菌物学报, 2019,38(3):381-392.
	Song ZL, Zhang W, Liao TG. Highly laccase-yielding strains of Pycnoporus sanguineus and their activities on tobacco stem biodegradation[J]. Mycosystema, 2019,38(3):381-392.
[25]	Long LK, Zhao HY, Ding DF, et al. Heterologous expression of two Aspergillus niger feruloyl esterases in Trichoderma reesei for the production of ferulic acid from wheat bran[J]. Bioprocess and Biosystems Engineering, 2018,41(5):593-601.
[26]	Mota TR, de Oliveira DM, Marchiosi R, et al. Plant cell wall composition and enzymatic deconstruction[J]. AIMS Bioengineering, 2018,5(1):63-77.