Optimization of Producing Cellulase by Aspergillus fumigatus A-16 and Its Enzymatic Properties

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

Abstract

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

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.

Figures/Tables 12

References 28

[1]	Souii A, Guesmi A, Ouertani R, et al. Carboxymethyl cellulase production by extremotolerant bacteria in low-cost media and application in enzymatic saccharification of Stevia biomass[J]. Waste Biomass Valorization, 2020, 11(5):2111-2122. doi: 10.1007/s12649-018-0496-2 URL
[2]	Mehboob N, Asad MJ, Asgher M, et al. Exploring thermophilic cellulolytic enzyme production potential of Aspergillus fumigatus by the solid-state fermentation of wheat straw[J]. Appl Biochem Biotechnol, 2014, 172(7):3646-3655. doi: 10.1007/s12010-014-0796-3 pmid: 24562980
[3]	Sohail M, Ahmad A, Khan SA. Production of cellulase from Aspergillus terreus MS105 on crude and commercially purified substrates[J]. 3 Biotech, 2016, 6(1):103. doi: 10.1007/s13205-016-0420-z URL
[4]	Mahmood RT, Asad MJ, Mehboob N, et al. Production, purification, and characterization of exoglucanase by Aspergillus fumigatus[J]. Appl Biochem Biotechnol, 2013, 170(4):895-908. doi: 10.1007/s12010-013-0227-x pmid: 23615734
[5]	Sharma HK, Xu CC, Qin WS. Co-culturing of novel Bacillus species isolated from municipal sludge and gut of red wiggler worm for improving CMCase activity[J]. Waste Biomass Valorization, 2020, 11(5):2047-2058. doi: 10.1007/s12649-018-0448-x URL
[6]	Zanirun Z, Bahrin EK, Lai-Yee P, et al. Effect of physical and chemical properties of oil palm empty fruit bunch, decanter cake and sago pith residue on cellulases production by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2[J]. Appl Biochem Biotechnol, 2014, 172(1):423-435. doi: 10.1007/s12010-013-0530-6 URL
[7]	Horn SJ, Vaaje-Kolstad G, Westereng B, et al. Novel enzymes for the degradation of cellulose[J]. Biotechnol Biofuels, 2012, 5(1):45. doi: 10.1186/1754-6834-5-45 pmid: 22747961
[8]	Zhang K, Pei ZJ, Wang DH. Organic solvent pretreatment of lignocellulosic biomass for biofuels and biochemicals:a review[J]. Bioresour Technol, 2016, 199:21-33. doi: 10.1016/j.biortech.2015.08.102 URL
[9]	Wang D, Sun J, Yu HL, et al. Maximum saccharification of cellulose complex by an enzyme cocktail supplemented with cellulase from newly isolated Aspergillus fumigatus ECU0811[J]. Appl Biochem Biotechnol, 2012, 166(1):176-186. doi: 10.1007/s12010-011-9414-9 pmid: 22086563
[10]	李乐, 李明星, 汤国雄, 等. 一株纤维素酶产生菌的筛选与产酶特性研究[J]. 环境科技, 2019, 32(1):24-29.
	Li L, Li MX, Tang GX, et al. Optimization of enzyme production conditions for a cellulase-producing strain[J]. Environ Sci Technol, 2019, 32(1):24-29.
[11]	Miao JX, Wang MM, Ma L, et al. Effects of amino acids on the lignocellulose degradation by Aspergillus fumigatus Z5:insights into performance, transcriptional, and proteomic profiles[J]. Biotechnol Biofuels, 2019, 12:4. doi: 10.1186/s13068-018-1350-2 URL
[12]	Srivastava N, Rawat R, Sharma R, et al. Effect of nickel-cobaltite nanoparticles on production and thermostability of cellulases from newly isolated thermotolerant Aspergillus fumigatus NS(class:Eurotiomycetes)[J]. Appl Biochem Biotechnol, 2014, 174(3):1092-1103. doi: 10.1007/s12010-014-0940-0 pmid: 24801407
[13]	Islam F, Roy N. Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses[J]. BMC Res Notes, 2018, 11(1):445. doi: 10.1186/s13104-018-3558-4 pmid: 29973263
[14]	El-Ghonemy DH, Ali TH, El-Bondkly AM, et al. Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase[J]. Antonie Van Leeuwenhoek, 2014, 106(5):853-864. doi: 10.1007/s10482-014-0255-8 pmid: 25119245
[15]	Grigorevski-Lima AL, da Vinha FNM, Souza DT, et al. Aspergillus fumigatus thermophilic and acidophilic endoglucanases[J]. Appl Biochem Biotechnol, 2009, 155(1-3):321-329. doi: 10.1007/s12010-008-8482-y pmid: 19127443
[16]	Das A, Paul T, Ghosh P, et al. Kinetic study of a glucose tolerant β-glucosidase from Aspergillus fumigatus ABK9 entrapped into alginate beads[J]. Waste Biomass Valorization, 2015, 6(1):53-61. doi: 10.1007/s12649-014-9329-0 URL
[17]	Sharma M, Soni R, Nazir A, et al. Evaluation of glycosyl hydrolases in the secretome of Aspergillus fumigatus and saccharification of alkali-treated rice straw[J]. Appl Biochem Biotechnol, 2011, 163(5):577-591. doi: 10.1007/s12010-010-9064-3 pmid: 20730507
[18]	于岚, 程芳, 邵文琦, 等. 一株嗜热纤维素酶生产菌的分离、鉴定及酶学研究[J]. 安徽农业科学, 2013, 41(17):7413-7417.
	Yu L, Cheng F, Shao WQ, et al. Study on isolation, identification and characterization of a thermophilic cellulase-producing strain[J]. J Anhui Agric Sci, 2013, 41(17):7413-7417.
[19]	Lin CY, Shen ZC, Qin WS. Characterization of xylanase and cellulase produced by a newly isolated Aspergillus fumigatus N2 and its efficient saccharification of barley straw[J]. Appl Biochem Biotechnol, 2017, 182(2):559-569. doi: 10.1007/s12010-016-2344-9 URL
[20]	Saroj P, Manasa P, Narasimhulu K. Characterization of thermophilic fungi producing extracellular lignocellulolytic enzymes for lignocellulosic hydrolysis under solid-state fermentation[J]. Bioresour Bioprocess, 2018, 5:31. doi: 10.1186/s40643-018-0216-6 URL
[21]	谢洁, 商必志, 任慧爽, 等. 一株纤维素酶产生菌B. cereus JYMB2菌株的筛选鉴定[J]. 西南大学学报:自然科学版, 2016, 38(5):45-51.
	Xie J, Shang BZ, Ren HS, et al. Screening and identification of a cellulase-producing strain of Bacillus cereus-JYMB2[J]. J Southwest Univ Nat Sci Ed, 2016, 38(5):45-51.
[22]	马振刚, 熊亮, 张真, 等. 高产碱性纤维素酶细菌的筛选鉴定及其酶学特性与发酵条件研究[J]. 南方农业学报, 2021, 52(3):722-731.
	Ma ZG, Xiong L, Zhang Z, et al. Screening and identification of a strain with high yield of alkaline cellulase and its enzyme characterizations and fermentation conditions[J]. J South Agric, 2021, 52(3):722-731.
[23]	魏姣, 万学瑞, 吴润, 等. 产纤维素酶真菌菌株的分离筛选及产酶条件优化[J]. 甘肃农业大学学报, 2016, 51(2):8-15.
	Wei J, Wan XR, Wu R, et al. Isolation and screening of fungi strains producing cellulase and optimization of conditions for enzyme production[J]. J Gansu Agric Univ, 2016, 51(2):8-15.
[24]	何楠, 令利军, 冯蕾, 等. 1株产纤维素酶细菌的筛选、鉴定及生长特性[J]. 微生物学杂志, 2017, 37(1):43-49.
	He N, Ling LJ, Feng L, et al. Isolation, identification and growth characteristics of a cellulase-producing bacterium[J]. J Microbiol, 2017, 37(1):43-49.
[25]	李婷, 王玥, 刘中珊, 等. 一株降解纤维素的低温放线菌Streptomyces azureus及产酶条件优化[J]. 中国农学通报, 2021, 37(32):25-33.
	Li T, Wang Y, Liu ZS, et al. A novel low temperature cellulose-degrading strain Streptomyces azureus and its enzymatic production condition optimization[J]. Chin Agric Sci Bull, 2021, 37(32):25-33.
[26]	赵龙妹, 陈林, 杜东晓, 等. 产纤维素酶细菌的筛选鉴定与特性分析[J]. 中国农学通报, 2021, 37(30):83-88.
	Zhao LM, Chen L, Du DX, et al. Screening, identification and characteristic analysis of cellulase-producing bacteria[J]. Chin Agric Sci Bull, 2021, 37(30):83-88.
[27]	蒋芳, 刘松青, 甄阳光, 等. 一株产高温纤维素酶菌株的分离筛选[J]. 纤维素科学与技术, 2015, 23(2):50-54.
	Jiang F, Liu SQ, Zhen YG, et al. Isolation and screening of a thermophilic cellulose bacterial strain[J]. J Cellul Sci Technol, 2015, 23(2):50-54.
[28]	金伟, 陈文静, 缪礼鸿, 等. 1株耐热纤维素酶产生菌的筛选及其酶学特性[J]. 江苏农业科学, 2017, 45(20):272-274.
	Jin W, Chen WJ, Miao LH, et al. Screening of a heat-resistant cellulase-producing strain and enzymatic specificity[J]. Jiangsu Agric Sci, 2017, 45(20):272-274.

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

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

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

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