产纤维素酶细菌的筛选鉴定与特性分析

赵龙妹, 陈林, 杜东晓, 董惠心, 李旺, 李元晓, 何万领, 曹平华

中国农学通报. 2021, 37(30): 83-88

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中国农学通报 ›› 2021, Vol. 37 ›› Issue (30) : 83-88. DOI: 10.11924/j.issn.1000-6850.casb2020-0855
生物科学

产纤维素酶细菌的筛选鉴定与特性分析

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Screening, Identification and Characteristic Analysis of Cellulase-Producing Bacteria

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摘要

为了筛选获得产纤维素酶细菌,为新型饲料添加剂的开发提供材料基础。本研究以玉米田土壤为样品,使用刚果红平板法筛选产纤维素酶细菌并分析其所产酶的特性。通过试验,筛选获得产纤维素酶菌株,经形态学和分子生物学法将其鉴定为巨大芽孢杆菌(Bacillus megaterium)。对巨大芽孢杆菌XT2所产纤维素酶进行酶学特性分析,发现其最适反应条件为50℃,pH 6.0,具有一定的热稳定性,K+对纤维素酶具有激活作用,Mg2+、Ca2+和Mn2+对酶活具有抑制作用。该菌生长至20 h时,所产纤维素酶活力最高,达到0.774 U/mL。该产纤维素酶芽孢杆菌的成功分离获取为饲料新资源的开发以及新型饲料添加剂的研制提供了菌种材料。

Abstract

To screen cellulase-producing bacteria and provide a material basis for the development of new feed additives, in this study, soil samples from corn fields were used to screen cellulase-producing bacteria by congo red plate method, and the characteristics of cellulase produced by it were analyzed. Through the test, the cellulase-producing strain was screened and identified as Bacillus megaterium by morphological and molecular biological methods. The enzymatic characteristics of cellulase produced by Bacillus megaterium XT2 was analyzed and the optimal reaction condition was found to be 50℃ and pH 6.0, and the cellulase showed certain thermal stability, K+ could activate the cellulase activity, and Mg2+, Ca2+ and Mn2+ had an inhibitory effect on enzyme activity. When the strain grew to 20 h, the cellulase activity reached the highest (0.774 U/mL). The successful isolation of cellulase-producing Bacillus could provide a strain material for the development of new feed resources and new feed additives.

关键词

纤维素酶 / 芽孢杆菌 / 筛选 / 鉴定 / 酶学特性

Key words

cellulase / Bacillus / screening / identification / enzymatic characteristics

引用本文

导出引用
赵龙妹 , 陈林 , 杜东晓 , 董惠心 , 李旺 , 李元晓 , 何万领 , 曹平华. 产纤维素酶细菌的筛选鉴定与特性分析. 中国农学通报. 2021, 37(30): 83-88 https://doi.org/10.11924/j.issn.1000-6850.casb2020-0855
Zhao Longmei , Chen Lin , Du Dongxiao , Dong Huixin , Li Wang , Li Yuanxiao , He Wanling , Cao Pinghua. Screening, Identification and Characteristic Analysis of Cellulase-Producing Bacteria. Chinese Agricultural Science Bulletin. 2021, 37(30): 83-88 https://doi.org/10.11924/j.issn.1000-6850.casb2020-0855

0 引言

纤维素是陆地环境中光合作用的初级产物,也是生物圈中最丰富的可再生生物资源[1,2,3]。纤维素是由葡萄糖单元通过β-1,4糖苷键连接而成的线性均聚物,它是木质纤维类物质中最重要的多聚物,与半纤维素、木质素共同构成木质纤维类物质[4]。在所有种类的植物生物质中,木质纤维类物质由于其大量可获取、低成本和环保性,非常适合用于能量供应[5,6],而酶解植物中的碳水化合物已经成为生物质发酵小分子化和能源化过程中最重要的技术[7,8,9]。自然界中植物、动物和微生物均能够产酶,微生物来源的酶因其易于获取和大规模生产而得到广泛的关注和研究。真菌所产酶种类多,产量高,但生产周期较长,且可能会产生霉味,细菌中的芽孢杆菌所产酶的种类也较多[10],除了蛋白酶和淀粉酶之外,芽孢杆菌还可分泌纤维素酶[11,12]、木聚糖酶[13]等木质纤维降解酶以及具备特殊功能的酶如低温酶[14],此外芽孢杆菌生产周期短,工业能耗低,因此,筛选获取产酶细菌已成为功能微生物研究的热点。本研究拟通过平板法和酶活测定法,从玉米田土壤中筛选获取产纤维素酶细菌,利用形态学和分子生物学法对其进行鉴定,并分析该菌的生长规律和产酶规律,研究所产酶的酶学特性,以期为新型饲料添加剂的研发及非常规饲料资源的利用奠定基础。

1 材料与方法

1.1 筛菌样品

采集于河南省洛阳市河南科技大学校园内玉米田地土壤,琴湖水样。

1.2 主要仪器和设备

立式高压蒸汽灭菌锅(型号:LDZX-50KBS)购于上海申安医疗器械厂;双人单面超净工作台(型号为SW-CJ-2FD)购于苏州净化设备有限公司;生物显微镜(型号为CX31)购于奥林巴斯有限公司;高速台式离心机(TGL-16B)购于上海安亭科学仪器厂;酶标仪(ReadMax 1000F)购于上海闪谱生物科技有限公司。

1.3 主要试剂和培养基

1.3.1 主要试剂 蛋白胨购于北京奥博星生物技术有限责任公司;葡萄糖、琼脂粉等均购于天津市科密欧化学试剂有限公司;羧甲基纤维素钠(CMC-Na)、氯化钠等均购于国药集团化学试剂有限公司。DNS试剂的配置参考Miller等[15]的方法。
1.3.2 筛选培养基 CMC-Na 10 g,K2HPO4 1.31 g,NaNO3 3 g,KCl 0.5 g,MgSO4·7H2O 0.5 g,FeSO4 0.01 g,加蒸馏水至1000 mL,pH 6.5,在121℃,0.11 MPa条件下灭菌20~25 min,冷却后使用。
1.3.3 发酵培养基 蛋白胨10 g,酵母粉0.5 g,KH2PO4 1 g,MgSO4·7H2O 0.2 g,NaCl 10 g,加蒸馏水至1000 mL,pH 6.5,在121℃,0.11 MPa条件下灭菌20~25 min,冷却后使用。
1.3.4 固体培养基 固体培养基在上述培养基基础上添加2.3%的琼脂粉,按照上述条件配制后,在121℃,0.11 MPa条件下灭菌20~25 min,冷却至常温或制备平板后接种使用。

1.4 产纤维素酶菌株的筛选

称取0.5 g样品,溶于50 mL无菌水中,置于摇床中200 r/min振荡4~6 h后取1 mL样品进行梯度稀释,取适度稀释的样品50 μL涂布于筛选培养基固体平板上,30℃条件下培养1~3天,直至长出单菌落。挑取生长情况良好的单菌落点接种于筛选培养基固体平板上,待菌落长出后,使用1 mg/mL刚果红对平板染色2 h,然后使用1 mol/L NaCl溶液洗脱1 h直至出现清晰透明圈,观察菌株周围透明圈情况,确定产纤维素酶菌株,并进行后续研究。

1.5 菌株的鉴定

观察固体平板上菌落的形态,革兰氏染色后进行镜检,观察菌体的形态特征,进行初步鉴定。利用细菌基因组DNA快速抽提试剂盒提取菌株的基因组,以基因组为模板,使用引物进行PCR获取16S rDNA序列,经生工生物工程股份有限公司测序后,将序列信息在NCBI网站上(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行比对分析,并使用MEGA X软件通过邻接法(Neighbor-Joining Method)构建系统发育树,对菌株进行鉴定。

1.6 菌株所产纤维素酶的酶学特性分析

1.6.1 粗酶液的制备和酶活力的测定
(1)粗酶液的制备:挑取单菌落接种于发酵培养基中,30 ℃,200 r/min培养16 h,将发酵菌液12000 r/min离心5 min,上清即为粗酶液。
(2)酶活力的测定:以羧甲基纤维素钠作为底物,使用DNS法[15]测定反应后还原糖的含量,计算酶活力大小。纤维素酶活力的定义:在一定温度和pH下,每分钟水解1%羧甲基纤维素钠生成1 μmol还原糖所需的酶量为一个酶活单位(U)。
1.6.2 温度对酶活力的影响 在一定pH下,分别将粗酶液与底物溶液在30、40、50、60、70、80℃条件下反应,测定酶活力大小,分析温度对纤维素酶活力的影响。
1.6.3 pH对酶活力的影响 在一定温度条件下,分别测定粗酶液在pH 5、pH 6、pH 7、pH 8、pH 9、pH 10条件下的酶活力,分析酸碱度对纤维素酶活力的影响。
1.6.4 耐热性分析 分别将粗酶液在80℃条件下处理10、20、30、40、50、60 min,然后测定粗酶液的残余酶活力大小,分析该纤维素酶的耐热性。
1.6.5 耐碱性分析 分别将粗酶液在pH 10的条件下处理10、20、30、40、50、60 min,然后测定粗酶液残余酶活力大小,分析碱性环境对该纤维素酶活力的影响。
1.6.6 不同金属离子对酶活力的影响 向粗酶液中加入终浓度为0.1 mol/L的Na+、Mg2+、Ca2+、K+和Mn2+金属离子盐溶液,测定纤维素酶活力,分析不同金属离子对酶活力的影响。
1.6.7 菌株生长规律和产酶情况的分析 挑取单菌落接种于液体发酵培养基中,30℃,200 r/min过夜培养,制备种子液,将种子液以1%的接种量接种于发酵培养基中,30℃,200 r/min振荡培养,每隔2 h取样一次,分别测定菌液在波长600 nm下的吸光度和上清液的纤维素酶活力,分析该菌的生长情况和产酶规律。

2 结果与分析

2.1 产纤维素酶菌株的筛选

挑取筛选培养基平板上长出的单菌落再次接种筛选固体培养基,培养至菌落长出,经过刚果红染色并脱色后,如图1所示,可见菌落XT2周围产生明显透明圈,判断该菌可产纤维素酶,而且XT2菌株为细菌,因此对其进行后续分析研究。
图1 刚果红染色后菌落周围的透明圈

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2.2 菌株的鉴定

图2A所示,该菌在固体平板上的菌落呈白色或微黄色,近圆形,带有少许光泽;如图2B所示,该菌为革兰氏阳性菌,菌体呈杆状,单杆或长链,芽孢卵圆形或柱形,位于菌体中央或稍偏一端。
图2 菌株XT2的形态学观察

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将测序获得的16S rDNA序列信息在NCBI网站上进行比对并使用MEGA X软件通过邻接法(Neighbor-Joining Method)构建系统发育树,结果如图3所示,该菌与巨大芽孢杆菌Bacillus megaterium(MH762123.1)聚为一支,故将其鉴定为巨大芽孢杆菌(Bacillus megaterium)。
图3 菌株XT2的系统发育树

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2.3 菌株所产纤维素酶的酶学特性分析

2.3.1 温度对酶活力的影响 如图4所示,该菌所产纤维素酶的最适反应温度为50℃,在40~70℃之间能够发挥75%以上的酶活,有望作为饲料添加剂,在动物胃肠道中发挥作用。
图4 温度对酶活力的影响

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2.3.2 pH对酶活力的影响 如图5所示,该纤维素酶的最适反应为pH 6,在pH 7时,能够发挥88%以上的酶活,在弱酸或中性条件下能够发挥较大的酶活,这也有利于其作为一种饲料添加剂在动物胃肠道内发挥作用。
图5 pH对酶活力的影响

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2.3.3 纤维素酶的耐热性 如图6所示,该纤维素酶在80℃条件下处理10 min后,能够剩余70%以上的酶活,处理20 min后能够剩余50%以上的酶活,随着处理时间的增加,残余酶活减少,处理30 min后残余酶活不足50%,高温处理会造成蛋白质的变性,从而使酶活力降低,而该酶表现出一定的耐热性。
图6 纤维素酶的耐热性

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2.3.4 纤维素酶的耐碱性 如图7所示,该纤维素酶在pH 10条件下处理10 min后,残余50%以上酶活,处理20 min后,残余不足40%的酶活,说明该酶在碱性条件下易失活。
2.3.5 不同金属离子对酶活力的影响 如图8所示,不同金属离子对酶活能够产生抑制或激活的作用,K+对该纤维素酶活力具有激活作用,Ca2+、Mg2+、Mn2+对酶活具有抑制作用。
图7 纤维素酶的耐碱性

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图8 不同金属离子对纤维素酶活力的影响

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2.3.6 该菌的生长情况和产酶规律 如图9所示,0~2 h是该菌生长的迟缓期,2~10 h进入对数生长期,菌体增殖迅速,10~20 h进入生长稳定期,菌体增殖的同时,部分菌体因生长环境的限制发生死亡,因此菌体数目变化不大,20~24 h进入衰亡期,由于培养基中营养物质消耗殆尽,大量代谢产物的积累,导致菌体死亡数目高于增殖数目,菌体浓度有所下降。由图9中酶活曲线可看出,该菌所产纤维素酶从生长初期开始分泌,到稳定期末时累积的酶量达到最高值,随后,由于环境中代谢产物的影响以及菌体数目的下降,酶活发生下降。
图9 菌株XT2的生长曲线和产酶规律

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3 讨论

土壤的成分非常复杂,包括水分、空气、氧化的腐殖质、岩石风化矿物质、动植物和微生物残骸分解产生的有机质以及土壤微生物,其中土壤微生物推动土壤生态系统中的物质循环,降解其中的有机质,如纤维素、半纤维素和木质素等木质纤维类物质[16]。因此,土壤中的微生物多数能够分泌纤维素酶、半纤维素酶等木质纤维类物质的降解酶,土壤也是筛选降解纤维物质菌株的重要样品。不同来源的土壤,由于植被生长的不同,其中微生物多样性有很大差异,所含功能菌也有所差异,如干旱区不同盐生植物群落微生物多样性具有显著差异,其中梭梭群落的土壤微生物群落具有较强的微生物总体活性和功能多样性[17],而极地陆域如南极、青藏高原和北极土壤中微生物多样性由于低温、养分贫乏而具有较大差异[18]。由于玉米植株中含量较高的纤维素和半纤维素,玉米田土壤中的纤维类有机质含量较丰富,可能含有较多的纤维降解菌,因此,本研究使用玉米田土壤作为样品,筛选产纤维素酶菌株。
纤维素酶在轻工业中具有较广泛的应用,在饲料业和畜牧养殖业中,主要用来提高动物对粗纤维物质的消化吸收率,维护动物胃肠道系统的健康,以及开发非常规饲料资源[19]。不同特点的纤维素酶有不同的应用,如碱性纤维素酶主要应用于增强洗涤剂的使用效果[20],酸性纤维素酶可用于牛仔洗涤、青贮制备[21],或者作为饲料添加剂,在动物肠道中发挥作用。本研究中筛选获得的纤维素酶来源于巨大芽孢杆菌,为酸性纤维素酶,可在单胃动物的消化道和反刍动物的瘤胃中发挥作用,因此可作为饲料用酶进行开发。
芽孢杆菌是一种益生菌,与其他益生菌相比,其最大的特点就是具有非常好的抗逆性[22]。此外,芽孢杆菌还能够产生多种酶类,可应用于发酵饲料的生产,有研究利用响应面法优化巨大芽孢杆菌产纤维素酶的发酵条件,结果发现优化后纤维素酶的活力提高了2倍[23]。也有研究指出巨大芽孢杆菌能够抑制黄曲霉毒素的生成,并发挥解磷的功能,安全性良好[24]。还有研究发现,在植物蛋白质饲料中添加巨大芽孢杆菌能够提高鱼体抗氧化能力以及非特异性免疫能力[25]。巨大芽孢杆菌在生长过程中还能够分泌一些抑菌物质,抑制致病菌的生长[26]。本研究中筛选到的巨大芽孢杆菌能够产生纤维素酶,后期可对其抑菌物质的分泌进行检测,为其作为新型饲料添加剂的开发和应用提供参考。

4 结论

本研究以玉米田土壤为筛菌材料,以羧甲基纤维素钠为唯一碳源,筛选获得产纤维素酶菌株;利用分子生物学法结合形态学观察将其鉴定为巨大芽孢杆菌(Bacillus megaterium)。该菌所产纤维素酶的最适反应条件为50℃,pH 6.0,具有一定的热稳定性,K+对纤维素酶具有激活作用,Mg2+、Ca2+和Mn2+对酶活具有抑制作用。该菌生长至20 h时,所产纤维素酶活力较高,达到0.774 U/mL。本研究所筛选到的产纤维素酶芽孢杆菌未来可通过发酵法和酶解法处理高纤维含量的非常规饲料,可拓宽饲料原料来源并解决饲料资源短缺的问题,对非常规饲料资源的开发以及新型饲料添加剂的研制提供了菌种材料并奠定了坚实的基础。

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Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.
[2]
Somerville C, Bauer S, Brininstool G, et al. Toward a systems approach to understanding plant cell walls[J]. Science, 2004, 306(5705):2206-2211.
One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses of the polysaccharide components, combined with high-resolution imaging, have provided the basis for much of the current understanding of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into a useful model will require a system-based approach.
[3]
Bischof R H, Ramoni J, Seiboth B. Cellulases and beyond: The first 70 years of the enzyme producer trichoderma reesei[J]. Microb Cell Fact, 2016, 15(1):106.
More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.
[4]
Foyle T, Jennings L, Mulcahy P. Compositional analysis of lignocellulosic materials: Evaluation of methods used for sugar analysis of waste paper and straw[J]. Bioresource Technology, 2007, 98(16):3026-3036.
[5]
Lynd L R, Zyl W H, McBride J E, et al. Consolidated bioprocessing of cellulosic biomass: An update[J]. Current Opinion in Biotechnology, 2005, 16(5):577-583.
Biologically mediated processes seem promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Although processes featuring a step dedicated to the production of cellulase enzymes have been the focus of most research efforts to date, consolidated bioprocessing (CBP)--featuring cellulase production, cellulose hydrolysis and fermentation in one step--is an alternative approach with outstanding potential. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system enabling cellulose utilization. Recent studies of the fundamental principles of microbial cellulose utilization support the feasibility of CBP.
[6]
Barbosa FC, Silvello MA, Goldbeck R. Cellulase and oxidative enzymes: New approaches, challenges and perspectives on cellulose degradation for bioethanol production[J]. Biotechnology Letters, 2020, 42(6):875-884.
[7]
Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—factors affecting enzymes, conversion and synergy[J]. Biotechnology Advances, 2012, 30(6):1458-1480.
Lignocellulose is a complex substrate which requires a variety of enzymes, acting in synergy, for its complete hydrolysis. These synergistic interactions between different enzymes have been investigated in order to design optimal combinations and ratios of enzymes for different lignocellulosic substrates that have been subjected to different pretreatments. This review examines the enzymes required to degrade various components of lignocellulose and the impact of pretreatments on the lignocellulose components and the enzymes required for degradation. Many factors affect the enzymes and the optimisation of the hydrolysis process, such as enzyme ratios, substrate loadings, enzyme loadings, inhibitors, adsorption and surfactants. Consideration is also given to the calculation of degrees of synergy and yield. A model is further proposed for the optimisation of enzyme combinations based on a selection of individual or commercial enzyme mixtures. The main area for further study is the effect of and interaction between different hemicellulases on complex substrates.Copyright © 2012 Elsevier Inc. All rights reserved.
[8]
Xin D, Yang M, Chen X, et al. Improving cellulase recycling efficiency by decreasing the inhibitory effect of unhydrolyzed solid on recycled corn stover saccharification[J]. Renewable energy, 2020, 145(1):215-221.
[9]
Singh S. Biological treatment of plant biomass and factors affecting bioactivity[J]. Journal of Cleaner Production, 2020: 279.
[10]
Su Y, Liu C, Fang H, et al. Bacillus subtilis: A universal cell factory for industry, agriculture, biomaterials and medicine[J]. Microbial Cell Factories, 2020, 19:173.
[11]
Elhameed E A, Sayed A R M, Radwan T E E , et al. Biochemical and molecular characterization of five bacillus isolates displaying remarkable carboxymethyl cellulase activities[J]. Current Microbiology, 2020, 77(10):3076-3084.
Cellulases have many useful applications in industry and biotechnology. So, identification of new bacterial strains expressing cellulases with better properties is desired. Five soil bacterial strains screened for high carboxymethyl cellulase (CMCase) activities were characterized and identified by 16S rRNA analysis as Bacillus amyloliquefaciens (FAY088), B. velezensis (FAY0103), B. tequilensis (FAY0117), B. subtilis (FAY0136), and B. subtilis (FAY0182). Their CMCase activities were 1.49, 1.26, 1.21, 1.21, and 1.24 U/ml, respectively. The maximum CMCase production was attained by growth at 35 °C, pH 6, and 180 rpm for 5 days. Residual activities of CMCases from FAY088 and FAY0117 were 88% or more after growth at 40 °C, which is same as FAY0182 CMCase at 40 and 45 °C. Additionally, FAY0182 retained 73% residual activity at 50 °C. FAY088 and FAY0182 retained more than 85% at pH 7 and 8. Conversely, residual activities from FAY0103 and FAY0136 declined a lot by increasing growth temperature beyond 40 °C and pH beyond 7. The maximum CMCase stability in all isolates was observed at pH 7, 3-h incubation, and 40 °C except for FAY0103 CMCase showed optimum temperature at 30 °C. More than 70% CMCase stability was retained in case of FAY088 at 50 °C, FAY0117 at 50-70 °C, and FAY0136 at 50-60 °C. FAY088 CMCase seemed to be the lest sensitive to temperature variation as it displayed residual activities 67, 72, 78, 84, 77, 74, and 72% at pH 3, 4, 5, 6, 8, 9, and 10, respectively. Finally, the five CMCase-producing isolates are recommended further enzyme applications in biotechnology and industry.
[12]
Dehghanikhah F, Shakarami J, Asoodeh A. Purification and biochemical characterization of alkalophilic cellulase from the symbiotic Bacillus subtilis BC1 of the leopard moth Zeuzera pyrina (L.) (Lepidoptera:cossidae)[J]. Current Microbiology, 2020, 77:1254-1261.
In the current study, an extracellular cellulase belonging to symbiotic Bacillus subtilis Bc1 of the leopard moth is purified and characterized. The molecular mass of enzyme was 47.8 kDa using SDS-PAGE. The purified enzyme had optimum activity in temperature and pH around 60 °C and 8, respectively. The purified cellulase was introduced as a stable enzyme in a wide variety of temperature (20-80 °C) and pH (4-10) and remained active to more than 74% at 80 °C for 1 h. Moreover, the cellulase extremely was stabled in the presence of metal ions and organic solvents and its activity was increased by acetone (20% v/v), CaCl and CoCl and inhibited by MnCl and NiCl. The values of enzyme's K and V were found to be 1.243 mg/mL and 271.3 µg/mL/min, respectively. The purified cellulase hydrolyzed cellulose, avicel and carboxymethyl cellulose (CMC) and the final product of CMC hydrolysis was cellobiose using thin-layer chromatography analysis. Consequently, owing to exo/endoglucanase activity and organic solvent, temperature and pH stability of the purified cellulase belong to B. subtilis BC1, it can be properly employed for various industrial purposes.
[13]
Baramee S, Siriatcharanon A K, Ketbot P, et al. Biological pretreatment of rice straw with cellulase-free xylanolytic enzyme-producing Bacillus firmus K-1: Structural modification and biomass digestibility[J]. Renewable energy, 2020, 160:555-563.
[14]
Ma L, Aizhan R, Wang X, et al. Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds[J]. AMB Express, 2020, 10(42):1-11.
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Bayer E A, Lamed R, Himmel M E. The potential of cellulases and cellulosomes for cellulosic waste management[J]. Current Opinion in Biotechnology, 2007, 18(3):237-245.
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Hou M, Gentu G, Liu T, et al. Silage preparation and fermentation quality of natural grasses treated with lactic acid bacteria and cellulase in meadow steppe and typical steppe[J]. Asian-Australasian Journal of Animal Sciences, 2017, 30(6).
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谷笑笑, 王振华, 潘康成. 益生芽孢杆菌对动物免疫功能影响研究进展[J]. 微生物学通报, 2016, 43(9):2079-2085.
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基金

河南省重点研发与推广专项(科技攻关)“酶解玉米芯新型饲料添加剂的开发”(212102110174)
河南科技大学博士科研启动基金项目“酶解玉米芯与益生素联用效果研究”(13480076)

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