耐热解淀粉芽孢杆菌BA-DES4产纤维素酶的分离纯化及酶学性质

张智; 温冬灼; 冯丽荣; 章圣龙; 杨可心; 张晓彤

doi:10.13386/j.issn1002-0306.2022070337

耐热解淀粉芽孢杆菌BA-DES4产纤维素酶的分离纯化及酶学性质

doi: 10.13386/j.issn1002-0306.2022070337

1.
东北林业大学林学院，黑龙江哈尔滨 150040
2.
黑龙江国宏节能环保有限公司，黑龙江哈尔滨 150040

基金项目: 黑龙江省“揭榜挂帅”科技攻关项目（2021ZXJ03B01）。

详细信息

作者简介:
张智（1964−），女，博士，教授，研究方向：食品发酵，植物生物转化，功能食品等研究，E-mail：Idzhangzhi@163.com

中图分类号: TS201.1
计量
- 文章访问数: 77
- HTML全文浏览量: 26
- PDF下载量: 4
- 被引次数: 0
出版历程
- 收稿日期: 2022-08-02
- 网络出版日期: 2023-04-20
- 刊出日期: 2023-06-01

Separation, Purification and Enzymatic Property of Cellulase Produced by Thermostable Bacillus amyloliquefaciens BA-DES4

1.
School of Forestry, Northeast Forestry University, Harbin 150040, China
2.
Heilongjiang Guohong Energy Conservation and Environmental Protection Co., Ltd., Harbin 150040, China

摘要

摘要: 目的：本研究以一株产纤维素酶的解淀粉芽孢杆菌BA-DES4为材料，纯化并研究了其纤维素酶的酶学性质。方法：研究采用硫酸铵分级沉淀及SephadexG-75凝胶过滤层析方式对其所产纤维素酶进行分离纯化，通过聚丙烯酰胺凝胶电泳（SDS-PAGE）确定其分子量，并对纯化后纤维素酶的酶液进行酶学性质研究。结果表明：发酵液中分离纯化获得纤维素酶系组分（内切葡聚糖酶），对纯化的电泳内切葡聚糖酶进行酶活测定，其比活力为51.08 U/mg。发现其分子量为22.4 kDa；初步酶学性质研究表明：该酶的最适反应温度和最适pH分别为65 ℃和6.0，且在pH5.0～7.0和温度55～65 ℃下稳定性较高；Mn²⁺对纤维素酶活力激活作用较为显著，Cu²⁺的抑制作用最大。结论：该菌株可作为产内切葡聚糖酶的潜在菌株，内切葡聚糖酶可在Mn²⁺、Fe²⁺的作用下促进酶活力，同时在高温及酸性环境中发挥作用，能够参与高效降解高温酸性环境中的纤维素，提高生产率，同时将分解成的葡萄糖供发酵使用，具有应用高温大曲发酵酒生产的潜力，为该酶的进一步研究奠定了基础。
- 解淀粉芽孢杆菌 /
- 纤维素酶 /
- 纯化 /
- 酶学性质
Abstract: Objects: This research was performed based on taking one Bacillus amyloliquefaciens BA-DES4 producing the cellulose as the material, and the enzymatic property of its cellulose was researched upon the purification. Methods: This research applied the ammonium sulfate fractional precipitation and SephadexG-75 gel filtration chromatography method to carry out the separation and purification to the cellulose it produced. Determined its molecular weight by means of polyacrylamide gel electrophoresis (SDS-PAGE), and carried out the research of enzymatic property to enzyme solution of cellulase upon purification. Results: The cellulase system components could be obtained in zymotic fluid upon separation and purification (endoglucanase), and its specific activity was 51.08 U/mg through carrying out the measurement of enzyme activity to purified electrophoresis endoglucanase. It was identified that its molecular weight was 22.4 kDa. The primary research of enzymatic property indicated that the optimal reaction temperature and optimal pH for this enzyme were 65 ℃ and 6.0 respectively with a high stability when pH was 5.0～7.0 and temperature was 55～65 ℃. The activation of Mn²⁺ to cellulase activities was significant with the highest inhibiting effect of Cu²⁺. Conclusion: This bacterial strain can be used as the potential strain for producing the endoglucanase with the enzymatic activities of endoglucanase can be promoted under the effects of Mn²⁺, Fe²⁺. Meanwhile, it can work under the high temperature and acid environment along with participating in the cellulose of high effective degradation and high-temperature acid environment so as to improve the production efficiency. The glucose to be decomposed can be used for fermentation with a potential of producing the fermented wine by applying the high-temperature Daqu, which has laid a foundation for further studying this cellulase.
- Bacillus amyloliquefaciens /
- cellulase /
- purification /
- enzymatic property

HTML全文

图 1 硫酸铵分级沉淀相对酶活

Figure 1. Ammonium sulfate graded precipitation relative enzyme activity

下载: 全尺寸图片幻灯片

图 2 凝胶过滤层析洗脱图谱

Figure 2. Elution spectrum of gel filtration chromatography

下载: 全尺寸图片幻灯片

图 3 粗制纤维素酶的SDS-PAGE凝胶电泳分析

Figure 3. SDS-PAGE gel electrophoresis analysis of crude cellulase

注：泳道1：低分子量蛋白Marker；泳道2：出发菌株BA-2；泳道3：诱变菌株BA-DES4；泳道4、5：平行试验；kDa：分子量。

下载: 全尺寸图片幻灯片

图 4 纯化纤维素酶的SDS-PAGE凝胶电泳分析

Figure 4. SDS-PAGE gel electrophoresis analysis of purified cellulase

注：MK：低分子量蛋白Marker；kDa：分子量；CMCase：内切葡聚糖酶。

下载: 全尺寸图片幻灯片

图 5 纤维素酶最适温度

Figure 5. Optimum temperature of cellulase

下载: 全尺寸图片幻灯片

图 6 纤维素酶最适pH

Figure 6. Optimum pH of cellulase

下载: 全尺寸图片幻灯片

图 7 纤维素酶热稳定性

Figure 7. Thermal stability of cellulase

下载: 全尺寸图片幻灯片

图 8 纤维素酶pH稳定性

Figure 8. pH stability of cellulase

下载: 全尺寸图片幻灯片

图 9 金属离子对酶促反应的影响

Figure 9. The effect of metal ions on enzymatic reactions

下载: 全尺寸图片幻灯片

表 1 蛋白质标准曲线不同溶液含量配制表

Table 1. Protein standard curve different solution content configuration table

试剂	编号
试剂	1	2	3	4	5	6
牛血清蛋白（mL）	0	0.2	0.4	0.6	0.8	1.0
蒸馏水（mL）	1.0	0.8	0.6	0.4	0.2	0
考马斯亮蓝G-250染色液（mL）	5	5	5	5	5	5
蛋白含量（mg）	0	0.02	0.04	0.06	0.08	0.10

下载: 导出CSV

表 2 菌株BA-2纤维素酶分离纯化结果

Table 2. Separation and purification results of cellulase from strain BA-2

指标	总酶活（U）	总蛋白（mg）	比酶活（U/mg）	纯化倍数	回收率（%）
粗酶液（BA-2）	196.62±0.23^a	18.57±0.12^a	10.59±0.07^c	1	100
硫酸铵沉淀	102.74±0.41^b	7.46±0.09^b	13.78±0.11^b	1.31	40.17
SephadexG-75	75.38±0.36^c	2.83±0.06^c	26.63±0.08^a	2.52	15.24
注：表中数据为平均值±标准差；同列不同小写字母表示差异显著（P<0.05），表3同。

下载: 导出CSV

表 3 菌株BA-DES4纤维素酶分离纯化结果

Table 3. Separation and purification results of cellulase from strain BA-DES4

指标	总酶活（U）	总蛋白（mg）	比酶活（U/mg）	纯化倍数	回收率（%）
粗酶液（BA-DES4）	319.51±0.25^a	23.35±0.11^a	13.68±0.06^c	1	100
硫酸铵沉淀	168.84±0.39^b	6.58±0.08^b	25.66±0.12^b	1.88	28.18
SephadexG-75	126.69±0.28^b	2.49±0.10^c	51.08±0.09^a	3.73	10.62

下载: 导出CSV

参考文献(29)

[1]	宫秀杰, 钱春荣, 于洋, 等. 近年纤维素降解菌株筛选研究进展[J]. 纤维素科学与技术,2021,29(2):68−77. [GONG X J, QIAN C R, YU Y, et al. Progress on screening of cellulose degrading strains in recent years[J]. Journal of Cellulose Science and Technology,2021,29(2):68−77. doi: 10.16561/j.cnki.xws.2021.02.07
[2]	徐忠, 冯昕宁, 高阳, 等. 改性纤维素在食品和调味品加工中应用进展[J]. 中国调味品,2021,46(1):177−179. [XU Z, FENG X N, GAO Y, et al. Application progress of modified cellulose in food and condiment processing[J]. China Condiment,2021,46(1):177−179. doi: 10.3969/j.issn.1000-9973.2021.01.036
[3]	邢飞, 钱嘉君, 刘慧, 等. 食品接触用再生纤维素薄膜的应用进展与法规研究[J]. 食品安全质量检测学报,2021,12(7):2607−2612. [XING F, QIAN J J, LIU H, et al. Research on the application progress and regulations of regenerated cellulose film for food contact[J]. Journal of Food Safety & Quality,2021,12(7):2607−2612.
[4]	章沙沙, 徐健峰, 柳增善. 纤维素降解菌的研究与应用进展[J]. 工业微生物,2021,51(2):46−52. [ZHANG S S, XU J F, LIU Z S. Progress in research and application of cellulose-degrading microorganisms[J]. Industrial Microbiology,2021,51(2):46−52. doi: 10.3969/j.issn.1001-6678.2021.02.007
[5]	江小妹, 林春伟, 王魁云, 等. 团头鲂肠道菌株MA35产纤维素酶分离纯化及性质分析[J]. 上海海洋大学学报,2020,29(2):313−320. [JIANG X M, LIN C W, WANG K Y, et al. Isolation, purification and characterization of cellulase produced from Aspergillus niveus MA35 in the gut of the Megalobrama amblycephala[J]. Joumal of Shanghai Oceam University,2020,29(2):313−320. doi: 10.12024/jsou.20190302564
[6]	王世伟, 王卿惠, 马兆魁, 等. Fusarium oxysporim M1菌株的rDNA ITS鉴定及其纤维素酶活性及纯化研究[J]. 微生物学杂志,2018,38(2):55−62. [WANG S W, WANG Q H, MA Z K, et al. rDNA its identification, its cellulase activity and purification of Fusarium oxysporum strain M1[J]. Journal of Microbiology,2018,38(2):55−62.
[7]	FAN X, LIANG Y, LI F, et al. Integrated purification of a nanobody using ammonium sulfate precipitation and capto MMC[J]. Journal of Chemical Technology & Biotechnology,2020,95(1):246−254.
[8]	张轶群, 林洪, 李振兴, 等. 虾过敏原蛋白纯化中硫酸铵沉淀法的改进[J]. 食品与药品,2008(11):50−52. [ZHANG Y Q, LIN H, LI Z X, et al. Improvement of shrimp (Penaeus vannamei) allergens purification by ammonium sulfate precipitation[J]. Food and Drug,2008(11):50−52. doi: 10.3969/j.issn.1672-979X.2008.06.017
[9]	吴静. 高产纤维素酶霉菌的筛选及纤维素酶系的分离纯化[D]. 贵阳: 贵州大学, 2020 WU J. Isolation of molds with high-activity of cellulase and the purification of cellulase system[D]. Guiyang: Guizhou University, 2020.
[10]	纪莹莹. 高温降解纤维素菌株的筛选及酶学性质的研究[D]. 沈阳: 沈阳农业大学, 2019 JI Y Y. Cellulase-producing actinomycetes with the capacity of high-temperature tolerance and characterization of the enzymatic properties of its cellulase[D]. Shenyang: Shenyang Agricultural University, 2019.
[11]	OKAWARAY, HIRANO H, KIMURA A, et al. Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in phos-tag SDS-PAGE[J]. Journal of Proteomics,2021,231:104005. doi: 10.1016/j.jprot.2020.104005
[12]	徐亚军, 李珂, 刘珂珂, 等. 大豆根瘤内生菌全细胞可溶性蛋白SDS-PAGE电泳图谱分析[J]. 食品研究与开发,2019,40(11):159−165. [XU Y J, LI K, LIU K K, et al. Analysis on whole cell soluble protein electrophoresis of SDS-PAGE electrophoresis profiles of endophytic bacteria from soybean nodules[J]. Food Research and Development,2019,40(11):159−165.
[13]	郑斌, 王舒, 王子迎, 等. 蓝莓酵素中蛋白质和蛋白酶含量测定[J]. 吉林农业,2018(19):68−70. [ZHENG B, WANG S, WANG Z Y, et al. Determination of protein and protease content in blueberry enzyme[J]. Agriculture of Jilin,2018(19):68−70.
[14]	温冬灼, 张智, 魏罡, 等. 纤维素降解芽孢杆菌筛选及产酶条件优化[J]. 中南林业科技大学学报,2022,42(2):169−180. [WEN D Z, ZHI Z, WEI G, et al. Screening of cellulose-degrading bacillus and optimization of enzyme production conditions[J]. Journal of Central South University of Forestry & Technology,2022,42(2):169−180. doi: 10.14067/j.cnki.1673-923x.2022.02.018
[15]	黄婉玉, 曹炜, 李菁, 等. 考马斯亮蓝法测定果汁中蛋白质的含量[J]. 食品与发酵工业,2009,35(5):160−162. [HUANG W Y, CAO W, LI J, et al. Determination of protein in fruit juices by kormas brilliant blue method[J]. Food and Fermentation Industries,2009,35(5):160−162. doi: 10.13995/j.cnki.11-1802/ts.2009.05.015
[16]	ZHANG T, ZHANG H, WEI M, et al. Effects of enzymatic treatment on the physicochemical properties and antioxidant activity of hawthorn pectin[J]. Materials Today Communications,2022,30:103225. doi: 10.1016/j.mtcomm.2022.103225
[17]	CUI L, MEDDEB-MOUEIHI F, LAFRAMBOISE F, et al. Effect of commercial cellulases and refining on kraft pulp properties: Correlations between treatment impacts and enzymatic activity components[J]. Carbohydrate Polymers,2015,115:193−199. doi: 10.1016/j.carbpol.2014.08.076
[18]	GENG D H, LIN Z, LIU L, et al. Effects of ultrasound-assisted cellulase enzymatic treatment on the textural properties and in vitro starch digestibility of brown rice noodles[J]. LWT,2021,146:111543. doi: 10.1016/j.lwt.2021.111543
[19]	KIELISZEK M, POBIEGA K, PIWOWAREK K, et al. Characteristics of the proteolytic enzymes produced by lactic acid bacteria[J]. Molecules,2021,26(7):1858. doi: 10.3390/molecules26071858
[20]	蔡正安, 刘素纯, 刘仲华, 等. 茯砖茶中冠突散囊菌纤维素酶的酶学性质研究[J]. 茶叶科学,2010,30(1):57−62. [CAI Z A, LIU S C, LIU Z H, et al. Cellulase enzymatic property of Eurotium cristatum from brick tea[J]. Journal of Tea Science,2010,30(1):57−62. doi: 10.3969/j.issn.1000-369X.2010.01.009
[21]	SUI M, LI C, LI G, et al. Study on enzyme properties of Bacillus subtilis LC-9 multifunction cellulose[C]//IOP Conference Series: Earth and Environmental Science. IOP Publishing, 2018, 170(2): 022106.
[22]	韩硕, 赵岩, 宋天顺, 等. 纤维素分解菌的筛选和酶学性质分析[J]. 西北农业学报,2013,22(4):172−177. [HAN S, ZHAO Y, SONG T S, et al. Isolation of cellulose-decomposing strains and analysis of enzymatic characteristics of cellulase[J]. Acta Agriculturae Boreali-occidentalis Sinica,2013,22(4):172−177. doi: 10.7606/j.issn.1004-1389.2013.04.030
[23]	王婷婷, 宋星, 李燕. 文蛤纤维素酶分离纯化及性质[J]. 食品与生物技术学报,2018,37(12):1324−1329. [WANG T T, SONG X, LI Y. Isolation, purification and characterization of cellulase from Meretrix meretrix L doi: 10.3969/j.issn.1673-1689.2018.12.015 J]. Journal of Food Science and Biotechnology,2018,37(12):1324−1329. doi: 10.3969/j.issn.1673-1689.2018.12.015
[24]	葛飞, 石贝杰, 唐尧, 等. 康氏木霉诱变菌株纤维素酶系的分离纯化与酶学特性研究[J]. 现代食品科技,2015,31(12):149−155, 184. [GE F, SHI B J, TANG X, et al. Isolation, purification, and enzymatic properties of cellulase system in mutant strain Trichoderma koningii SG0026[J]. Modern Food Science and Technology,2015,31(12):149−155, 184.
[25]	戴圻霏. 低温纤维素酶高产菌筛选和酶学性质研究[D]. 长春: 吉林农业大学, 2020 DAI K F. Breeding of low-temperature cellulase high-producing bacteria and study on its properties[D]. Changchun: Jilin Agricultural University, 2020.
[26]	董明杰, 杨云娟, 唐湘华, 等. 脂环酸芽孢杆菌D-1的耐盐内切葡聚糖酶基因克隆、表达与酶学性质[J]. 微生物学报,2016,56(10):1626−1637. [DONG M J, YANG Y J, TANG X H, et al. Gene cloning, heterologous expression and enzyme characterizations of halo-tolerant endoglucanase from Alicyclobacillus D-1[J]. Acta Microbiologica Sinica,2016,56(10):1626−1637.
[27]	常鑫园, 谢占玲, 张凤梅, 等. 镰刀菌Q7-31T内切葡聚糖酶Egn21的分离纯化及酶学性质[J]. 微生物学报,2017,57(1):33−42. [CHANG X Y, XIE Z L, ZHANG F M, et al. Purification and characterization of endoglucanase Egn21 from Fusarium sp. Q7-31T[J]. Acta Microbiologica Sinica,2017,57(1):33−42.
[28]	崔海洋, 程仕伟, 黄田红, 等. 产纤维素酶的解淀粉芽孢杆菌分离鉴定及酶学性质研究[J]. 食品科学技术学报,2014,32(3):43−47, 53. [CUI H Y, CHENG S W, HUANG T H, et al. Screening and identifying of Bacillus amyloliquefaciens for cellulase production and its enzymatic characters[J]. Journal of Food Science and Technology,2014,32(3):43−47, 53. doi: 10.3969/j.issn.2095-6002.2014.03.008
[29]	孙会刚, 徐慧敏, 黄天姿, 等. 产酸性纤维素酶细菌的筛选鉴定及其酶学性质[J]. 食品科技,2021,46(7):55−59. [SUN H G, XU H M, HUANG T Z, et al. Screening, isolating and enzymatic properties of bacterium producing acidic cellulase[J]. Food Science and Technology,2021,46(7):55−59.