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海洋细菌来源 β-琼胶酶的生物信息学分析与高效制备

尤钰娴 解文嫣 班宵逢 孔昊存 李才明 李兆丰

尤钰娴, 解文嫣, 班宵逢, 孔昊存, 李才明, 李兆丰. 海洋细菌来源 β-琼胶酶的生物信息学分析与高效制备[J]. 南方水产科学, 2022, 18(2): 1-12. doi: 10.12131/20210330
引用本文: 尤钰娴, 解文嫣, 班宵逢, 孔昊存, 李才明, 李兆丰. 海洋细菌来源 β-琼胶酶的生物信息学分析与高效制备[J]. 南方水产科学, 2022, 18(2): 1-12. doi: 10.12131/20210330
YOU Yuxian, XIE Wenyan, BAN Xiaofeng, KONG Haocun, LI Caiming, LI Zhaofeng. Bioinformatics analysis and efficient preparation of β-agarase from marine bacteria[J]. South China Fisheries Science, 2022, 18(2): 1-12. doi: 10.12131/20210330
Citation: YOU Yuxian, XIE Wenyan, BAN Xiaofeng, KONG Haocun, LI Caiming, LI Zhaofeng. Bioinformatics analysis and efficient preparation of β-agarase from marine bacteria[J]. South China Fisheries Science, 2022, 18(2): 1-12. doi: 10.12131/20210330

海洋细菌来源 β-琼胶酶的生物信息学分析与高效制备

doi: 10.12131/20210330
基金项目: 国家重点研发计划项目 (2019YFD0901901)
详细信息
    作者简介:

    尤钰娴 (1996—),女,博士研究生,研究方向为淀粉生物技术。E-mail: yuxianyou@stu.jiangnan.edu.cn

    通讯作者:

    李兆丰 (1979—),男,教授,博士,从事淀粉生物技术研究。E-mail: zfli@jiangnan.edu.cn

  • 中图分类号: Q 814.9

Bioinformatics analysis and efficient preparation of β-agarase from marine bacteria

  • 摘要: 为丰富优质琼胶酶品类,充分利用海藻资源,实现功能性琼胶寡糖的高效制备,采用基因组挖矿技术挖掘得到一个来源于海洋细菌——淡黄色噬琼胶菌 (Agarivorans gilvus) WH0801的β-琼胶酶 (β-AGA酶),利用生物信息学分析对该酶的理化性质和结构特征进行预测,发现该酶为非分泌性蛋白。在此基础上,采用分子生物学手段引入信号肽,实现了该酶在大肠杆菌 (Escherichia coli) 中的胞外表达,并通过发酵调控策略优化提高其生产效率。结果表明,采用种龄5 h的种子培养液进行发酵,发酵出发培养基为TB (pH 7.0),碳源为6 g·L−1的果糖,氮源为30 g·L−1的酵母提取液Ⅱ,于25 ℃培养2 h后加入终浓度为0.025 mmol·L−1的IPTG诱导48 h,此条件下发酵所得酶活为16.72 U·mL−1,相比初始酶活提高了约5倍,证明β-AGA酶具有良好的工业应用潜力。
  • 图  1  β-琼胶酶基因系统进化树

    Figure  1.  Phylogenetic tree of β-agarase

    图  2  β-AGA酶二级结构预测

    Figure  2.  Secondary structure prediction of β-AGAase

    图  3  β-AGA酶三级结构预测

    Figure  3.  Tertiary structure prediction of β-AGAase

    图  4  β-AGA酶亲水性/疏水性预测

    Figure  4.  Hydropathicity/hydrophobicity prediction of β-AGAase

    图  5  β-AGA酶亚细胞定位预测

    Figure  5.  Subcellular localization prediction of β-AGAase

    图  6  β-AGA酶信号肽分析

    Figure  6.  Signal peptide prediction of β-AGAase

    图  7  β-AGA酶功能结构域预测

    Figure  7.  Conserved domain prediction of β-AGAase

    图  8  表达载体pET-20b(+)/β-aga的构建

    Figure  8.  Construction of expression plasmid pET-20b(+)/β-aga

    图  9  重组β-AGA酶的种子生长曲线 (a) 和接种时间对重组β-AGA酶发酵的影响 (b)

    Figure  9.  Seed growth curve of β-AGAase (a) and effect of inoculation time on fermentation (b) of recombinant β-AGAase

    图  10  发酵培养基类型对重组β-AGA酶发酵的影响

    Figure  10.  Effect of culture medium on fermentation of recombinant β-AGAase

    图  11  IPTG浓度和添加时间对重组β-AGA酶发酵的影响

    Figure  11.  Effects of IPTG concentration and additive time on fermentation of recombinant β-AGAase

    图  12  时间、温度和pH对重组β-AGA酶发酵的影响

    Figure  12.  Effects of time, temperature and initial pH on fermentation of recombinant β-AGAase

    图  13  碳源种类和添加量对重组β-AGA酶发酵的影响

    Figure  13.  Effect of carbon sources and their additive amount on fermentation of recombinant β-AGAase

    表  1  β-AGA酶的氨基酸组成

    Table  1.   Amino acid composition of β-AGAase

    氨基酸
    Amino acid
    数量
    Amount
    占比
    Proportion/%
    丙氨酸 Alanine 82 8.6
    精氨酸 Arginine 27 2.8
    天冬酰胺 Aspartic acid 51 5.3
    天冬氨酸 Asparagine 81 8.5
    半胱氨酸 Cysteine 1 0.1
    谷氨酰胺 Glutamine 38 4.0
    谷氨酸 Glutamic acid 54 5.7
    甘氨酸 Glycine 73 7.6
    组氨酸 Histidine 15 1.6
    异亮氨酸 Isoleucine 40 4.2
    亮氨酸 Leucine 70 7.3
    赖氨酸 Lysine 57 6.0
    蛋氨酸 Methionine 20 2.1
    苯丙氨酸 Phenylalanine 48 5.0
    脯氨酸 Proline 40 4.2
    丝氨酸 Serine 74 7.7
    苏氨酸 Threonine 56 5.9
    色氨酸 Tryptophane 26 2.7
    酪氨酸 Tyrosine 39 4.1
    缬氨酸 Valine 63 6.6
    下载: 导出CSV

    表  2  有机氮源对重组β-AGA酶发酵的影响

    Table  2.   Effect of organic nitrogen source on fermentation of recombinant β-AGAase U·mL−1

    氮源种类
    Nitrogen source
    添加量 Additive amount/(g·L−1)
    0612182430
    1.37±0.03a2.14±0.02b2.94±0.03c3.79±0.04d4.48±0.03e5.83±0.02f
    1.37±0.03a3.03±0.02b4.26±0.04c6.22±0.03d8.04±0.04e9.21±0.09f
    1.37±0.03a3.28±0.02b4.35±0.02c6.19±0.03d8.47±0.06e8.64±0.07e
    1.37±0.03a3.65±0.03b4.80±0.02c6.73±0.04d8.51±0.05e9.58±0.07f
    1.37±0.03a4.10±0.02b6.67±0.05c8.92±0.06d10.44±0.05e11.87±0.08f
    1.37±0.03a4.76±0.02b7.59±0.04c10.15±0.05d12.48±0.06e15.33±0.10f
    1.37±0.03a4.28±0.02b7.02±0.04c9.58±0.04d11.57±0.07e13.25±0.08f
    1.37±0.03a3.82±0.01b5.04±0.03c6.96±0.03d8.81±0.04e9.94±0.03f
    注:数值为 3 次平行实验的平均值,同一行中不同上标字母表示显著性差异(P<0.05)。
    ①. 分子级蛋白胨;②. 蛋白胨(鱼粉);③. 牛肉浸膏;④. 大豆蛋白胨;Ⅰ. 分子级酵母提取物;Ⅱ. 酵母提取物 H07014;Ⅲ. 酵母提取物 H07002;Ⅳ. 酵母浸膏。 Note: Each value represents the average value of three independent measurements, and those with different letters within the same row are significantly different (P<0.05).
    ①. Molecular level peptone; ②. Peptone (fish meal); ③. Beef extract; ④. Soya peptone; I. Molecular level yeast extract; II. Yeast extract H07014; III. Yeast extract H07002; IV. Yeast extract.
    下载: 导出CSV

    表  3  正交试验因素和水平表

    Table  3.   Factors and levels in orthogonal array design

    水平
    Level
    A:碳源添加量
    Carbon source
    additive amount/
    (g·L−1)
    B:发酵温度
    Fermentation
    temperature/
    C:IPTG添加时间
    IPTG additive
    time/h
    15201.0
    26251.5
    37302.0
    下载: 导出CSV

    表  4  发酵条件优化正交试验结果

    Table  4.   Orthogonal array design layout and experimental results

    试验号
    Test No.
    A:碳源
    添加量
    Carbon source
    additive amount/
    (g·L−1)
    B:发酵
    温度
    Fermentation
    temperature/
    C:IPTG
    添加时间
    IPTG additive
    time/h
    酶活
    Enzyme
    activity/
    (U·mL−1)
    11 (5)1 (20)1 (1.0)13.99±0.04e
    212 (25)2 (1.5)14.15±0.05f
    313 (30)3 (2.0)13.02±0.07b
    42 (6)1215.63±0.06g
    522316.72±0.06h
    623113.39±0.04c
    73 (7)1313.64±0.05d
    832114.18±0.03f
    933212.67±0.02a
    K113.7214.4213.85
    K215.2515.0214.15
    K313.5013.0314.46
    R1.751.990.61
    注:同一列中不同上标字母表示显著性差异 (P<0.05)。 Note: Different superscript letters within the same column indicate significant difference (P<0.05).
    下载: 导出CSV
  • [1] 慕欣. 三株海洋新菌的鉴定及细菌O17~T和TS1~T的琼胶酶研究[D]. 济南: 山东大学, 2017: 1.
    [2] 王露楠, 杨少玲, 戚勃, 等. 3种改性方法对琼胶理化性质的影响[J]. 南方水产科学, 2021, 17(2): 97-103. doi: 10.12131/20200197
    [3] 戚勃, 杨贤庆, 李来好, 等. 琼胶寡糖对冻虾仁和罗非鱼片品质的影响[J]. 南方水产科学, 2012, 8(6): 73-79.
    [4] XU X Q, SU B M, XIE J S, et al. Preparation of bioactive neoagaroligosaccharides through hydrolysis of Gracilaria lemaneiformi sagar: a comparative study[J]. Food Chem, 2018, 240(1): 330-337.
    [5] LEE M H, JANG J H, YOON G Y, et al. Neoagarohexaose-mediated activation of dendritic cells via Toll-like receptor 4 leads to stimulation of natural killer cells and enhancement of antitumor immunity[J]. Bmb Rep, 2017, 50(5): 263-268. doi: 10.5483/BMBRep.2017.50.5.014
    [6] WANG W, LIU P, HAO C, et al. Neoagaro-oligosaccharide monomers inhibit inflammation in LPS-stimulated macrophages through suppression of MAPK and NF-κB pathways[J]. Sci Rep, 2017, 7: 44252. doi: 10.1038/srep44252
    [7] JOO H S, JE-HYEON L, JOO K E, et al. Anti-obesity and anti-diabetic effect of neoagarooligosaccharides on high-fat diet-induced obesity in mice[J]. Mar Drugs, 2017, 15(4): 90-101. doi: 10.3390/md15040090
    [8] HONG S J, LEE J H, KIM E J, et al. In vitro and in vivo investigation for biological activities of neoagarooligosaccharides prepared by hydrolyzing agar with β-agarase[J]. Biotechnol Bioeng, 2017, 22(4): 489-496.
    [9] 杨绍青, 刘学强, 刘瑜, 等. 酶法制备几种功能性低聚糖的研究进展[J]. 生物产业技术, 2019(4): 10.
    [10] ZHANG N, HOU E, SONG J, et al. Neoagarotetraose-modulated gut microbiota and alleviated gut inflammation in antibiotic treatment mice[J]. Food Agr Immunol, 2017, 28(6): 1408-1423. doi: 10.1080/09540105.2017.1346063
    [11] YANG M, ZHANG Z L, HE Y, et al. Study on the structure characterization and moisturizing effect of Tremella polysaccharide fermented from GCMCC5.39[J]. Food Sci Hum Well, 2021, 10(4): 471-479. doi: 10.1016/j.fshw.2021.04.009
    [12] 李静. 琼脂降解菌的筛选及其琼胶酶的研究[D]. 无锡: 江南大学, 2020: 6.
    [13] KAZLOWSKI B, PAN C L, KO Y T. Monitoring and preparation of neoagaro- and agaro-oligosaccharide products by high performance anion exchange chromatography systems[J]. Carbohyd Polym, 2015, 122: 351-358. doi: 10.1016/j.carbpol.2014.09.003
    [14] ZHANG W, XU J, LIU D, et al. Characterization of an α-agarase from Thalassomonas sp. LD5 and its hydrolysate[J]. Appl Microbiol Biot, 2018, 102(5): 2203-2212. doi: 10.1007/s00253-018-8762-6
    [15] 林福娣. 新琼寡糖的酶法制备及其生物活性研究[D]. 厦门: 华侨大学, 2020: 2-5.
    [16] LI J, XIE M, GAO Y. Identification and biochemical characterization of a novel exo-type β-agarase Aga3463 from an Antarctic Pseudoalteromonas sp. strain[J]. Int J Biol Macromol, 2019, 129: 162-170.
    [17] CHEN X, HOU Y, JIN M, et al. Expression and characterization of a novel thermostable and pH-stable β-agarase from deep-sea bacterium Flammeovirga sp. OC4[J]. J Agric Food Chem, 2016, 64(38): 7251-7258. doi: 10.1021/acs.jafc.6b02998
    [18] CHEN Y, WU H, WANG G, et al. Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring[J]. J Biosci Bioeng, 2019, 127(4): 403-410. doi: 10.1016/j.jbiosc.2018.10.001
    [19] CUI X, JIANG Y, CHANG L, et al. Heterologous expression of an agarase gene in Bacillus subtilis, and characterization of the agarase[J]. Int J Biol Macromol, 2018, 120: 657-664. doi: 10.1016/j.ijbiomac.2018.07.118
    [20] LI R, YING X, CHEN Z, et al. Expression and characterization of a GH16 family β-agarase derived from the marine bacterium Microbulbifer sp. BN3 and its efficient hydrolysis of agar using raw agar-producing red seaweeds Gracilaria sjoestedtii and Gelidium amansii as substrates[J]. Catalysts, 2020, 10(8): 885.
    [21] LEE J, HONG S, LEE C, et al. Production of ethanol from agarose by unified enzymatic saccharification and fermentation in recombinant yeast[J]. J Microbiol Biotech, 2019, 29(4): 625-632. doi: 10.4014/jmb.1902.02012
    [22] LI R, CHEN Z, YING X, et al. A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris[J]. Int J Biol Macromol, 2018, 119: 1164-1170.
    [23] 李才明, 黄敏, 石建中, 等. 金属离子协同氨基酸提高重组β-环糊精葡萄糖基转移酶在枯草芽孢杆菌中的表达[J]. 食品与发酵工业, 2016, 42(7): 1-8.
    [24] LEE D, PARK G, KIM N, et al. Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate[J]. Biotechnol Lett, 2006, 28(23): 1925-1932. doi: 10.1007/s10529-006-9171-y
    [25] DONG J, TAMARU Y, ARAKI T. A unique β-agarase, AgaA, from a marine bacterium, Vibrio sp. strain PO-303[J]. Appl Microbiol Biot, 2007, 74(6): 1248. doi: 10.1007/s00253-006-0781-z
    [26] CHEN X, LIN H, JIN M, et al. Characterization of a novel alkaline β-agarase and its hydrolysates of agar[J]. Food Chem, 2019, 295(15): 311-319.
    [27] 班宵逢. 静电相互作用对淀粉分支酶热稳定性影响的研究[D]. 无锡: 江南大学, 2017: 50.
    [28] 杨平, 张边江, 王立科, 等. 豆科家族中的木糖异构酶基因分析[J]. 食品与发酵工业, 2020, 46(19): 23-27.
    [29] 杨艳北, 许晶, 沈城辉, 等. N-酰基高丝氨酸内酯酶的生物信息学分析[J]. 甘肃农业科技, 2021, 52(2): 31-37.
    [30] 李兆丰. 软化类芽孢杆菌α-环糊精葡萄糖基转移酶在大肠杆菌中的表达及其产物特异性分析[D]. 无锡: 江南大学, 2009: 22-25.
    [31] LIU J, LIU Z, JIANG C, et al. Biochemical characterization and substrate degradation mode of a novel α-agarase from Catenovulum agarivorans[J]. J Agric Food Chem, 2019, 67(37): 10373-10379. doi: 10.1021/acs.jafc.9b03073
    [32] DONG Q, RUAN L W, SHI H. A β-agarase with high pH stability from Flammeovirga sp. SJP92[J]. Carbohydr Res, 2016, 432(2): 1-8.
    [33] 文霞, 周少璐, 杨秀茳, 等. 海洋微生物多糖降解酶的研究进展[J]. 生物技术通报, 2016, 32(11): 38-46.
    [34] 王晨, 赵雨佳, 李春, 等. 动态转录调控微生物代谢途径研究进展[J]. 化工进展, 2019, 38(9): 4238-4246.
    [35] 冉凡娇, 孙谧, 包静, 等. Bohai sea-9145重组脂肪酶基因工程菌的发酵表达条件优化[J]. 食品工业科技, 2016, 37(16): 178-183.
    [36] 韩来闯. 生物元件的挖掘、改造及在基因表达调控系统中的应用[D]. 无锡: 江南大学, 2020: 6.
    [37] 于平, 陈凯飞, 朱祺, 等. 重组大肠杆菌生物合成γ-氨基丁酸的发酵条件优化[J]. 中国食品学报, 2018, 18(6): 112-120.
    [38] ZHU Y, ZHAO R, XIAO A, et al. Characterization of an alkaline β-agarase from Stenotrophomonas sp. NTa and the enzymatic hydrolysates[J]. Int J Biol Macromol, 2016, 86: 525-534. doi: 10.1016/j.ijbiomac.2016.01.106
    [39] RAMOSA K R M, VALDEHUESAA K N G, NISOLA G M, et al. Identification and characterization of a thermostable endolytic β-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C[J]. New Biotechnol, 2018, 40: 261-267. doi: 10.1016/j.nbt.2017.09.006
    [40] AN K, SHI X, CUI F. Characterization and overexpression of a glycosyl hydrolase family 16 beta-agarase YM01-1 from marine bacterium Catenovulum agarivorans YM01T[J]. Protein Expres Purif, 2018, 143: 1-8. doi: 10.1016/j.pep.2017.10.002
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  • 收稿日期:  2021-11-03
  • 修回日期:  2021-12-27
  • 录用日期:  2022-01-10
  • 网络出版日期:  2022-01-28
  • 刊出日期:  2022-04-01

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