高效褐藻胶降解菌的筛选与产酶条件优化

doi:10.13209/j.0479-8023.2017.122

北京大学学报自然科学版 ›› 2017, Vol. 53 ›› Issue (6): 1115-1121.DOI: 10.13209/j.0479-8023.2017.122

高效褐藻胶降解菌的筛选与产酶条件优化

李婷婷^1,², 陈倩², 石萍¹, 耿旭³()

1. 深圳市重金属污染控制与资源化重点实验室, 北京大学深圳研究生院环境与能源学院, 深圳 518055
2. 北京市新型污水深度处理工程技术研究中心, 北京大学环境工程系, 北京 100871
3. 中美食品安全联合研究中心, 西北农林科技大学, 杨凌 712100

收稿日期:2016-05-12 修回日期:2016-05-31 出版日期:2017-11-20 发布日期:2017-11-20
基金资助:
北京市科技专项(Z161100005016034)资助

Screening of Alginate Degrading Bacteria and Optimization of Its Enzyme-Producing Conditions

Tingting LI^1,², Qian CHEN², Ping SHI¹, Shu GENG³()

1. Shenzhen Key Laboratory for Heavy Metal Pollution Control and Reutilization, School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen 518055
2. Beijing Engineering Research Center for Advanced Wastewater Treatment, Department of Environmental Engineering , Peking University, Beijing 100871
3. Sino-US Joint Food Safety Research Center, Northwest A&F University, Yangling 712100;

Received:2016-05-12 Revised:2016-05-31 Online:2017-11-20 Published:2017-11-20

摘要/Abstract

摘要：

以海藻酸钠为唯一碳源, 从天然腐烂海带中筛选得到一株高效褐藻胶降解菌株53#, 经形态学观察和16S rRNA鉴定, 确定为类芽孢杆菌Paenibacillus agaridevorans。采用正交试验方法, 以pH、温度、NaCl浓度和海藻酸钠初始浓度为影响因素, 对该菌株的产酶条件进行优化, 得到53#菌的最佳产酶条件: pH=8, 25℃, NaCl浓度15 g/L, 海藻酸钠初始浓度15 g/L。在最佳产酶条件下, 褐藻胶裂解酶最大酶活可达390.53±17.32 U/mL。筛选得到的类芽孢杆菌Paenibacillus agaridevorans 53#具有易于培养、产酶速度快和酶活力高等优点, 能够实现褐藻胶的高效糖化, 在褐藻生产生物乙醇领域具有潜在利用价值。

关键词: 褐藻胶降解, 褐藻胶裂解酶, 类芽孢杆菌, 生物乙醇

Abstract:

53#, a strain of efficient alginate degrading bacteria, is isolated from rotted kelp using sodium alginate as the only carbon resource. Strain 53# is identified as Paenibacillus agaridevorans based on physiological characteristics and 16S rRNA sequencing. The optimaltion yield condition of strain 53# is identified at pH=8, T=25ºC, NaCl 15 g/L, and sodium alginate 15 g/L by orthogonal experiment and analysis, and the highest enzyme activity is 390.53±17.32 U/mL. Strain 53# has the advantages such as being cultivated easily, producing enzyme fast and having high enzyme activity. It can achieve a high efficiency for saccharification of alginate and thus has potential value to be utilized in the production of bioethanol from brown algae.

Key words: alginate degradation, alginate lyase, Paenibacillus, biofuel

李婷婷, 陈倩, 石萍, 耿旭. 高效褐藻胶降解菌的筛选与产酶条件优化[J]. 北京大学学报自然科学版, 2017, 53(6): 1115-1121.

Tingting LI, Qian CHEN, Ping SHI, Shu GENG. Screening of Alginate Degrading Bacteria and Optimization of Its Enzyme-Producing Conditions[J]. Acta Scientiarum Naturalium Universitatis Pekinensis, 2017, 53(6): 1115-1121.

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链接本文: https://xbna.pku.edu.cn/CN/10.13209/j.0479-8023.2017.122

https://xbna.pku.edu.cn/CN/Y2017/V53/I6/1115

图/表 8

参考文献 25

[1]	Daroch M, Geng S, Wang G.Recent advances in liquid biofuel production from algal feedstocks. Applied Energy, 2013, 102: 1371-1381
[2]	Horn S, Aasen I, Østgaard K.Production of ethanol from mannitol by Zymobacter palmae. Journal of Industrial Microbiology and Biotechnology, 2000, 24(1): 51-57
[3]	陈姗姗, 潘诗翰, 董蓉, 等. 褐藻燃料乙醇研究进展及其应用前景. 中国酿造, 2011, 30(4): 11-15
[4]	Horn S, Aasen I, Østgaard K.Ethanol production from seaweed extract. Journal of Industrial Micro-biology and Biotechnology 2000, 25(5): 249-254
[5]	张洪荣, 王长云, 刘斌, 等. 一种饱和褐藻胶寡糖的制备方法. 中国海洋药物, 2006, 25(3): 1-6
[6]	Wargacki A J, Leonard E, Win M N, et al.An engineered microbial platform for direct biofuel production from brown macroalgae. Science, 2012, 335: 308-313
[7]	Enquist-Newman M, Faust A M E, Bravo D D, et al. Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature, 2014, 505: 239-243
[8]	Wall D, Douglas S, Ferro V, et al.Characterisation of the anticoagulant properties of a range of structu-rally diverse sulfated oligosaccharides. Thrombosis Research, 2001, 103(4): 325-335
[9]	王庭欣, 王庭祥, 庞佳宏. 海带多糖降血糖, 血脂作用的研究. 营养学报, 2007, 29(1): 99-100
[10]	Iwamoto Y, Xu X, Tamura T, et al.Enzymatically depolymerized alginate oligomers that cause cytotoxic cytokine production in human mononuclear cells. Bioscience, Biotechnology, and Biochemistry, 2003, 67(2): 258-263
[11]	Fujihara M, Nagumo T.An influence of the structure of alginate on the chemotactic activity of macrophages and the antitumor activity. Carbohydrate Research, 1993, 243(1): 211-216
[12]	孙丽萍, 薛长湖, 许家超, 等. 褐藻胶寡糖体外清除自由基活性的研究. 中国海洋大学学报(自然科学版), 2005, 35(5): 811-814
[13]	Akiyama H, Endo T, Nakakita R, et al.Effect of depolymerized alginates on the growth of bifidobac-teria. Bioscience, Biotechnology, and Biochemistry, 1992, 56(2): 355-356
[14]	Rahman M M, Inoue A, Tanaka H, et al.Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the common sea hare Aplysia kurodai. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2010, 157(4): 317-325
[15]	Li L, Jiang X, Guan H, et al.Preparation, purification and characterization of alginate oligosaccharides degraded by alginate lyase from Pseudomonas sp. HZJ 216. Carbohydrate Research, 2011, 346(6): 794-800
[16]	Wong T Y, Preston L A, Schiller N L.Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and app-lications. Annual Reviews in Microbiology, 2000, 54(1): 289-340
[17]	胡晓珂. 褐藻胶裂合酶工程化研究与应用[D]. 青岛: 中国海洋大学, 2004
[18]	汪立平, 刘玉佩, 孙晓红, 等. 褐藻胶裂解酶基因在大肠杆菌中的表达及其酶学性质.食品与生物技术学报, 2012(2): 83-88
[19]	李安雪, 汪立平, 赵勇. 重组褐藻胶裂解酶基因工程菌的高密度培养. 食品工业科技, 2013, 34(7): 202-205
[20]	Tavernier M L, Petit E, Delattre C, et al.Production of oligoglucuronans using a monolithic enzymatic microreactor. Carbohydrate Research, 2008, 343(15): 2687-2691
[21]	Qiao M, Ouyang L M.Recombinant Production of alginate lyase for improved stress resistance in plants. Journal of Pure and Applied Microbiology, 2013, 7(3): 1615-1624
[22]	侯士昌, 温少红, 唐志红, 等. 一株高效褐藻酸降解菌的筛选, 鉴定及其发酵条件的优化. 海洋科学, 2014, 38(7): 20-26
[23]	Wang Y, Guo E W, Yu W G, et al.Purification and characterization of a new alginate lyase from a marine bacterium Vibrio sp. Biotechnology Letters, 2013, 35(5): 703-708
[24]	Iwamoto Y, Araki R, Iriyama K I, et al.Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates. Bioscience, Biotech-nology,and Biochemistry, 2001, 65(1): 133-142
[25]	Li J W, Dong S, Song J, et al.Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524. Marine Drugs, 2011, 9(1): 109-123

编号	pH	T/ºC	NaCl浓度/(g·L^-1)	海藻酸钠初始浓度/(g·L^-1)	最大酶活/(U·mL^-1)
1	6	22	15	5	203.93
2	6	25	30	10	257.50
3	6	28	45	15	283.80
4	7	22	30	15	359.54
5	7	25	45	5	132.27
6	7	28	15	10	214.40
7	8	22	45	10	268.33
8	8	25	15	15	390.33
9	8	28	30	5	61.60
k₁	238.41	266.26	259.55	132.93
k₂	242.40	267.03	229.54	247.74
k₃	244.42	189.93	236.13	344.55
极差	6.01	77.10	30.01	211.62

编号	pH	T/ºC	NaCl浓度/(g·L^-1)	海藻酸钠初始浓度/(g·L^-1)	最大酶活/(U·mL^-1)
1	6	22	15	5	203.93
2	6	25	30	10	257.50
3	6	28	45	15	283.80
4	7	22	30	15	359.54
5	7	25	45	5	132.27
6	7	28	15	10	214.40
7	8	22	45	10	268.33
8	8	25	15	15	390.33
9	8	28	30	5	61.60
k₁	238.41	266.26	259.55	132.93
k₂	242.40	267.03	229.54	247.74
k₃	244.42	189.93	236.13	344.55
极差	6.01	77.10	30.01	211.62

酶来源	处理手段	粗酶活/(U · mL^-1)	纯化后酶活/(U · mL^-1)	文献来源
交替单胞菌Alteromonas H4	无	146.45^a		[22]
Vibrio sp. 510-64	复合诱变	289.1^b		[17]
蜡样芽胞杆菌F1-5-10	转基因	12.20^b	26.37	[18-19]
Vibrio sp. QY105	硫酸铵盐析, 琼脂糖凝胶色谱纯化	15.86^b	76.18	[23]
Alteromonas sp strain No. 272	二乙胺基乙基纤维素株层析, 琼脂糖凝胶色谱纯化	8.3 U/mg^b	1122.8 U/mg	[24]
Pseudoalteromonas sp. SM0524	离子交换柱, 凝胶过滤	62.6^b	244.94	[25]
Paenibacillus sp strain 8-5	转基因	30.0^b(胞外酶) 61^b(胞内酶)		[21]
53#	驯化	390.53±17.32^b		本研究

酶来源	处理手段	粗酶活/(U · mL^-1)	纯化后酶活/(U · mL^-1)	文献来源
交替单胞菌Alteromonas H4	无	146.45^a		[22]
Vibrio sp. 510-64	复合诱变	289.1^b		[17]
蜡样芽胞杆菌F1-5-10	转基因	12.20^b	26.37	[18-19]
Vibrio sp. QY105	硫酸铵盐析, 琼脂糖凝胶色谱纯化	15.86^b	76.18	[23]
Alteromonas sp strain No. 272	二乙胺基乙基纤维素株层析, 琼脂糖凝胶色谱纯化	8.3 U/mg^b	1122.8 U/mg	[24]
Pseudoalteromonas sp. SM0524	离子交换柱, 凝胶过滤	62.6^b	244.94	[25]
Paenibacillus sp strain 8-5	转基因	30.0^b(胞外酶) 61^b(胞内酶)		[21]
53#	驯化	390.53±17.32^b		本研究

高效褐藻胶降解菌的筛选与产酶条件优化

Screening of Alginate Degrading Bacteria and Optimization of Its Enzyme-Producing Conditions

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编号	pH	T/ºC	NaCl浓度/ (g·L^-1)	海藻酸钠初始浓度/(g · L^-1)
1	6	22	15	5
2	6	25	30	10
3	6	28	45	15
4	7	22	30	15
5	7	25	45	5
6	7	28	15	10
7	8	22	45	10
8	8	25	15	15
9	8	28	30	5

编号	pH	T/ºC	NaCl浓度/ (g·L^-1)	海藻酸钠初始浓度/(g · L^-1)
1	6	22	15	5
2	6	25	30	10
3	6	28	45	15
4	7	22	30	15
5	7	25	45	5
6	7	28	15	10
7	8	22	45	10
8	8	25	15	15
9	8	28	30	5

编号	pH	T/ºC	NaCl浓度/ (g·L^-1)	海藻酸钠初始浓度/(g · L^-1)
1	6	22	15	5
2	6	25	30	10
3	6	28	45	15
4	7	22	30	15
5	7	25	45	5
6	7	28	15	10
7	8	22	45	10
8	8	25	15	15
9	8	28	30	5

编号	pH	T/ºC	NaCl浓度/ (g·L^-1)	海藻酸钠初始浓度/(g · L^-1)
1	6	22	15	5
2	6	25	30	10
3	6	28	45	15
4	7	22	30	15
5	7	25	45	5
6	7	28	15	10
7	8	22	45	10
8	8	25	15	15
9	8	28	30	5

编号	pH	T/ºC	NaCl浓度/ (g·L^-1)	海藻酸钠初始浓度/(g · L^-1)
1	6	22	15	5
2	6	25	30	10
3	6	28	45	15
4	7	22	30	15
5	7	25	45	5
6	7	28	15	10
7	8	22	45	10
8	8	25	15	15
9	8	28	30	5

编号	pH	T/ºC	NaCl浓度/ (g·L^-1)	海藻酸钠初始浓度/(g · L^-1)
1	6	22	15	5
2	6	25	30	10
3	6	28	45	15
4	7	22	30	15
5	7	25	45	5
6	7	28	15	10
7	8	22	45	10
8	8	25	15	15
9	8	28	30	5