高效产褐藻胶裂解酶菌株产酶条件优化及降解寡糖结构分析
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摘要
为筛选高效产褐藻胶裂解酶菌株,以褐藻胶作为唯一碳源,在海星Asteroidea sp.内脏中筛选出一株产胞外褐藻胶裂解酶的菌株AlgX2,并进行了菌株鉴定、产酶条件优化及降解寡糖结构分析。结果表明:经过菌株形态、生理生化及16S rRNA基因鉴定,该菌株属于盐单胞菌属,命名为Cobetia AlgX2;以3,5-二硝基水杨酸法(DNS)测定的菌株酶活性为指标,对褐藻胶裂解酶菌株产酶活性进行正交试验显示,产褐藻胶裂解酶的菌株最适产酶培养基成分为褐藻胶14 g/L、硫酸铵4 g/L、NaCl 30 g/L、初始pH 6.0,优化产酶培养条件为培养温度22℃、接种量2%、装液量50 mL/250 mL、转速150 r/min;在最优产酶培养条件下进行10 L发酵罐产酶试验显示,发酵液酶活力最高为0.176 U/mL;利用粗酶液酶解制备褐藻胶寡糖,并对寡糖进行HPLC色谱检测显示,样品的聚合度为dp2～dp5。研究表明,Cobetia AlgX2菌株可用于扩大发酵生产,产酶效率高且褐藻胶裂解效果显著。
A alginate-producing lyase bacterial strain AlgX2 was selected in the viscera of starfish Asteroidea sp. using sodium alginate as sole carbon source in order to screen biodegradable alginate lyase microbial strain which was identified to belong to the genus of Cobetia, named as Cobetia AlgX2, upon on the identification of strain morphology, physiology, biochemistry and 16 S rRNA gene. The enzyme activity of the strain determined by the DNS method was used as an indicator, and the culture medium and conditions of the stain were optimized by orthogonal experiment. The results showed that the optimum enzyme-producing medium for the stain was observed under conditions of 14 g/L of alginate, 4 g/L of ammonium sulfate, 30 g/L of NaCl, initial pH of 6.0, culture temperature of 22 ℃, inoculation volume of 2%, liquid volume of 50 mL/250 mL and speed of 150 r/min. Under the optimal conditions, an enlarged volume of 10 L fermentation was applied in culturing and producing the enzyme, with the enzyme activity of 0.176 U/mL. HPLC analysis of alginate oligosaccharides prepared by enzymatic hydrolysis of crude enzyme liquid revealed that the samples had the polymerization degree of dp2-dp5. The findings indicate that this strain can be used to expand fermentation production, high enzyme yielding efficiency and significant alginate cracking effect.
A alginate-producing lyase bacterial strain AlgX2 was selected in the viscera of starfish Asteroidea sp. using sodium alginate as sole carbon source in order to screen biodegradable alginate lyase microbial strain which was identified to belong to the genus of Cobetia, named as Cobetia AlgX2, upon on the identification of strain morphology, physiology, biochemistry and 16 S rRNA gene. The enzyme activity of the strain determined by the DNS method was used as an indicator, and the culture medium and conditions of the stain were optimized by orthogonal experiment. The results showed that the optimum enzyme-producing medium for the stain was observed under conditions of 14 g/L of alginate, 4 g/L of ammonium sulfate, 30 g/L of NaCl, initial pH of 6.0, culture temperature of 22 ℃, inoculation volume of 2%, liquid volume of 50 mL/250 mL and speed of 150 r/min. Under the optimal conditions, an enlarged volume of 10 L fermentation was applied in culturing and producing the enzyme, with the enzyme activity of 0.176 U/mL. HPLC analysis of alginate oligosaccharides prepared by enzymatic hydrolysis of crude enzyme liquid revealed that the samples had the polymerization degree of dp2-dp5. The findings indicate that this strain can be used to expand fermentation production, high enzyme yielding efficiency and significant alginate cracking effect.
引文
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[20] 刘旭梅,李恒,蒋敏,等.产褐藻胶裂解酶菌株Cobetia sp. WG-007的筛选及发酵优化[J].食品与生物技术学报,2014,33(9):903-909.
[21] 陈艳红,杨帆,肖安风,等.褐藻胶裂解酶发酵工艺优化及其中试放大[J].集美大学学报:自然科学版,2016,21(3):184-190.
[22] Sim S J,Baik K S,Park S C,et al.Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone[J].Journal of Industrial Microbiology & Biotechnology,2012,39(4):585-593.
[2] 韩伟,林娟,谢勇,等.褐藻胶裂解酶基因的克隆表达及重组酶酶学性质[J].微生物学通报,2017,44(5):1074-1080.
[3] 张玉娟,罗福文,姚子昂,等.海藻酸钠寡糖生物活性的研究进展[J].中国酿造,2014,33(1):5-8.
[4] Yamamoto Y,Kurachi M,Yamaguchi K,et al.Induction of multiple cytokine secretion from RAW264.7 cells by alginate oligosaccharides[J].Bioscience,Biotechnology,and Biochemistry,2007,71(1):238-241.
[5] Iwasaki K I,Matsubara Y.Purification of alginate oligosaccharides with root growth-promoting activity toward lettuce[J].Bioscience,Biotechnology,and Biochemistry,2000,64(5):1067-1070.
[6] 刘瑞志.褐藻寡糖促进植物生长与抗逆效应机理研究[D].青岛:中国海洋大学,2009.
[7] 邰宏博,唐丽薇,陈带娣,等.褐藻胶寡糖制备的研究进展[J].生命科学研究,2015,19(1):75-79.
[8] 汤海青,欧昌荣,郑晓冬.1株产褐藻胶裂解酶海洋细菌的分离鉴定及其酶学性质[J].浙江大学学报:农业与生命科学版,2013,39(4):387-395.
[9] Rahman M M,Wang Ling,Inoue A,et al.cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula[J].Carbohydrate Research,2012,360:69-77.
[10] Huang Lishuxin,Zhou Jungang,Li Xiao,et al.Characterization of a new alginate lyase from newly isolated Flavobacterium sp. S20[J].Journal of Industrial Microbiology & Biotechnology,2013,40(1):113-122.
[11] Gacesa P,Wusteman F S.Plate assay for simultaneous detection of alginate lyases and determination of substrate specificity[J].Applied & Environmental Microbiology,1990,56(7):2265-2267.
[12] 陈朋,朱玥明,韩文佳,等.产褐藻胶裂解酶菌种的筛选、鉴定及发酵条件优化[J].食品科学,2015,36(15):105-111.
[13] Zhou Jiushun,Cai Menghao,Jiang Tao,et al.Mixed carbon source control strategy for enhancing alginate lyase production by marine Vibrio sp. QY102[J].Bioprocess and Biosystems Engineering,2014,37(3):575-584.
[14] 李恒,朱思婷,刘旭梅,等.褐藻胶裂解酶产生菌的分离鉴定及产酶发酵优化[J].中国生物工程杂志,2014,34(9):94-101.
[15] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[16] Li Changqing,Liu Wenchao,Zhu Ping,et al.Phylogenetic diversity of bacteria associated with the marine sponge Gelliodes carnosa collected from the Hainan island coastal waters of the South China Sea[J].Microbial Ecology,2011,62(4):800-812.
[17] 李云涛,汪立平,张孟,等.鲍鱼来源褐藻胶裂解酶菌株的筛选及发酵条件优化[J].大连海洋大学学报,2017,32(5):574-583.
[18] 张真庆.褐藻胶酶解寡糖的制备、分离和结构鉴定[D].青岛:中国海洋大学,2003.
[19] 黄李淑馨,刘钢,岳敏,等.产褐藻胶裂解酶菌株的分离与鉴定[J].食品工业科技,2013,34(23):147-151.
[20] 刘旭梅,李恒,蒋敏,等.产褐藻胶裂解酶菌株Cobetia sp. WG-007的筛选及发酵优化[J].食品与生物技术学报,2014,33(9):903-909.
[21] 陈艳红,杨帆,肖安风,等.褐藻胶裂解酶发酵工艺优化及其中试放大[J].集美大学学报:自然科学版,2016,21(3):184-190.
[22] Sim S J,Baik K S,Park S C,et al.Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone[J].Journal of Industrial Microbiology & Biotechnology,2012,39(4):585-593.