低温乳糖酶产生菌的分离鉴定及酶的提纯与特性研究
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摘要
从新疆低温环境中筛选到一株可产低温乳糖酶的菌株14-1,利用形态学方法,生理生化实验、16S rDNA基因序列同源性比对及系统发育树构建对菌株进行鉴定,结果显示该菌属于水生拉恩氏菌(Rahnella aquatilis),命名为Rahnella aquatilis sp.14-1。
对低温乳糖酶产生菌Rahnella aquatilis sp.14-1进行了摇瓶条件下产低温乳糖酶的研究,确定了摇瓶最佳发酵工艺:乳糖含量为2.5%,接种量3%,培养基初始pH为6.5,装液量为在500 mL三角瓶装液体培养基100 mL。20℃,200 r·min~(-1)摇床培养48 h,14~(-1)菌产低温乳糖酶活性4.51 U·mL~(-1)。
Rahnella aquatilis14-1 sp.乳糖酶为胞内酶,将菌体用超声波破壁提取粗酶液,用硫酸铵分级沉淀,Sephadex G-25和DEAE-Cellulose柱层析进一步纯化。经过纯化的酶用SDS-PAGE检验其纯度,结果为电泳单条带,该酶的单亚基表观分子量约为60 KDa。酶的纯化倍数为4.19,回收率25%。
Rahnella aquatilis sp.14-1乳糖酶的酶学性质研究表明,以ONPG为底物,酶的最适反应温度为35℃,对热敏感,45℃保温2 h,酶完全失去活力;最适反应pH值为6.5～7.0,在pH值低于5和高于9酶不稳定。。金属离子Mn2+对酶有激活作用,活力提高了19.8%。而Cu~(2+)、Zn~(2+)、Fe~(3+)、Al~(3+)离子对酶活力有明显的抑制作用,其活力分别减少了94.35%、92.76%、71.98%和88.93%。K~+、Li~+、Mg~(2+)、Na~+和Ca~(2+)对酶活力的影响很小。
Rahnella aquatilis sp.14-1乳糖酶水解牛乳中的乳糖,35℃水解2 h,乳糖水解率达到80%以上。
One cold-adapted lactase producing strain 14-1 was isolated from its low temperature environment in XINJINAG province. The strain was identified by studying its characteristics of morphology, cultivation, physiology and biochemistry and 16S rDNA sequence alignment. The results showed that the strain belongs to Rahnella aquatilis.
Shake-flask culture conditions of cold-adapted lactase were studied with psychrotrophic strain14-1. The fermentation conditions were optimized: lactose concentration 2.5%, initial pH 6.5, seed inoculation volume 3%, 500 mL flask contained 100mL medium. The maximumβ-Galactosidase activity, about 4.51 U·mL~(-1) was obtained at 20℃when cultured for 48 hours.
The lactase was intracellular enzyme,so the cell pellets were disrupted with a sonfier sonicator.The crude enzyme was purified by column chromatography and a single band was obtained on SDS-PAGE. Apparent molecular weight of subunits of the purified enzyme estimated by SDS-PAGE were about 60 KDa.The specific activity of crude enzyme was increase up to 321.7 U·mg~(-1) with purifying fold of 4.19 and the yield was 25%.
Research about the properties of the enzyme showed that the optimal temperature was 35℃, and the optimal pH value was 6.5 to 7.0 for the enzyme with ONPG as a substrate. The enzyme activity was sensitive to temperatures above 40℃and was undetectable after 2h incubation at 45℃. The metal ions Mn~(2+), K~+ activated the enzyme activity while Cu~(2+), Zn~(2+) , Fe~(3+) and Al~(3+) inhibited activity.
When milk was treated with the lactase at 35℃for 2 hours, the degree of hydrolysis for lactose can reached 80%.
对低温乳糖酶产生菌Rahnella aquatilis sp.14-1进行了摇瓶条件下产低温乳糖酶的研究,确定了摇瓶最佳发酵工艺:乳糖含量为2.5%,接种量3%,培养基初始pH为6.5,装液量为在500 mL三角瓶装液体培养基100 mL。20℃,200 r·min~(-1)摇床培养48 h,14~(-1)菌产低温乳糖酶活性4.51 U·mL~(-1)。
Rahnella aquatilis14-1 sp.乳糖酶为胞内酶,将菌体用超声波破壁提取粗酶液,用硫酸铵分级沉淀,Sephadex G-25和DEAE-Cellulose柱层析进一步纯化。经过纯化的酶用SDS-PAGE检验其纯度,结果为电泳单条带,该酶的单亚基表观分子量约为60 KDa。酶的纯化倍数为4.19,回收率25%。
Rahnella aquatilis sp.14-1乳糖酶的酶学性质研究表明,以ONPG为底物,酶的最适反应温度为35℃,对热敏感,45℃保温2 h,酶完全失去活力;最适反应pH值为6.5～7.0,在pH值低于5和高于9酶不稳定。。金属离子Mn2+对酶有激活作用,活力提高了19.8%。而Cu~(2+)、Zn~(2+)、Fe~(3+)、Al~(3+)离子对酶活力有明显的抑制作用,其活力分别减少了94.35%、92.76%、71.98%和88.93%。K~+、Li~+、Mg~(2+)、Na~+和Ca~(2+)对酶活力的影响很小。
Rahnella aquatilis sp.14-1乳糖酶水解牛乳中的乳糖,35℃水解2 h,乳糖水解率达到80%以上。
One cold-adapted lactase producing strain 14-1 was isolated from its low temperature environment in XINJINAG province. The strain was identified by studying its characteristics of morphology, cultivation, physiology and biochemistry and 16S rDNA sequence alignment. The results showed that the strain belongs to Rahnella aquatilis.
Shake-flask culture conditions of cold-adapted lactase were studied with psychrotrophic strain14-1. The fermentation conditions were optimized: lactose concentration 2.5%, initial pH 6.5, seed inoculation volume 3%, 500 mL flask contained 100mL medium. The maximumβ-Galactosidase activity, about 4.51 U·mL~(-1) was obtained at 20℃when cultured for 48 hours.
The lactase was intracellular enzyme,so the cell pellets were disrupted with a sonfier sonicator.The crude enzyme was purified by column chromatography and a single band was obtained on SDS-PAGE. Apparent molecular weight of subunits of the purified enzyme estimated by SDS-PAGE were about 60 KDa.The specific activity of crude enzyme was increase up to 321.7 U·mg~(-1) with purifying fold of 4.19 and the yield was 25%.
Research about the properties of the enzyme showed that the optimal temperature was 35℃, and the optimal pH value was 6.5 to 7.0 for the enzyme with ONPG as a substrate. The enzyme activity was sensitive to temperatures above 40℃and was undetectable after 2h incubation at 45℃. The metal ions Mn~(2+), K~+ activated the enzyme activity while Cu~(2+), Zn~(2+) , Fe~(3+) and Al~(3+) inhibited activity.
When milk was treated with the lactase at 35℃for 2 hours, the degree of hydrolysis for lactose can reached 80%.
引文
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[2] 陈秀兰,张玉忠等. 海洋适冷菌SM9913产生的低温蛋白酶[J].海洋科学,2001,25(1):4-8
[3] 辛明秀,周培谨等. 冷适应微生物产生的冷活性酶[J]微生物学报,2000,40(6):83-88
[4] 曾旷新,俞勇等. 低温微生物及其酶类的研究概况[J].微生物学杂志, 2004,(9):83-87
[5] 张树政. 酶制剂工业[M].北京:科学出版社,1998,6.
[6] 吴晖,牛晨艳等.乳糖不耐受症的现状及解决办法[J].现代食品科技, 2006,87(22):152-155
[7] 王敏,檀建新. 乳糖酶的应用及发展现状[J].食品与发酵工业, 2003(29):89-92
[8] 何梅. 乳糖酶缺乏和乳糖不耐受[M] 国外医学卫生学分册,1996,26(6):339-342
[9] 李达,刘兆庆等. 乳糖不耐受与功能性液体乳——低乳糖奶 农产品加工学[J].2005,47 (11):33-36
[10] 秦立虎,谭源. 低乳糖学生营养奶的研究[J]. 乳品导刊 2006年2月
[11] 郭虹良,黄艾祥. 温度对低乳糖牛奶褐变的影响[J].食品科技,2004,
[12] 陈铁涛. 低乳糖乳制品[J] .中国食品,1999,(10):29-31
[13] 卢丽丽,肖敏,徐小东. 转糖基β-半乳糖苷酶产生菌Enterobacter agglomerans B1:筛选鉴定、发酵产酶及其合成低聚半乳糖的研究[J].微生物学报,2008,(1):38-44
[14] 蔡永进. 酶解制备低乳糖牛奶的优化研究[J]. 四川食品与发酵, 2007 ,43(5):32-34
[15] 王敏,檀建新. 微生物乳糖酶的特性和发展现状[J]. 河北农业大学学报,2003,26
[16] LOVELAND.Characterization of Psychrotrophic Microorganisms Producing β-Galactosidase Activities[J].APPLIED AND ENVIRONMENTAL MICROBIOLOGY,1993,60:12-18
[17] DONALD E.TRIMBUR. Characterization of a Psychrotrophic Arthrobacter Gene and Its Cold-Activeβ-Galactosidase[J].APPLIED AND ENVIRONMENAL MICROBIOLOGY, 1994,60:4544-4552
[18] T.Nakagawa, Y.Fujimoto. Isoloation and characterization of psychrophiles producing cold-active β-galactosidase [J].Letters in Applied Microbiology, 2003, 37:154-157
[19] James A.Coker. Biochemical Characterization of a β-galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate , [J].JOURNAL OF BACTEROLOGY, 2003,185:5478-5482
[20] A.Hoyoux .Cold-Adapted β-Galactosidase from the Antarctic psychrophile pseudoalteromonas haloplanktis [J] Appl MicrobioBiotechnol,2001,67:1529-1535
[21] S.fernandes. β-Galactosidase from a cold –adapted bacterium : purification , characterization and application for lactase hydrolysis[J] . Appl Microbial Biotechnol,2002,58:313-321
[22]Turkiewica . Antarctic marine bacterium Pseudoalteromonas sp.22b as a source of cold-adapted β-Galactosidase[J].Biomolecular Engineering,2003,20:317-324
[23] Karasov. The cloning ,purification and characterization of a cold –acative β-Galactosidase from the psychrotoletrant Antarctic bacterium Arthrobacter sp.C2-2[J]. Enzyme MicrobTechnol, 2003,33:836-844.
[24] Yuji Fujimoto . Ryoko Ikehata. Purification and molecular characterization of cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2 [J]. Appl Microbiol Biotechnol. Springer-Verlag 2006,72(4):720-725
[25] James A. Protein engineering of a cold-active β-galactosidase from Arthrobacter sp.SB to increase lactose hydrolysis reveals new sites affecting low temperature activity[J].Extremophiles,2006,10:512-524.
[26] R.H.Jacobson.Three-dimensional structure of β-galactosidase from E.coli, [J]. Nature, 1994, 369(30):761-766
[27] PETRA NICHOLAS. Beta-Galactosidase Activity in psychrotrophic Microorganisms and their Potential use in Food Industry [J]. Czech Food Sci, 2002,20:43-47
[28] Qin Y. Ning Z X. Sythesis of Galacto-oligosaccharides with Immobilizedβ-galactosidase[J]. Food and Fermentation Industries ,2001,27(11):12-16
[29] 秦燕,宁正祥. β-半乳糖苷酶的应用研究进展[J].食品研究与开发,2000,21(2):3-6
[30] 黄北阳. 开发低乳制品促进我国乳品产业发展[J] .当代畜牧,2001,(3):1-2
[31] 尤新.功能性低聚糖[J].食品工业科技,2001,22(6):1-4
[32] 胡学智.功能性低聚糖及其制造概要[J].工业微生物,1997,(1):30-3
[33] 迟乃玉,张庆方,窦少华. 海洋低温BS041208菌株选育及发酵培养基的研究 食品与发酵工业2006 32卷3 4-6
[34] 张锐. 极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究[J]. 微生物学通报, 2001,28(4):5-9
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