一株产低温β-半乳糖苷酶微杆菌的筛选鉴定、产酶条件及其酶学特性
|
摘要
【背景】低温β-半乳糖苷酶能在低温下仍保持较高的乳糖水解活性,筛选酶学特性适合在牛乳体系中高效水解乳糖的β-半乳糖苷酶生产菌株,是低乳糖牛乳加工产业关注的焦点。【目的】对天山中国一号冰川沉积物中分离的一株产低温β-半乳糖苷酶菌株的产酶条件和酶学特性进行研究。【方法】结合X-Gal平板法初筛和测定粗酶液酶活复筛,获得产低温β-半乳糖苷酶的菌株。通过形态学、生理生化试验及16S rRNA基因测序分析对筛选菌株进行鉴定,单因素摇瓶实验优化菌株的产酶条件,硫酸铵分级沉淀初步纯化β-半乳糖苷酶并对其酶学特性进行分析。【结果】通过形态学、生理生化特征和16S rRNA基因鉴定,确定菌株LW106为微杆菌属(Microbacterium)菌株;该菌株最适产酶温度为25°C,最佳产酶碳源为可溶性淀粉,培养基初始pH为7.0,接种量为3%;对初步纯化的低温β-半乳糖苷酶酶学性质的研究表明,LW106所产β-半乳糖苷酶的最适pH为6.0,最适反应温度为35°C,4°C时酶活为最大酶活的78%,4°C和pH 7.0时的稳定性最好,10 mmol/L的Na+对酶活性基本没有抑制作用,Ca~(2+)对酶活性具有一定的激活作用。【结论】菌株LW106所产低温β-半乳糖苷酶的酶学特性表明该酶在乳品低温加工领域具有进一步研究和应用的价值。
[Background] Cold-active β-galactosidases are typically characterized by a high lactose hydrolysis activity at low temperature. Such a characteristic makes cold-active β-galactosidase highly attractive for application in low-lactose milk production industry, and how to mine novel cold-activeβ-galactosidase with enzymatic properties perfectly suitable for lactose hydrolysis in milk solution condition becomes a research focus. [Objective] This study is aiming to screen novel strains producing cold-active β-galactosidase from glacier sediments at China No.1 glacier in Tianshan Mountains and to evaluate if the enzymatic properties of the cold-active β-galactosidase is suitable for further application in low-lactose milk production. [Methods] Primary screening using X-Gal culture plate, and secondary screening by assaying the relative β-galactosidase activity at low temperature were carried out. The target strain selected was identified by physiological, biochemical and 16 S rRNA gene sequencing. The growth condition and enzyme production condition of the isolate were optimized by single-factor experiment.Enzymatic properties of the partially purified β-galactosidase produced by the isolate were also investigated. [Results] LW106, a strain with high β-galactosidase activity, was identified as Microbacterium sp. LW106. The optimal temperature for LW106 producing β-galactosidase is 25 °C.Soluble starch was the optimal carbon source for LW106 to produce β-galactosidase, the optimum medium initial pH was 7.0, and the optimal inoculum size was 3%. Preliminary study on its enzymatic properties showed that the optimal pH of the β-galactosidase of strain LW106 was 6.0, and the optimal reaction temperature was 35 °C with a relative activity of 78% at 4 °C. The β-galactosidase produced by LW106 showed the best stability at 4 °C and pH 7.0. Metal ion Na+ at a level of 10 mmol/L did not inhibit the activity of β-galactosidase of LW106, while 10 mmol/L of Ca~(2+) activated the β-galactosidase activity.[Conclusion] The enzymatic properties of β-galactosidase produced by Microbacterium sp. LW106 indicated it may be suitable for lactose hydrolysis at low temperature, showing good application potential in low-lactose milk production industry.
[Background] Cold-active β-galactosidases are typically characterized by a high lactose hydrolysis activity at low temperature. Such a characteristic makes cold-active β-galactosidase highly attractive for application in low-lactose milk production industry, and how to mine novel cold-activeβ-galactosidase with enzymatic properties perfectly suitable for lactose hydrolysis in milk solution condition becomes a research focus. [Objective] This study is aiming to screen novel strains producing cold-active β-galactosidase from glacier sediments at China No.1 glacier in Tianshan Mountains and to evaluate if the enzymatic properties of the cold-active β-galactosidase is suitable for further application in low-lactose milk production. [Methods] Primary screening using X-Gal culture plate, and secondary screening by assaying the relative β-galactosidase activity at low temperature were carried out. The target strain selected was identified by physiological, biochemical and 16 S rRNA gene sequencing. The growth condition and enzyme production condition of the isolate were optimized by single-factor experiment.Enzymatic properties of the partially purified β-galactosidase produced by the isolate were also investigated. [Results] LW106, a strain with high β-galactosidase activity, was identified as Microbacterium sp. LW106. The optimal temperature for LW106 producing β-galactosidase is 25 °C.Soluble starch was the optimal carbon source for LW106 to produce β-galactosidase, the optimum medium initial pH was 7.0, and the optimal inoculum size was 3%. Preliminary study on its enzymatic properties showed that the optimal pH of the β-galactosidase of strain LW106 was 6.0, and the optimal reaction temperature was 35 °C with a relative activity of 78% at 4 °C. The β-galactosidase produced by LW106 showed the best stability at 4 °C and pH 7.0. Metal ion Na+ at a level of 10 mmol/L did not inhibit the activity of β-galactosidase of LW106, while 10 mmol/L of Ca~(2+) activated the β-galactosidase activity.[Conclusion] The enzymatic properties of β-galactosidase produced by Microbacterium sp. LW106 indicated it may be suitable for lactose hydrolysis at low temperature, showing good application potential in low-lactose milk production industry.
引文
[1]Zhang SZ.Enzyme Preparation Industry[M].Beijing:Science Press,1984:655-670(in Chinese)张树政.酶制剂工业[M].北京:科学出版社,1984:655-670
[2]Li YQ.Studies onβ-galactosidase produced by filamentous fungi[D].Tianjin:Master’s Thesis of Tianjin Institute of Light Industry,2000(in Chinese)李玉强.丝状真菌β-半乳糖苷酶的研究[D].天津:天津轻工业学院硕士学位论文,2000
[3]Qin Y,Ning ZX,Hu XY.Advance of application and research inβ-galactoxidase[J].Journal of Shenyang Agricultural University,2000,31(6):595-599(in Chinese)秦燕,宁正祥,胡新宇.β-半乳糖苷酶的应用研究进展[J].沈阳农业大学学报,2000,31(6):595-599
[4]Shen LL.Screening and mutation breeding of a strain producing cold-adapted lactase and the study on optimization of fermentation conditions and its crude enzymatic properties[D].Wuxi:Master’s Thesis of Jiangnan University,2013(in Chinese)沈莲莲.低温乳糖酶产生菌株的选育、产酶条件优化及其粗酶性质研究[D].无锡:江南大学硕士学位论文,2013
[5]KarasováP,Spiwok V,Malá?,et al.β-galactosidase activity in psychrotrophic microorganisms and their potential use in food industry[J].Czech Journal of Food Sciences,2002,20(2):43-47
[6]Cie?liński H,Kur J,Bia?kowska A,et al.Cloning,expression,and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp.22b[J].Protein Expression and Purification,2005,39(1):27-34
[7]Ghosh M,Pulicherla KK,Rekha VPB,et al.Cold activeβ-galactosidase from Thalassospira sp.3SC-21 to use in milk lactose hydrolysis:a novel source from deep waters of Bay-of-Bengal[J].World Journal of Microbiology and Biotechnology,2012,28(9):2859-2869
[8]Alikkunju AP,Sainjan N,Silvester R,et al.Screening and characterization of cold-activeβ-galactosidase producing psychrotrophic Enterobacter ludwigii from the sediments of Arctic Fjord[J].Applied Biochemistry and Biotechnology,2016,180(3):477-490
[9]Nakagawa T,Fujimoto Y,Uchino M,et al.Isolation and characterization of psychrophiles producing cold-active-galactosidase[J].Letters in Applied Microbiology,2003,37(2):154-157
[10]Zhang M,Gu YL,Xu YL,et al.Phylogenetic and physiological diversity of cold-adapted bacteria producingβ-galactosidase from permafrost sediments of the bottom layer of the Glacier No.1 in the Tianshan Mountains[J].Acta Microbiologica Sinica,2011,51(12):1605-1615(in Chinese)张明,顾燕玲,徐宇丽,等.天山1号冰川底部沉积层产β-半乳糖苷酶低温菌株的系统发育分析及生理多样性[J].微生物学报,2011,51(12):1605-1615
[11]Buchana RE,Gibbons NE.Bergey’s Manual of Determinative Bacteriology[M].8th ed.Beijing:Science Press,1984:382-533(in Chinese)布坎南RE,吉本斯NE.伯杰氏细菌鉴定手册[M].8版.北京:科学出版社,1984:382-533
[12]Li DY,Shang A,Zhang D,et al.Screening of a glycolipid biosurfactant-producing strain for kerogen biodegradation[J].Microbiology China,2017,44(11):2606-2614(in Chinese)李德雨,商奥,张迪,等.一株糖脂表面活性剂产生菌的筛选及干酪根降解[J].微生物学通报,2017,44(11):2606-2614
[13]Guan B,Zhang X,Hu YC,et al.Isolation and identification of pathogenic fungi in rotten sweet cherry from Shihezi,Xinjiang[J].Microbiology China,2018,45(11):2480-2487(in Chinese)关波,张璇,胡玉婵,等.新疆石河子地区甜樱桃采后腐烂致病真菌的分离鉴定[J].微生物学通报,2018,45(11):2480-2487
[14]Zhang YJ.Isolation and identification of a Microbacterium sp.LW106 strain producing cold-activeβ-galactosidase and its enzymatic properties[D].Shihezi:Master’s Thesis of Shihezi University,2018(in Chinese)张宇洁.一株产低温β-半乳糖苷酶菌株Microbacterium sp.LW106的筛选鉴定、产酶条件及其酶学特性[D].石河子:石河子大学硕士学位论文,2018
[15]Coker JA,Sheridan PP,Loveland-Curtze J,et al.Biochemical characterization of aβ-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate[J].Journal of Bacteriology,2003,185(18):5473-5482
[16]Hoyoux A,Jennes I,Dubois P,et al.Cold-adaptedβ-galactosidase from the antarctic psychrophile Pseudoalteromonas haloplanktis[J].Applied and Environmental Microbiology,2001,67(4):1529-1535
[17]Pawlak-Szukalska A,Wanarska M,Popinigis AT,et al.A novel cold-activeβ-D-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp.32cB-Gene cloning,purification and characterization[J].Process Biochemistry,2014,49(12):2122-2133
[18]Turkiewicz M,Kur J,Bia?kowska A,et al.Antarctic marine bacterium Pseudoalteromonas sp.22b as a source of cold-adaptedβ-Galactosidase[J].Biomolecular Engineering,2003,20(4/6):317-324
[19]Fan YT,Hua X,Zhang YZ,et al.Cloning,expression and structural stability of a cold-adaptedβ-galactosidase from Rahnella sp.R3[J].Protein Expression and Purification,2015,115:158-164
[20]Zhang X,Hua X,Xu Q,et al.Purification and characterization of a novel cold-adaptedβ-galactosidase[J].Food&Machinery,2014,30(3):174-178(in Chinese)张雪,华霄,许琪,等.低温β-半乳糖苷酶分离纯化及酶学性质研究[J].食品与机械,2014,30(3):174-178
[21]Wang GX,Gao Y,Hu B,et al.A novel cold-adaptedβ-galactosidase isolated from Halomonas sp.S62:gene cloning,purification and enzymatic characterization[J].World Journal of Microbiology and Biotechnology,2013,29(8):1473-1480
[22]Zhou CL,Yu Y,Li HR,et al.Screening and identification of cold-activeβ-galactosidase-producing bacterium and characterization of theβ-galactosidase[J].Chinese Journal of Polar Research,2010,22(1):48-55(in Chinese)周春雷,俞勇,李会荣,等.低温β-半乳糖苷酶产生菌的筛选、鉴定、生长特性及酶学性质的初步研究[J].极地研究,2010,22(1):48-55
[23]Zhang B.Screening of psychrotolerant bacterium producing cold-activeβ-galactosidase and optimization for producing conditions ofβ-galactosidase[D].Guangzhou:Master’s Thesis of Guangdong Pharmaceutical University,2011(in Chinese)张博.产冷活性β-半乳糖苷酶低温菌株的筛选及产酶条件的优化[D].广州:广东药学院硕士学位论文,2011
[24]Zeng Q,Wang YS,Sun K,et al.Preliminary studies on the screening,identification and optimum fermentative conditions of a strain Marinomonas sp.BSi20414 isolated from arctic sea ice producingβ-galactosidase[J].Chinese Journal of Polar Research,2011,23(2):108-114(in Chinese)曾倩,王毓舒,孙鲲,等.一株分离自北极海冰的产β-半乳糖苷酶的海单胞菌(Marinomonas sp.BSi20414)的筛选、鉴定和产酶条件研究[J].极地研究,2011,23(2):108-114
[2]Li YQ.Studies onβ-galactosidase produced by filamentous fungi[D].Tianjin:Master’s Thesis of Tianjin Institute of Light Industry,2000(in Chinese)李玉强.丝状真菌β-半乳糖苷酶的研究[D].天津:天津轻工业学院硕士学位论文,2000
[3]Qin Y,Ning ZX,Hu XY.Advance of application and research inβ-galactoxidase[J].Journal of Shenyang Agricultural University,2000,31(6):595-599(in Chinese)秦燕,宁正祥,胡新宇.β-半乳糖苷酶的应用研究进展[J].沈阳农业大学学报,2000,31(6):595-599
[4]Shen LL.Screening and mutation breeding of a strain producing cold-adapted lactase and the study on optimization of fermentation conditions and its crude enzymatic properties[D].Wuxi:Master’s Thesis of Jiangnan University,2013(in Chinese)沈莲莲.低温乳糖酶产生菌株的选育、产酶条件优化及其粗酶性质研究[D].无锡:江南大学硕士学位论文,2013
[5]KarasováP,Spiwok V,Malá?,et al.β-galactosidase activity in psychrotrophic microorganisms and their potential use in food industry[J].Czech Journal of Food Sciences,2002,20(2):43-47
[6]Cie?liński H,Kur J,Bia?kowska A,et al.Cloning,expression,and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp.22b[J].Protein Expression and Purification,2005,39(1):27-34
[7]Ghosh M,Pulicherla KK,Rekha VPB,et al.Cold activeβ-galactosidase from Thalassospira sp.3SC-21 to use in milk lactose hydrolysis:a novel source from deep waters of Bay-of-Bengal[J].World Journal of Microbiology and Biotechnology,2012,28(9):2859-2869
[8]Alikkunju AP,Sainjan N,Silvester R,et al.Screening and characterization of cold-activeβ-galactosidase producing psychrotrophic Enterobacter ludwigii from the sediments of Arctic Fjord[J].Applied Biochemistry and Biotechnology,2016,180(3):477-490
[9]Nakagawa T,Fujimoto Y,Uchino M,et al.Isolation and characterization of psychrophiles producing cold-active-galactosidase[J].Letters in Applied Microbiology,2003,37(2):154-157
[10]Zhang M,Gu YL,Xu YL,et al.Phylogenetic and physiological diversity of cold-adapted bacteria producingβ-galactosidase from permafrost sediments of the bottom layer of the Glacier No.1 in the Tianshan Mountains[J].Acta Microbiologica Sinica,2011,51(12):1605-1615(in Chinese)张明,顾燕玲,徐宇丽,等.天山1号冰川底部沉积层产β-半乳糖苷酶低温菌株的系统发育分析及生理多样性[J].微生物学报,2011,51(12):1605-1615
[11]Buchana RE,Gibbons NE.Bergey’s Manual of Determinative Bacteriology[M].8th ed.Beijing:Science Press,1984:382-533(in Chinese)布坎南RE,吉本斯NE.伯杰氏细菌鉴定手册[M].8版.北京:科学出版社,1984:382-533
[12]Li DY,Shang A,Zhang D,et al.Screening of a glycolipid biosurfactant-producing strain for kerogen biodegradation[J].Microbiology China,2017,44(11):2606-2614(in Chinese)李德雨,商奥,张迪,等.一株糖脂表面活性剂产生菌的筛选及干酪根降解[J].微生物学通报,2017,44(11):2606-2614
[13]Guan B,Zhang X,Hu YC,et al.Isolation and identification of pathogenic fungi in rotten sweet cherry from Shihezi,Xinjiang[J].Microbiology China,2018,45(11):2480-2487(in Chinese)关波,张璇,胡玉婵,等.新疆石河子地区甜樱桃采后腐烂致病真菌的分离鉴定[J].微生物学通报,2018,45(11):2480-2487
[14]Zhang YJ.Isolation and identification of a Microbacterium sp.LW106 strain producing cold-activeβ-galactosidase and its enzymatic properties[D].Shihezi:Master’s Thesis of Shihezi University,2018(in Chinese)张宇洁.一株产低温β-半乳糖苷酶菌株Microbacterium sp.LW106的筛选鉴定、产酶条件及其酶学特性[D].石河子:石河子大学硕士学位论文,2018
[15]Coker JA,Sheridan PP,Loveland-Curtze J,et al.Biochemical characterization of aβ-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate[J].Journal of Bacteriology,2003,185(18):5473-5482
[16]Hoyoux A,Jennes I,Dubois P,et al.Cold-adaptedβ-galactosidase from the antarctic psychrophile Pseudoalteromonas haloplanktis[J].Applied and Environmental Microbiology,2001,67(4):1529-1535
[17]Pawlak-Szukalska A,Wanarska M,Popinigis AT,et al.A novel cold-activeβ-D-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp.32cB-Gene cloning,purification and characterization[J].Process Biochemistry,2014,49(12):2122-2133
[18]Turkiewicz M,Kur J,Bia?kowska A,et al.Antarctic marine bacterium Pseudoalteromonas sp.22b as a source of cold-adaptedβ-Galactosidase[J].Biomolecular Engineering,2003,20(4/6):317-324
[19]Fan YT,Hua X,Zhang YZ,et al.Cloning,expression and structural stability of a cold-adaptedβ-galactosidase from Rahnella sp.R3[J].Protein Expression and Purification,2015,115:158-164
[20]Zhang X,Hua X,Xu Q,et al.Purification and characterization of a novel cold-adaptedβ-galactosidase[J].Food&Machinery,2014,30(3):174-178(in Chinese)张雪,华霄,许琪,等.低温β-半乳糖苷酶分离纯化及酶学性质研究[J].食品与机械,2014,30(3):174-178
[21]Wang GX,Gao Y,Hu B,et al.A novel cold-adaptedβ-galactosidase isolated from Halomonas sp.S62:gene cloning,purification and enzymatic characterization[J].World Journal of Microbiology and Biotechnology,2013,29(8):1473-1480
[22]Zhou CL,Yu Y,Li HR,et al.Screening and identification of cold-activeβ-galactosidase-producing bacterium and characterization of theβ-galactosidase[J].Chinese Journal of Polar Research,2010,22(1):48-55(in Chinese)周春雷,俞勇,李会荣,等.低温β-半乳糖苷酶产生菌的筛选、鉴定、生长特性及酶学性质的初步研究[J].极地研究,2010,22(1):48-55
[23]Zhang B.Screening of psychrotolerant bacterium producing cold-activeβ-galactosidase and optimization for producing conditions ofβ-galactosidase[D].Guangzhou:Master’s Thesis of Guangdong Pharmaceutical University,2011(in Chinese)张博.产冷活性β-半乳糖苷酶低温菌株的筛选及产酶条件的优化[D].广州:广东药学院硕士学位论文,2011
[24]Zeng Q,Wang YS,Sun K,et al.Preliminary studies on the screening,identification and optimum fermentative conditions of a strain Marinomonas sp.BSi20414 isolated from arctic sea ice producingβ-galactosidase[J].Chinese Journal of Polar Research,2011,23(2):108-114(in Chinese)曾倩,王毓舒,孙鲲,等.一株分离自北极海冰的产β-半乳糖苷酶的海单胞菌(Marinomonas sp.BSi20414)的筛选、鉴定和产酶条件研究[J].极地研究,2011,23(2):108-114