海洋低温葡萄糖氧化酶细菌选育及其酶学性质研究
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摘要
为获得产低温葡萄糖氧化酶(glucose oxidase,GOD)的细菌,提高其产酶能力,研究GOD酶学性质,通过平板显色法从海泥样品中筛选低温GOD生产菌株,经形态特征、生理生化鉴定及分子生物学鉴定确定菌株分类。经诱变育种获得高产GOD菌株,并对所产GOD进行酶学性质研究。研究获得1株编号8-III的高产低温GOD细菌,鉴定为柠檬酸杆菌属(Citrobacters sp. 8-III),产GOD酶活力为0. 75 U/m L。经诱变得到可稳定遗传的突变菌株8-III-a44,产GOD酶活力为1. 77 U/m L,为原始菌株的2. 36倍。最适p H为6. 5,最适作用温度为15℃,在0～35℃有较高酶活力,为低温酶。并且添加K+、Ni2+对GOD活性有明显促进作用。该研究为其在海产品保鲜、饲料防腐方面的应用奠定基础。
This study aimed to obtain low-temperature glucose oxidase( GOD) producing bacteria,improve their enzyme-producing ability and study the properties of GOD. The low-temperature GOD producing strains were screened from sea sediments and identified. The strain with high yield of GOD was obtained by mutation breeding,and the properties of such GOD were studied. A strain 8-III with high yield of cold-active GOD was identified as Citrobacters sp. 8-III,and the activity of the GOD was 0. 75 U/m L. Moreover,a mutant strain 8-III-a44 with high stability was obtained,the produced GOD activity was 1. 77 U/m L,which was 2. 36 times of that of strain 8-III. Furthermore,the optimal temperature and pH for the GOD produced by 8-III-a44 strain were 15 ℃ and 6. 5,respectively. Besides,the enzyme was stable at 0-35 ? C,which indicated that it was a low-temperature enzyme. In addition,K+and Ni2 +significantly promoted its activity. In conclusion,this study expands the source of GOD and lays a foundation for its applications in seafoods and feeds preservation.
This study aimed to obtain low-temperature glucose oxidase( GOD) producing bacteria,improve their enzyme-producing ability and study the properties of GOD. The low-temperature GOD producing strains were screened from sea sediments and identified. The strain with high yield of GOD was obtained by mutation breeding,and the properties of such GOD were studied. A strain 8-III with high yield of cold-active GOD was identified as Citrobacters sp. 8-III,and the activity of the GOD was 0. 75 U/m L. Moreover,a mutant strain 8-III-a44 with high stability was obtained,the produced GOD activity was 1. 77 U/m L,which was 2. 36 times of that of strain 8-III. Furthermore,the optimal temperature and pH for the GOD produced by 8-III-a44 strain were 15 ℃ and 6. 5,respectively. Besides,the enzyme was stable at 0-35 ? C,which indicated that it was a low-temperature enzyme. In addition,K+and Ni2 +significantly promoted its activity. In conclusion,this study expands the source of GOD and lays a foundation for its applications in seafoods and feeds preservation.
引文
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[21] LIANG J,WANG J,ZHANG L,et al. Glucose oxidaseloaded liposomes for in situ amplified signal of electrochemical immunoassay on a handheld p H meter[J]. New Journal of Chemistry,2019,43.
[22]石征宇,易弋,黄德生,等.一株弗氏柠檬酸杆菌的分离鉴定及致病性研究[J].中国畜牧兽医,2018,45(2):479-485.
[23] MADIGAN M T,MARTINKO J M,PARKER J. Microbial Biology[M]. Beijing:Science Press,2001:390.
[24] MIRN J,GONZLEZ MP,PASTRANA L,et al. Diauxic production of glucose oxidase by Aspergillus niger in submerged culture[J]. Enzyme and Microbial Technology,2002,31(5):615-620.
[25] TAMURA K,PETERSON D,PETERSON N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Molecular Biology and Evolution,2011,28(10):2 731-2 739.
[26] WEISBURG W G,BARNS S M,PELLETIER D A. 16S ribosomal DNA amplification for phylogenetic study[J].Journal of Bacteriology,1991,173(2):697-703.
[2] HATZINIKOLAOU D G,HANSEN O C,MACRIS B J,et al. A new glucose oxidase from Aspergillus niger characterization and regulation studies of enzyme and gene[J]. Applied Microbiology and Biotechnology,1996,46(4):371-381.
[3] WANG Z,ZHOU X,ZHANG J,et al. Direct electrochemical reduction of single-layer graphene oxide and subsequent functionalization with glucose oxidase[J]. Journal of Physical Chemistry C,2009,113(32):14 071-14 075.
[4] DUBEY M K,ZEHRA A,AAMIR M,et al. Improvement strategies,cost effective production,and potential applications of fungal glucose oxidase(GOD):Current updates[J]. Frontiers in Microbiology,2017,8(8):89-90.
[5] HUGGETT A S G,NIXON D A. Use of glucose oxidase,peroxidase,and O-dianisidine in determination of blood and urinary glucose[J]. The Lancet,1957,270(6 991):368-370.
[6] GOUDA M D,SINGH S A,RAO A G,et al. Thermal inactivation of glucose oxidase:mechanism and stabilization using additives[J]. Journal of Biological Chemistry,2003,278(27):24 324-24 333.
[7] GU T,ZHANG Y,DENG F,et al. Direct electrochemistry of glucose oxidase and biosensing for glucose based on DNA/chitosan film[J]. Journal of Environmental Sciences,2011,23(11):S66-S69.
[8] SISAK C,CSANDI Z,RNAY E,et al. Elimination of glucose in egg using immobilized glucose oxidase[J]. Enzyme and Microbial Technology,2016,39(5):1 002-1 007.
[9] MISLOVICOVD,MICHALKOVE,VIKARTOVSKA.Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures[J]. Process Biochemistry,2007,42(4):704-709.
[10] FIEDUREK J. Glucose oxidase synthesis by Aspergillus niger GIV-10 on starch[J]. Acta Microbiologica Polonica,1991,40(3-4):197-203.
[11] LIU J Z,YANG H Y,WANG L P,et al. Synthesis of glucose oxidase and catalase by Aspergillus niger in resting cell culture system[J]. Letters in Applied Microbiology,2010,29(5):33-41.
[12] NGUYEN L T,YANG K L. Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions[J]. Enzyme&Microbial Technology,2017,100(Complete):52-59.
[13]狄乾斌,韩增林.我国海洋资源开发综合效益的评价探讨[J].国土与自然资源研究,2003(3):16-18.
[14]陈祖军,谭显英,韦鹤平.论我国海洋资源与环境的可持续发展[J].水资源保护,2001(1):7-10.
[15]楼东,谷树忠,钟赛香.中国海洋资源现状及海洋产业发展趋势分析[J].资源科学,2005,27(5):20-26.
[16]冯梁.“加快建设海洋强国”下我国经略海洋重大战略问题[J].亚太安全与海洋研究,2018(4):13-15.
[17]叶日英,徐德峰,孙力军,等.海洋源高产葡萄糖氧化酶细菌的筛选和主要酶学性质[J].浙江农业学报,2018,30(4):672-678.
[18]石淑钰,张庆芳,迟乃玉,等.一株海洋低温葡萄糖氧化酶菌株的筛选、鉴定及部分酶学性质[J].微生物学通报,2014,41(5):832-838.
[19] GANADU M L,ANDREOTTI L,VITALI I,et al. Glucose oxidase catalyses the reduction of O2to H2O2in the presence of irradiated Ti O2and isopropyl alcohol[J]. Photochemical&Photobiological Sciences,2002,1(12):951-954.
[20] FUJITA M,IKE M,TACHIBANA S,et al. Characterization of a bioflocculant produced by Citrobacter sp. TKF04 from acetic and propionic acids[J]. Journal of Bioscience&Bioengineering,2000,89(1):40-46.
[21] LIANG J,WANG J,ZHANG L,et al. Glucose oxidaseloaded liposomes for in situ amplified signal of electrochemical immunoassay on a handheld p H meter[J]. New Journal of Chemistry,2019,43.
[22]石征宇,易弋,黄德生,等.一株弗氏柠檬酸杆菌的分离鉴定及致病性研究[J].中国畜牧兽医,2018,45(2):479-485.
[23] MADIGAN M T,MARTINKO J M,PARKER J. Microbial Biology[M]. Beijing:Science Press,2001:390.
[24] MIRN J,GONZLEZ MP,PASTRANA L,et al. Diauxic production of glucose oxidase by Aspergillus niger in submerged culture[J]. Enzyme and Microbial Technology,2002,31(5):615-620.
[25] TAMURA K,PETERSON D,PETERSON N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Molecular Biology and Evolution,2011,28(10):2 731-2 739.
[26] WEISBURG W G,BARNS S M,PELLETIER D A. 16S ribosomal DNA amplification for phylogenetic study[J].Journal of Bacteriology,1991,173(2):697-703.