海洋高产尿酸氧化酶菌株筛选鉴定及酶学性质研究
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摘要
尿酸氧化酶广泛用于常规临床分析和临床药物,在医学治疗和诊断中的重要性日益增加。为得到高产尿酸氧化酶的优良菌株,以大连黄海海泥、海水为材料,依次分别采用透明圈法、酶偶联分光光度法进行初筛和复筛,获一株高产尿酸氧化酶菌株Z7,根据菌株Z7的16S rDNA基因序列和形态学、生理生化特征,该菌株被鉴定为苛求芽孢杆菌(Bacillus fastidiosus)。酶学性质研究表明:尿酸氧化酶蛋白的分子量约为33.1 kD;最适作用温度是25℃,酶活高达673.7 U/mg;最适作用pH为8.0,在pH 8-9表现出较强的稳定性;Fe~(3+)、Ca~(2+)对尿酸氧化酶具有激活作用。Ag+、Hg~(2+)对该酶抑制性较强,使其几乎丧失活性。该菌株产酶活性及稳定性良好,可为尿酸氧化酶的工业化生产奠定理论基础,并具有潜在开发价值。
Uricase is used in routine clinical analysis and clinical medicine widely. Its importance in medical treatment and diagnosis is increasing. A high uricase-producting strain Z7 was screened from the Dalian Huanghai Sea mud and seawater by transparent circle method and enzyme coupling spectrophotometry,and identified as Bacillus fastidiosus by 16 S rDNA phylogenetic analysis,morphology and physiological and biochemical characteristics. Results showed that the molecular weight of the protein was about 33 kD. The optimum temperature for uricase was 25℃,and specific activity was 673.7 U/mg. The optimum pH for uricase was 8.0,and it was stable in the pH range of 8.0 to 9.0. Its relative enzyme activity could still maintain more than 90% after 1 h's incubation. The activation of Fe~(3+) and Ca~(2+) on uricase was stimulative effects,while Ag+,Hg~(2+) showed strong inhibitory effects. Owing to the excellent chill enzyme activity and stability,Z7 can lay a theoretical foundation for the industrialized production of uricase and has potential development value.
Uricase is used in routine clinical analysis and clinical medicine widely. Its importance in medical treatment and diagnosis is increasing. A high uricase-producting strain Z7 was screened from the Dalian Huanghai Sea mud and seawater by transparent circle method and enzyme coupling spectrophotometry,and identified as Bacillus fastidiosus by 16 S rDNA phylogenetic analysis,morphology and physiological and biochemical characteristics. Results showed that the molecular weight of the protein was about 33 kD. The optimum temperature for uricase was 25℃,and specific activity was 673.7 U/mg. The optimum pH for uricase was 8.0,and it was stable in the pH range of 8.0 to 9.0. Its relative enzyme activity could still maintain more than 90% after 1 h's incubation. The activation of Fe~(3+) and Ca~(2+) on uricase was stimulative effects,while Ag+,Hg~(2+) showed strong inhibitory effects. Owing to the excellent chill enzyme activity and stability,Z7 can lay a theoretical foundation for the industrialized production of uricase and has potential development value.
引文
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[2]周继朋. ADMA代谢酶基因多态性与中国汉族人群动脉硬化性心脑血管疾病易感性及机制研究[D].长沙:中南大学,2014.
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[6]Richette P, Perezruiz F, Doherty M, et al. Improving cardiovascular and renal outcomes in gout:what should we target?[J]. Nature Reviews Rheumatology, 2014, 10(11):654-661.
[7]逄飞,周新尚,肖景惠,等.海洋尿酸氧化酶菌株发酵条件响应面优化[J].中国酿造, 2018, 37(8):68-74.
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[11]陶磊,裴德宁,等.液质联用法分析重组假丝酵母尿酸氧化酶的二硫键[J].中国生物制品学杂志, 2015, 28(7):746-748.
[12]Shaaban MI, Abdelmegeed E, Ali YM. Cloning, expression, and purification of recombinant Uricase Enzyme from Pseudomonas aeruginosa Ps43 using Escherichia coli[J]. J Microbiol Biotechnol, 2015, 25(6):887-892.
[13]Dabbagh F, Ghoshoon MB, et al. Engineering human urate oxidase:Towards reactivating it as an important therapeutic enzyme[J].Current Pharmaceutical Biotechnology, 2015, 17(2):141-146.
[14]Kratzer JT, Lanaspa MA, Murphy MN, et al. Evolutionary history and metabolic insights of ancient mammalian uricases[J]. Proc Natl Acad Sci USA, 2014, 111(10):3763-3768.
[15]黄琳秋,江筠.尿酸氧化酶临床疗效和安全性评价[J].国际生物制品学杂志, 2016, 39(2):92-96.
[16]Miao Z, Li C, Chen Y, et al. Dietary and lifestyle changes associated with high prevalence of hyperuricemia and gout in the Shandong coastal cities of Eastern China[J]. Journal of Rheumatology,2008, 35(9):1859-1864.
[17]李文杰.枯草芽孢杆菌尿酸氧化酶的定向进化[D].北京:中国科学技术大学, 2017.
[18]陈志禹.微生物来源的尿酸氧化酶的研究进展及应用前景[J].微生物学通报, 2007, 34(6):1205-1208.
[19]Ghosh T, Sarkar P. Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme[J]. J Biosci, 2014, 39(5):805-819.
[20]Huang SH, Wu TK. Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis[J]. European Journal of Biochemistry, 2010,271(3):517-523.
[21]Abdel-Fattah YR, Saeed HM, Gohar YM, et al. Improved production of Pseudomonas aeruginosa uricase by optimization of process parameters through statistical experimental designs[J].Process Biochemistry, 2005, 40(5):1707-1714.
[22]汪年松,桂定坤.尿酸性肾病的中西医结合治疗进展[J].中华肾病研究电子杂志, 2015, 4(2):10-14.
[23]王一恬,沈微,陈献忠,等.一种来源于克劳氏芽孢杆菌的高碱性尿酸氧化酶的异源表达及重组酶性质分析[J].生物学杂志, 2013, 30(2):1-4.
[24]韩鸿鹏,王丁,等.产朊假丝酵母菌的筛选及其尿酸酶活力分析[J].河南教育学院学报:自然科学版, 2013, 22(1):14-18.
[25]张玉然,纪楠楠.诊断用耐热尿酸氧化酶菌株的筛选及条件优化[J].中国生化药物杂志, 2016, 36(9):154-157.
[26]张鹏程,卢向锋,李倩延,等.微杆菌属ZZJ4-1菌株的耐热尿酸氧化酶基因的克隆及重组酶性质[J].生物工程学报,2012, 28(7):813-822.
[27]阮继生.“伯杰氏系统细菌学手册(第二版)”第5卷与我国的放线菌系统学研究[J].微生物学报, 2013, 53(6):521-530.
[28]徐敬昭,陈贝,杜秉海,等.一株嗜麦芽寡养单胞菌的分离及其生物学特性[J].生物技术通报, 2019, 35(3):71-77.
[29]石群,张庆芳,杨丽娜,等.海洋肌酐水解酶菌株的筛选鉴定及其酶学性质研究[J].中国酿造, 2017, 36(3):19-23.
[30]张建新,刘起丽,胡文波,等.内生枯草芽孢杆菌HD-1β-甘露聚糖酶基因的克隆及结构生物学分析[J].河南师范大学学报:自然版, 2013, 41(1):126-129.
[31]熊科,熊苏玥,崔晓亭,等.链霉菌L10608产木聚糖酶纯化及特异水解底物生成益生元型产物研究[J].食品与发酵工业, 2017, 43(5):49-55.
[32]ElnaggarEA.Isolation,screeningandidentificationof actinobacteria with uricase activity:statistical optimization of fermentation conditions for improved production of uricase by NEAE-25[J]. Int J Pharmacol, 2007, 11(7):644-658.
[33]Wang LWC, Marzluf GA. Purification and characterization of uricase, a nitrogen-regulated enzyme, from Neurospora crassa[J].Arch Biochem Biophys, 1980, 201(1):185-193.
[34]Lucas K, Boland MJ, Schubert KR. Uricase from soybean root nodules:Purification, properties, and comparison with the enzyme from cowpea[J]. Arch Biochem Biophys, 1983, 226(1):190-197.
[35]孟尧,辛渝,谭晓晶,等.朊圆酵母尿酸酶的基本特性研究[J].四川大学学报:自然科学版, 2006, 43(2):414-419.
[36]姚明明.双水相萃取尿酸酶和磷酸甘油氧化酶[D].成都:四川大学, 2007.
[37]武文明,曾昭淳,李小彦,等.产朊假丝酵母尿酸酶的纯化和特性研究[J].西南大学学报:自然科学版, 2008, 30(3):84-89.
[2]周继朋. ADMA代谢酶基因多态性与中国汉族人群动脉硬化性心脑血管疾病易感性及机制研究[D].长沙:中南大学,2014.
[3]Petr Němec. Gout and cardiovascular risk[J]. VnitrníLékarství,2014, 60(10):893-901.
[4]刘荔,倪海祥.糖尿病高尿酸血症中西医治疗研究进展[J].吉林中医药, 2014, 34(2):214-216.
[5]肖慧捷,李倩.高尿酸血症与肾脏疾病[J].中华实用儿科临床杂志, 2014, 29(17):1284-1287.
[6]Richette P, Perezruiz F, Doherty M, et al. Improving cardiovascular and renal outcomes in gout:what should we target?[J]. Nature Reviews Rheumatology, 2014, 10(11):654-661.
[7]逄飞,周新尚,肖景惠,等.海洋尿酸氧化酶菌株发酵条件响应面优化[J].中国酿造, 2018, 37(8):68-74.
[8]杨莉,谭唱,王莉,等.最新血尿酸检测方法概述[J].吉林医学, 2014, 35(1):144-145.
[9]Folin O, Macallum AB. A new method for the(colorimetric)determination of uric acid in urine[J]. J Biol Chem, 1912, 13(3):363-369.
[10]Ahrari S, Dabbagh F, Ahrari S, et al. Rational design and engineering of a mutant variant of urate oxidase as a therapeutic enzyme:a molecular dynamics simulation approach[J]. Current Computer-Aided Drug Design, 2016, 13(1):30-38.
[11]陶磊,裴德宁,等.液质联用法分析重组假丝酵母尿酸氧化酶的二硫键[J].中国生物制品学杂志, 2015, 28(7):746-748.
[12]Shaaban MI, Abdelmegeed E, Ali YM. Cloning, expression, and purification of recombinant Uricase Enzyme from Pseudomonas aeruginosa Ps43 using Escherichia coli[J]. J Microbiol Biotechnol, 2015, 25(6):887-892.
[13]Dabbagh F, Ghoshoon MB, et al. Engineering human urate oxidase:Towards reactivating it as an important therapeutic enzyme[J].Current Pharmaceutical Biotechnology, 2015, 17(2):141-146.
[14]Kratzer JT, Lanaspa MA, Murphy MN, et al. Evolutionary history and metabolic insights of ancient mammalian uricases[J]. Proc Natl Acad Sci USA, 2014, 111(10):3763-3768.
[15]黄琳秋,江筠.尿酸氧化酶临床疗效和安全性评价[J].国际生物制品学杂志, 2016, 39(2):92-96.
[16]Miao Z, Li C, Chen Y, et al. Dietary and lifestyle changes associated with high prevalence of hyperuricemia and gout in the Shandong coastal cities of Eastern China[J]. Journal of Rheumatology,2008, 35(9):1859-1864.
[17]李文杰.枯草芽孢杆菌尿酸氧化酶的定向进化[D].北京:中国科学技术大学, 2017.
[18]陈志禹.微生物来源的尿酸氧化酶的研究进展及应用前景[J].微生物学通报, 2007, 34(6):1205-1208.
[19]Ghosh T, Sarkar P. Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme[J]. J Biosci, 2014, 39(5):805-819.
[20]Huang SH, Wu TK. Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis[J]. European Journal of Biochemistry, 2010,271(3):517-523.
[21]Abdel-Fattah YR, Saeed HM, Gohar YM, et al. Improved production of Pseudomonas aeruginosa uricase by optimization of process parameters through statistical experimental designs[J].Process Biochemistry, 2005, 40(5):1707-1714.
[22]汪年松,桂定坤.尿酸性肾病的中西医结合治疗进展[J].中华肾病研究电子杂志, 2015, 4(2):10-14.
[23]王一恬,沈微,陈献忠,等.一种来源于克劳氏芽孢杆菌的高碱性尿酸氧化酶的异源表达及重组酶性质分析[J].生物学杂志, 2013, 30(2):1-4.
[24]韩鸿鹏,王丁,等.产朊假丝酵母菌的筛选及其尿酸酶活力分析[J].河南教育学院学报:自然科学版, 2013, 22(1):14-18.
[25]张玉然,纪楠楠.诊断用耐热尿酸氧化酶菌株的筛选及条件优化[J].中国生化药物杂志, 2016, 36(9):154-157.
[26]张鹏程,卢向锋,李倩延,等.微杆菌属ZZJ4-1菌株的耐热尿酸氧化酶基因的克隆及重组酶性质[J].生物工程学报,2012, 28(7):813-822.
[27]阮继生.“伯杰氏系统细菌学手册(第二版)”第5卷与我国的放线菌系统学研究[J].微生物学报, 2013, 53(6):521-530.
[28]徐敬昭,陈贝,杜秉海,等.一株嗜麦芽寡养单胞菌的分离及其生物学特性[J].生物技术通报, 2019, 35(3):71-77.
[29]石群,张庆芳,杨丽娜,等.海洋肌酐水解酶菌株的筛选鉴定及其酶学性质研究[J].中国酿造, 2017, 36(3):19-23.
[30]张建新,刘起丽,胡文波,等.内生枯草芽孢杆菌HD-1β-甘露聚糖酶基因的克隆及结构生物学分析[J].河南师范大学学报:自然版, 2013, 41(1):126-129.
[31]熊科,熊苏玥,崔晓亭,等.链霉菌L10608产木聚糖酶纯化及特异水解底物生成益生元型产物研究[J].食品与发酵工业, 2017, 43(5):49-55.
[32]ElnaggarEA.Isolation,screeningandidentificationof actinobacteria with uricase activity:statistical optimization of fermentation conditions for improved production of uricase by NEAE-25[J]. Int J Pharmacol, 2007, 11(7):644-658.
[33]Wang LWC, Marzluf GA. Purification and characterization of uricase, a nitrogen-regulated enzyme, from Neurospora crassa[J].Arch Biochem Biophys, 1980, 201(1):185-193.
[34]Lucas K, Boland MJ, Schubert KR. Uricase from soybean root nodules:Purification, properties, and comparison with the enzyme from cowpea[J]. Arch Biochem Biophys, 1983, 226(1):190-197.
[35]孟尧,辛渝,谭晓晶,等.朊圆酵母尿酸酶的基本特性研究[J].四川大学学报:自然科学版, 2006, 43(2):414-419.
[36]姚明明.双水相萃取尿酸酶和磷酸甘油氧化酶[D].成都:四川大学, 2007.
[37]武文明,曾昭淳,李小彦,等.产朊假丝酵母尿酸酶的纯化和特性研究[J].西南大学学报:自然科学版, 2008, 30(3):84-89.
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