菌株LYH18发酵条件优化及肌氨酸氧化酶的分离纯化
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摘要
以海洋来源的芽孢杆菌(Bacillus sp.)LYH18为出发菌株,优化其发酵条件,并对其所产肌氨酸氧化酶(SOX)进行分离纯化。结果表明,其最适产酶发酵条件为肌酸添加量1.0%,酵母膏添加量0.8%,Mg SO_4·7H_2O添加量0.05%,KH_2PO_4添加量0.2%,K_2HPO_4添加量0.05%,初始pH值为7.0,培养温度25℃,装液量40 m L/100 m L。在此最佳发酵条件下,肌氨酸氧化酶酶活为4.45 U/m L。通过十二烷基硫酸钠-聚丙酰胺凝胶电泳(SDS-PAGE)分析肌氨酸氧化酶,其纯化后的分子质量为43 kDa。
Using Bacillus sp. LYH18 from ocean as the starting strain, the fermentation conditions were optimized, and the sarcosine oxidase(SOX)was isolated and purified. The results showed that the optimum fermentation conditions were creatine 1.0%, yeast extract 0.8%, Mg SO_4·7 H_2 O 0.05%,KH_2 PO_40.2%, K_2 HPO_4 0.05%, initial pH 7.0, culture temperature 25 ℃, and loading volume 40 ml/100 ml. Under the optimum fermentation conditions, the activity of SOX was 4.45 U/ml. The molecular weight of the purified SOX was 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis.
Using Bacillus sp. LYH18 from ocean as the starting strain, the fermentation conditions were optimized, and the sarcosine oxidase(SOX)was isolated and purified. The results showed that the optimum fermentation conditions were creatine 1.0%, yeast extract 0.8%, Mg SO_4·7 H_2 O 0.05%,KH_2 PO_40.2%, K_2 HPO_4 0.05%, initial pH 7.0, culture temperature 25 ℃, and loading volume 40 ml/100 ml. Under the optimum fermentation conditions, the activity of SOX was 4.45 U/ml. The molecular weight of the purified SOX was 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis.
引文
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[2]杨谢兰.使用微量白蛋白/尿肌酐和血糖联合检测法诊断糖尿病早期肾损伤的效果探析[J].当代医药论丛,2016,14(13):131-132.
[3]王正印,尹元,王伟灵.肌酐检测方法及常见药物对肌酐检测干扰的研究进展[J].检验医学,2018,33(4):370-373.
[4]ZHAO H,MA S,SHANG Y,et al.MicroRNAs in chronic kidney disease[J].Clin Chim Acta,2019,491(3):59-65.
[5]欧阳涓,姜傥.肾脏疾病的诊断进展[J].实用医院临床杂志,2012,9(3):25-29.
[6]陈文清.肾脏疾病的生化学诊断及进展[J].安徽卫生职业技术学院学报,2007(1):37-58.
[7]MORI N,KAWAKAMI B,HYAKUTOME K,et al.Characterization of betaine aldehyde dehydrogenase from Cylindrocarpon didymum m-1[J].J Agr Chem Soc Jpn,2014,44(12):3015-3016.
[8]刘辉,孙桂琴,马晓航,等.芽胞杆菌BSD-8肌氨酸氧化酶的纯化与性质[J].生物工程学报,2010,26(3):335-340.
[9]SUZUKI K,OGISHIMA M,SUGUYAMA M,et al.Molecular cloning and expression of a Streptomyces sarcosine oxidase gene in Streptomyces lividans[J].Biosci Biotech Bioche,1992,56(3):432-436.
[10]KIM J M,SHIMIZU S,YAMADA H.Sarcosine oxidase involved in creatinine degradation in Alcaligenes denitrificans subsp.denitrificans J9 and Arthrobacter sub.J5 and J11[J].J Agr Chem Soc Jpn,2014,50(11):2811-2816.
[11]包国祥,杨国灿,谢海英.紫外酶法和肌氨酸氧化酶法测定血清肌酐时对酚磺乙胺抗干扰性研究[J].中国卫生检验杂志,2018,28(1):64-67.
[12]孙谧,洪义国,李勃生,等.海洋微生物低温酶特性及其在工业中的潜在用途[J].海洋水产研究,2002(3):44-49.
[13]王萌,刘明,郭健,等.肌氨酸氧化酶法检测尿肌氨酸的方法学性能评价及参考值分析[J].临床检验杂志,2013,31(6):401-405.
[14]贾国辉,王耀荣.酶法肌酐检测的方法学改进探讨[J].中国冶金工业杂志,2015,32(3):369-270.
[15]石群,张庆芳,迟乃玉,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.
[16]赵更锋,马晓航,贾小明,等.产肌氨酸氧化酶菌株的分离及发酵条件研究[J].微生物学报,2003,42(2):235-240.
[17]GUO K,MA X,SUN G,et al.Expression and characterization of a thermostable sarcosine oxidase(SOX)from Bacillus in Escherichia coli[J].Appl Microbiol Biot,2006,73(3):559-566.
[18]蒲静,王瑞,姚明东,等.重组肌氨酸氧化酶的制备和鉴定[J].生物医学工程学杂志,2014,31(5):1090-1096.
[19]石群,张庆芳,迟乃玉,等.肌酐水解酶菌株S-09发酵条件优化及酶的分离纯化[J].中国酿造,2017,36(4):20-25.
[20]BEGOVICH K,WILHEIM J,et al.RNA structure drives stress granule assembly in yeast extracts[J].Mol Biol Cell,2017,28:M200.
[21]PRIYODIP P,PRAKASH P,BALAJI S.Phytases of probiotic bacteria:Characteristics and beneficial aspects[J].Indian J Microbiol,2017,57(2):148-154.
[22]王宁宁,吴振,江建梅,等.酵母类有机氮源及其在发酵行业的应用[J].产业与科技论坛,2014,13(2):69-71.
[23]罗子怡.详解pH值对酶活性的影响[J].农家参谋,2017(21):154.
[24]王超颖.浅析温度和pH值对酶活性的影响关系[J].技术与市场,2016,23(11):232.
[25]ANDERSEN K,BERGE T,GONCALVES J.Characteristic sizes of life in the oceans,from bacteria to whales[J].Annu Rev Mar Sci,2016,8:217-241.