不同干燥方法对菌体蛋白氨基酸分离鉴定的影响
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摘要
分别采用氮吹法、烘干法和真空冷冻干燥法对淀粉酶产色链霉菌的菌体蛋白氨基酸进行干燥处理,使用气相色谱与质谱联用对相应氨基酸衍生物的出峰时间及峰面积进行分析,结果表明,相较于氮吹法和烘干法,经真空冷冻干燥法预处理后,所需的16种氨基酸衍生物均可被检出,因此,真空冷冻干燥法是淀粉酶产色链霉菌中菌体蛋白氨基酸的可靠制备方法。该方法的建立为今后开展13C同位素代谢通量分析,深入阐明ε-聚赖氨酸生产机制,提供了技术保障。
Streptomyces diastatochromogenes cell protein isolates were dried by nitrogen-purge,oven-drying and vacuum lyophilization,respectively. The amino acid components of the cell protein isolates were analyzed by gas chromatography and mass spectrometry( GC-MS). The results showed that 16 kinds of amino acids could only be detected with vacuum lyophilization,indicating the vacuum lyophilization was a suitable method for preparing protein-bound amino acids from S. diastatochromogenes cell protein isolates for13 C-labeled metabolic flux analysis during ε-poly-L-lysine biosynthesis.
Streptomyces diastatochromogenes cell protein isolates were dried by nitrogen-purge,oven-drying and vacuum lyophilization,respectively. The amino acid components of the cell protein isolates were analyzed by gas chromatography and mass spectrometry( GC-MS). The results showed that 16 kinds of amino acids could only be detected with vacuum lyophilization,indicating the vacuum lyophilization was a suitable method for preparing protein-bound amino acids from S. diastatochromogenes cell protein isolates for13 C-labeled metabolic flux analysis during ε-poly-L-lysine biosynthesis.
引文
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[2] EL-SERSY N A,ABDELWAHAB A E,ABOUELKHIIR S S,et al. Antibacterial and anticancer activity ofε-poly-L-lysine(ε-PL)produced by a marine Bacillus subtilis sp.[J].Journal of Basic Microbiology,2012,52(5):513-522.
[3] SHUKLA S C,SINGH A,PANDEY A K,et al. Review on production and medical applications ofε-polylysine[J]. Biochemical Engineering Journal,2012,65:70-81.
[4] HAMANO Y. Amino-acid Homopolymers Occurring in Nature[M]. Berlin Heidelberg:Springer,2010:61-75.
[5] HIRAKI J,ICHIKAWA T,NINOMIYA S,et al. Use of ADME studies to confirm the safety ofε-polylysine as a preservative in food[J]. Regulatory Toxicology and Pharmacology,2003,37(2):328-340.
[6] BO Tao,LIU Miao,ZHONG Cheng,et al. Metabolomic analysis of antimicrobial mechanisms ofε-poly-L-lysine on Saccharomyces cerevisiae[J]. Journal of Agricultural and Food Chemistry,2014,62(19):4 454-4 465.
[7] BO Tao,HAN Pei-pei,SU Qin-zhi,et al. Antimicrobialε-poly-L-lysine induced changes in cell membrane compositions and properties of Saccharomyces cerevisiae[J]. Food Control,2016,61:123-134.
[8] HYLDGAARD M,MYGIND T,VAD B S,et al. The antimicrobial mechanism of action ofε-poly-L-lysine[J]. Applied and Environmental Microbiology,2014,80(24):7 758-7 770.
[9]宋庆超,李秀雯,王洪翠,等.ε-聚赖氨酸与纳他霉素复配剂抑菌性能的研究[J].中国酿造,2012,31(7):80-83.
[10] YOSHIDA T,NAGASAWA T.ε-Poly-L-lysine:microbial production,biodegradation and application potential[J].Applied Microbiology and Biotechnology,2003,62(1):21-26.
[11] RAJAPAKSHA D S W,KODITHUWAKKU K A H T,SILVA K F S T,et al. Evaluation of potassium sorbate andε-polylysine for their inhibitory activity on post-acidification of set yoghurt under cold storage for 20 days[J]. International Journal of Scientific and Research Publications,2013,3:1-7.
[12]张雪,赵娟,谭之磊,等.生物食品添加剂在草莓保鲜中的应用[J].中国食品添加剂,2013(3):158-162.
[13]贾士儒,谭之磊,罗晶,等.一种简单快速、货架期长的馒头的制作方法[P].中国专利:ZL 201310182022. X,2014-07-14.
[14] MIYA S,TAKAHASHI H,HASHIMOTO M,et al. Development of a controlling method for Escherichia coli O157:H7 and Salmonella spp. in fresh market beef by using polylysine and modified atmosphere packaging[J]. Food Control,2014,37(1):62-67.
[15] HATA T,SATO T,ICHIKAWA T,et al. Antifungal activity of protamine salmine hydrochloride andε-poly-L-lysine in actual food systems,rice or wheat-based confectioneries[J]. Journal of Food Processing and Preservation,2016,40(6):1 180-1 187.
[16] ZHU Hui-xia,JIA Shi-ru,YANG Hong-jiang,et al. Characterization of bacteriostatic sausage casing:A composite of bacterial cellulose embedded withε-polylysine[J].Food Science and Biotechnology,2010,19(6):1 479-1 484.
[17] PANDEY A K,KUMAR A. Improved microbial biosynthesis strategies and multifarious applications of the natural biopolymerε-poly-L-lysine[J]. Process Biochemistry,2014,49(3):496-505.
[18]关于批准ε-聚赖氨酸等4种食品添加剂新品种等的公告(2014年第5号)[EB/OL].中华人民共和国国家卫生和计划生育委员会,2014:213-221.
[19]贾士儒.生物防腐剂[M].北京:中国轻工业出版社,2009.
[20] WIECHERT W.13C metabolic flux analysis[J]. Metabolic Engineering,2001,3(3):195-206.
[21]钟其顶,李国辉,王道兵,等.气相色谱-燃烧-同位素质谱仪(GC-C-IRMS)测定游离氨基酸的δ13C值[J].酿酒科技,2013(9):7-10.
[22]申铁.13C代谢通量分析平台的建立与改良[D].合肥:中国科学技术大学,2010.
[23] ZAMBONI N,FENDT S,RUHL M,et al.13C-based metabolic flux analysis[J]. Nature Protocols,2009,4(6):878-892.
[24]赵东东,钟其顶,李国辉,等.低丰度13C标记的酿酒酵母代谢通量分析探讨研究[J].酿酒科技,2014(3):13-16.
[25]石召华,黄文芳,陈立军,等.不同干燥方式对地龙提取物中17种氨基酸的影响[J].中成药,2015,37(5):1 135-1 138.
[26] QUEK L E,DIETMAIR S,KROMER J,et al. Metabolic flux analysis in mammalian cell culture[J]. Metabolic Engineering,2010,12(2):161-171.