角蛋白降解能力差异菌株筛选体系的建立
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摘要
以微生物降解为主的羽毛生物炼制是资源化利用其的重要手段之一,但机理不明是限制其进一步应用的瓶颈。突变体库筛选性状变异菌株的正向遗传学是研究分子机理的常用方案,该方案要求高效可靠的筛选方法。然而,目前依靠测酶活方法来决定降解能力,费时费力,难以用于大规模筛选。本文通过优化羽毛培养基成分及接种菌浓度,建立了一种用于筛选不同角蛋白降解能力微生物的筛选体系。利用该体系分析由mini Tn10构建的S. maltophinia DHHJ突变体库,得到了6株羽毛角蛋白降解能力减弱的突变体,筛选结果与酶活测试100%一致。该研究结果为寻找与角蛋白降解相关基因奠定了基础。
Feather biorefinery is one of the important means of resource utilization,but the unclear degrading mechanism is the bottleneck of its further application. The forward genetics of mutant library screening mutant strains is a common scheme to study the molecular mechanism,which requires efficient and reliable screening methods. However,at present,it is time-consuming and laborious to determine the degradation ability by measuring enzyme activity,and it is difficult to be used for large-scale screening. In this paper,a screening system for screening microorganisms with different keratin degradation ability was established by optimizing the composition of the feather medium and the concentration of the inoculum. The S. maltophinia DHHJ mutant library constructed by mini Tn 10 was analyzed by this system,and six mutants with weakened degradation ability of feather keratin were obtained. The screening results were consistent with the enzyme activity test. The results of this study laid a foundation for the search for genes related to keratin degradation.
Feather biorefinery is one of the important means of resource utilization,but the unclear degrading mechanism is the bottleneck of its further application. The forward genetics of mutant library screening mutant strains is a common scheme to study the molecular mechanism,which requires efficient and reliable screening methods. However,at present,it is time-consuming and laborious to determine the degradation ability by measuring enzyme activity,and it is difficult to be used for large-scale screening. In this paper,a screening system for screening microorganisms with different keratin degradation ability was established by optimizing the composition of the feather medium and the concentration of the inoculum. The S. maltophinia DHHJ mutant library constructed by mini Tn 10 was analyzed by this system,and six mutants with weakened degradation ability of feather keratin were obtained. The screening results were consistent with the enzyme activity test. The results of this study laid a foundation for the search for genes related to keratin degradation.
引文
[1]BRANDELLI A.Bacterial Keratinases:Useful Enzymes for Bioprocessing Agroindustrial Wastes and Beyond[J].Food Bioprocess Tech,2008,1(2):105-116.
[2]傅红梅.羽毛粉水解制取可溶性蛋白的工艺研究[J].四川联合大学学报:工程科学版,1997,(6):65-71.
[3]KHOSA M A,ULLAH A.In-situ modification,regeneration,and application of keratin biopolymer for arsenic removal[J].JHazard Mater,2014,278:360-371.
[4]MEADE S J,DYER J M,CALDWELL J P,et al.Covalent Modification of the Wool Fiber Surface:Removal of the Outer Lipid Layer[J].Text Res J,2008,78(11):943-957.
[5]李江,姚斌,范云六.微生物角蛋白酶的分子生物学研究进展[J].工业微生物,2006,36(3):37-42.
[6]CHITTE R,NALAWADE V K,DEY S.Keratinolytic activety from the broth of a feather degrading the mophilic Streptomyces thermoviolaceus strain SD8[J].Lett Appl Microbiol,1999,28(2):131-136.
[7]ALUIGI A,ZOCCOLA M,VINEIS C,et al.Study on the structure and properties of wool keratin regenerated from formic acid[J].Int J Biol Macromol,2007,41(3):266-273.
[8]YIN J,RASTOGI S,TERRY A E,et al.Self-organization of oligopeptides obtained on dissolution of feather keratins in superheated water[J].Biomacromolecules,2007,8(3):800-806.
[9]朱茜.角蛋白单体诱导Stenotrophomonas maltophilia DHHJ菌株产角蛋白酶解析[D].东华大学,2016:9.
[10]王晓云.高产蛋白酶枯草芽孢杆菌的筛选与诱变选育研究[D].济南:山东农业大学,2015:7.
[11]GUPTA R,RAMNANI P.Microbial keratinases and their prospective applications:an overview[J].Appl Microbiol Biotechnol,2006,70(1):21-33.
[12]KORNILLOWICZ-KOWALSKA T,BOHACZ J.Biodegradation of keratin waste:theory and practical aspects[J].Waste Manag,2011,31(8):1689-1701.
[13]吴丽娜,赵小惠,郭凯晴.金枪鱼肉降解用高产蛋白酶菌株的筛选及鉴定[J].湖北农业科学,2017,56(13):2507-2511.
[14]GRADISAR H,KERN S,FRIEDRICH J.Keratinase of Doratomyces microsporu[J].Appl Microbiol Biotechnol,2000,53(2):196-200.
[2]傅红梅.羽毛粉水解制取可溶性蛋白的工艺研究[J].四川联合大学学报:工程科学版,1997,(6):65-71.
[3]KHOSA M A,ULLAH A.In-situ modification,regeneration,and application of keratin biopolymer for arsenic removal[J].JHazard Mater,2014,278:360-371.
[4]MEADE S J,DYER J M,CALDWELL J P,et al.Covalent Modification of the Wool Fiber Surface:Removal of the Outer Lipid Layer[J].Text Res J,2008,78(11):943-957.
[5]李江,姚斌,范云六.微生物角蛋白酶的分子生物学研究进展[J].工业微生物,2006,36(3):37-42.
[6]CHITTE R,NALAWADE V K,DEY S.Keratinolytic activety from the broth of a feather degrading the mophilic Streptomyces thermoviolaceus strain SD8[J].Lett Appl Microbiol,1999,28(2):131-136.
[7]ALUIGI A,ZOCCOLA M,VINEIS C,et al.Study on the structure and properties of wool keratin regenerated from formic acid[J].Int J Biol Macromol,2007,41(3):266-273.
[8]YIN J,RASTOGI S,TERRY A E,et al.Self-organization of oligopeptides obtained on dissolution of feather keratins in superheated water[J].Biomacromolecules,2007,8(3):800-806.
[9]朱茜.角蛋白单体诱导Stenotrophomonas maltophilia DHHJ菌株产角蛋白酶解析[D].东华大学,2016:9.
[10]王晓云.高产蛋白酶枯草芽孢杆菌的筛选与诱变选育研究[D].济南:山东农业大学,2015:7.
[11]GUPTA R,RAMNANI P.Microbial keratinases and their prospective applications:an overview[J].Appl Microbiol Biotechnol,2006,70(1):21-33.
[12]KORNILLOWICZ-KOWALSKA T,BOHACZ J.Biodegradation of keratin waste:theory and practical aspects[J].Waste Manag,2011,31(8):1689-1701.
[13]吴丽娜,赵小惠,郭凯晴.金枪鱼肉降解用高产蛋白酶菌株的筛选及鉴定[J].湖北农业科学,2017,56(13):2507-2511.
[14]GRADISAR H,KERN S,FRIEDRICH J.Keratinase of Doratomyces microsporu[J].Appl Microbiol Biotechnol,2000,53(2):196-200.