| 摘要: |
| 【目的】从土壤中分离筛选羽毛降解菌株，检测其产酶条件，研究其所产酶的酶学性质，以丰富角蛋白降解的菌株资源。【方法】从土壤样品中,以牛奶培养基和羽毛培养基筛选羽毛降解菌株，失重法测定羽毛降解率，并对筛选菌株进行形态观察、生理生化检测及16S rRNA序列鉴定。筛选菌株于37 ℃、180 r/min条件下，在以羽毛为唯一碳氮源的培养基中发酵，采用福林酚法测定其角蛋白酶活力，对培养时间（3，6，9，12，15，18，21和24 h）、接种量（体积分数1.5%，3.0%和6.0%）、发酵温度（22，27，32，37和42 ℃）及培养基初始pH（6.0，6.5，7.0，7.5和8.0）进行优化，并研究温度（30，40，50，60，70和80 ℃）、pH（6.0，7.0，8.0，9.0和10.0）、金属离子（K+，Mg2+，Ca2+，Fe3+，Zn2+，Mn2+，Cu2+和Ni2+）、化学试剂（二硫基苏糖醇（DTT）、乙二胺四乙酸（EDTA）、苯甲基磺酰氟（PMSF）、十二烷基硫酸钠（SDS）、β-巯基乙醇、异丙醇和二甲基亚砜（DMSO））和不同底物（酪蛋白、角蛋白、牛血清蛋白、牛血红蛋白、天青角蛋白和羽毛粉）对角蛋白酶活力的影响。【结果】从高温处理的土壤样品中筛选到1株羽毛降解菌DHW 06，形态观察、生理生化检测及16S rRNA序列分析初步鉴定为蜡样芽孢杆菌（Bacillus cereus）。在培养时间10 h、接种量为体积分数3.0%、发酵温度37 ℃和培养基初始pH 6.5的条件下发酵，最大酶活力达到129.47 U/mL。酶学特性结果表明，该酶的最适反应温度为60 ℃，最适反应pH为8.0，在30~50 ℃时具有较好的热稳定性。10 mmol/L的Mn2+使相对酶活力提高300%，10 mmol/L的Cu2+使相对酶活力提高120%；而1 mmol/L的Zn2+使相对酶活力丧失12%，1 mmol/L的Fe3+使相对酶活力丧失53%。体积分数为10%的β 巯基乙醇使相对酶活力提高1 658.95%，10 mmol/L的DTT使相对酶活力提高577.99%；而10 mmol/L的PMSF使相对酶活力丧失35.88%。该酶具有广泛的底物适应能力，对角蛋白的降解能力最强。【结论】筛选出1株可降解羽毛的菌株DHW-06，明确了其最优的产酶条件和酶学特性。 |
| 关键词: 蜡样芽孢杆菌 羽毛降解 发酵产酶 酶学性质 |
| DOI： |
| 分类号: |
| 基金项目:微生物代谢国家重点实验室开放资金项目（MMLKF14-09）；国家自然科学基金项目（31601700） |
|
| Isolation and identification and enzyme producing characteristics of feather-degrading bacteria DHW-06 |
|
DONG Mengmeng,HAO Pengze,WANG Jialiu,
|
| Abstract: |
| 【Objective】This study screened bacteria strains from soil samples with ability of feather degradation，determined its enzyme production conditions and investigated kinetics of isolated enzymes to expand strain resources for keratin degradation.【Method】The milk agar and feather culture medium were used to cultivate and screen feather-degrading strains from soil samples,and degrading rate was identified by weight losing assay.The morphology,physiology and biochemistry of the isolated strain was studied and 16S rRNA sequencing was performed to identify its taxonomy.The strain was cultivated in medium with feather as sole source of carbon and nitrogen at 37 ℃ and 180 r/min.Foline-phenol method was used to measure the keratinase activity.The optimal conditions for keratinase production was determined after tests with different incubation periods (3,6,9,12,15,18,21 and 24 h), inoculum (volume fractions 1.5%,3.0% and 6.0%),fermentation temperatures (22,27,32,37 and 42 ℃) and initial pH (6.0,6.5,7.0,7.5 and 8.0) of culture medium.The effects of temperature (30，40，50，60，70 and 80 ℃),pH (6.0，7.0，8.0，9.0 and 10.0),metal ions (K+,Mg2+,Ca2+,Fe2+,Zn2+,Mn2+,Cu2+ and Ni2+),chemical reagents (dithiothreitol (DTT),ethylenediaminetetraacetic acid (EDTA),phenylmethanesulfonyl fluoride (PMSF),sodium dodecyl sulfate (SDS),β-mercaptoethanol,isopropanol and dimethyl sulfoxide (DMSO)) and substrates (casein,keratin,BSA,hemoglobin,azure keratin and feather meal) on keratinase activity were also studied.【Result】A feather degrading bacterial strain was isolated from high temperature treated soil samples,and preliminary study on its morphology,physiology,biochemistry and 16S rRNA sequencing classified it as a novel strain of Bacillus cereus,which was named DHW 06.The optimal growth conditions for maximum enzymatic activity of 129.47 U/mL were cultivation time of 10 h,inoculum volume fraction of 3.0%,fermentation temperature of 37 ℃ and initial pH of 6.5.The kinetics study showed that the optimal temperature and pH were 60 ℃ and 8.0,and it had good thermal stability from 30 ℃ to 50 ℃.Additionally,10 mmol/L Mn2+ and 10 mmol/L Cu2+ increased the relative enzyme activity by 300% and 120%,but 1 mmol/L Zn2+ and 1 mmol/L Fe3+ reduced it by 12% and 53%,respectively.Furthermore,β-mercaptoethanol at volume fraction of 10% and 10 mmol/L DTT increased the relative enzyme activity by 1 658.95% and 577.99%,while 10 mmol/L PMSF caused a decrease of 35.88%.The enzyme was able to degrade a wide range of substrates and keratin was the most effective substrate.【Conclusion】This study isolated a novel Bacillus cereus strain DHW-06 with feather degrading ability and its optimal enzyme production conditions and enzymatic characteristics were determined. |
| Key words: <8>Bacillus cereus feather degradation enzyme production by fermentation enzymatic properties |
