产脂肪酶木霉的分离筛选鉴定及酶学性质研究
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摘要
从北京、河北任丘、保定、张家口、新疆、云南、蒙古等地采集了23份土壤样品分离出木霉菌株114株,筛选到4株产脂肪酶的木霉菌株,编号为:153-1、13-2、30425、西1-1,根据菌株的菌落形态、显微镜观察和ITSrDNA序列测定,鉴定该4菌株分别为哈茨木霉(Trichoderma harzianum)、长枝木霉(T. longibrachiatum)、哈茨木霉(T.harzianum)、长枝木霉(T. longibrachiatum),其酶活分别为4.79 U/ml、2.41 U/ml、5.32 U/ml、3.30 U/ml。
研究了脂肪酶的酶学性质,菌株153-1产生的脂肪酶最适反应温度为45℃,最适反应pH为9.5,金属离子Ca~(2+)、Mg~(2+)、Cu~(2+)、Pb~(2+)等对其活力有抑制作用,而Fe~(2+)、Ba~(2+)、Li~(2+)对其有激活作用;菌株13-2产生的脂肪酶最适反应温度为40℃,最适反应pH为10.0,金属离子Mn~(2+)、Cu~(2+)等对其活力有抑制作用,而K~+、Fe~(2+)、Ba~(2+)对其有激活作用;菌株30425产生的脂肪酶最适反应温度为20℃,最适反应pH为7.5,金属离子Li~(2+)、Mn~(2+)、Cu~(2+)等对其活力有抑制作用,而K~+、Ca~(2+)、Mg~(2+)对其有激活作用;菌株西1-1产生的脂肪酶最适反应温度为20℃,最适反应pH为9.5,金属离子Ca~(2+)、Cu~(2+)、Pb~(2+)等对其活力有抑制作用,而K~+、Ba~(2+)对其有激活作用。
对4个菌株所产脂肪酶进行了分离纯化。菌株发酵液经离心浓缩、硫酸铵沉淀、HiPrep DEAE Fast Flow离子交换层析或Resource Q离子交换层析、Superose12凝胶过滤层析等步骤处理,153-1得到组分单一的脂肪酶,13-2得到两个组分的脂肪酶,30425和西1-1未得到有活性的组分,需试验进一步分析。
本试验得到2株产低温脂肪酶的木霉,30425和西1-1;3株产碱性脂肪酶的木霉,153-1、13-2和西1-1;其中西1-1产生的脂肪酶是低温碱性脂肪酶,具有良好的开发前景。
114 strains of Trichoderma spp. were isolated from 23 soil samples collected from Beijing,Renqiu、Baoding and Zhangjiakou of Hebei Province,Xinjiang,Yunnan,Menggu.Four strains of lipase-producing Trichoderma were screened.They were identified as T.harzianum、T. longibrachiatum、T.harzianum、T. longibrachiatum by observation of colony morphology and microscope、determination of ITSrDNA sequence and named as 153-1、13-2、30425、Xi1-1. The lipase activity were 4.79 U/ml、2.41 U/ml、5.32 U/ml、3.30 U/ml.
The enzymatic characteristics of lipase showed that,the optimum temperature and pH of lipase produced by 153-1 for the enzymatic reaction was 45℃and 9.5.The lipase activity was stimulated by Fe~(2+)、Ba~(2+)、Li~(2+),but was inhibited by Ca~(2+)、Mg~(2+)、Cu~(2+)、Pb~(2+) .The optimum temperature and pH of lipase produced by 13-2 for the enzymatic reaction was 40℃and 10.0.The lipase activity was stimulated by K~+、Fe~(2+)、Ba~(2+),but was inhibited by Mn~(2+)、Cu~(2+). The optimum temperature and pH of lipase produced by 30425 for the enzymatic reaction was 20℃and 7.5.The lipase activity was stimulated by K~+、Ca~(2+)、Mg~(2+),but was inhibited by Li~(2+)、Mn~(2+)、Cu~(2+). The optimum temperature and pH of lipase produced by Xi1-1 for the enzymatic reaction was 20℃and 9.5.The lipase activity was stimulated by K~+、Ba~(2+),but was inhibited by Ca~(2+)、Cu~(2+)、Pb~(2+).
The lipase of four strains were purified by centrifuge enrichment,ammonium sulfate precipitation,ion-exchange chromatography on HiPrep DEAE Fast Flow or Resource Q and Superose12 gel-chromatography.The lipase produced by 153-1 got single component,and the lipase produced by 13-2 got two components,but the lipase produced by 30425 and Xi1-1 abtained no active components though purification.It need further test to analysis.
Low temperature lipase from two strains of Trichoderma 30425 and Xi1-1,and alkaline lipase from three strains of Trichoderma 153-1、13-2、Xi1-1were got in this study.The strain of Xi1-1 was low temperature alkaline lipase,it have prospective development.
研究了脂肪酶的酶学性质,菌株153-1产生的脂肪酶最适反应温度为45℃,最适反应pH为9.5,金属离子Ca~(2+)、Mg~(2+)、Cu~(2+)、Pb~(2+)等对其活力有抑制作用,而Fe~(2+)、Ba~(2+)、Li~(2+)对其有激活作用;菌株13-2产生的脂肪酶最适反应温度为40℃,最适反应pH为10.0,金属离子Mn~(2+)、Cu~(2+)等对其活力有抑制作用,而K~+、Fe~(2+)、Ba~(2+)对其有激活作用;菌株30425产生的脂肪酶最适反应温度为20℃,最适反应pH为7.5,金属离子Li~(2+)、Mn~(2+)、Cu~(2+)等对其活力有抑制作用,而K~+、Ca~(2+)、Mg~(2+)对其有激活作用;菌株西1-1产生的脂肪酶最适反应温度为20℃,最适反应pH为9.5,金属离子Ca~(2+)、Cu~(2+)、Pb~(2+)等对其活力有抑制作用,而K~+、Ba~(2+)对其有激活作用。
对4个菌株所产脂肪酶进行了分离纯化。菌株发酵液经离心浓缩、硫酸铵沉淀、HiPrep DEAE Fast Flow离子交换层析或Resource Q离子交换层析、Superose12凝胶过滤层析等步骤处理,153-1得到组分单一的脂肪酶,13-2得到两个组分的脂肪酶,30425和西1-1未得到有活性的组分,需试验进一步分析。
本试验得到2株产低温脂肪酶的木霉,30425和西1-1;3株产碱性脂肪酶的木霉,153-1、13-2和西1-1;其中西1-1产生的脂肪酶是低温碱性脂肪酶,具有良好的开发前景。
114 strains of Trichoderma spp. were isolated from 23 soil samples collected from Beijing,Renqiu、Baoding and Zhangjiakou of Hebei Province,Xinjiang,Yunnan,Menggu.Four strains of lipase-producing Trichoderma were screened.They were identified as T.harzianum、T. longibrachiatum、T.harzianum、T. longibrachiatum by observation of colony morphology and microscope、determination of ITSrDNA sequence and named as 153-1、13-2、30425、Xi1-1. The lipase activity were 4.79 U/ml、2.41 U/ml、5.32 U/ml、3.30 U/ml.
The enzymatic characteristics of lipase showed that,the optimum temperature and pH of lipase produced by 153-1 for the enzymatic reaction was 45℃and 9.5.The lipase activity was stimulated by Fe~(2+)、Ba~(2+)、Li~(2+),but was inhibited by Ca~(2+)、Mg~(2+)、Cu~(2+)、Pb~(2+) .The optimum temperature and pH of lipase produced by 13-2 for the enzymatic reaction was 40℃and 10.0.The lipase activity was stimulated by K~+、Fe~(2+)、Ba~(2+),but was inhibited by Mn~(2+)、Cu~(2+). The optimum temperature and pH of lipase produced by 30425 for the enzymatic reaction was 20℃and 7.5.The lipase activity was stimulated by K~+、Ca~(2+)、Mg~(2+),but was inhibited by Li~(2+)、Mn~(2+)、Cu~(2+). The optimum temperature and pH of lipase produced by Xi1-1 for the enzymatic reaction was 20℃and 9.5.The lipase activity was stimulated by K~+、Ba~(2+),but was inhibited by Ca~(2+)、Cu~(2+)、Pb~(2+).
The lipase of four strains were purified by centrifuge enrichment,ammonium sulfate precipitation,ion-exchange chromatography on HiPrep DEAE Fast Flow or Resource Q and Superose12 gel-chromatography.The lipase produced by 153-1 got single component,and the lipase produced by 13-2 got two components,but the lipase produced by 30425 and Xi1-1 abtained no active components though purification.It need further test to analysis.
Low temperature lipase from two strains of Trichoderma 30425 and Xi1-1,and alkaline lipase from three strains of Trichoderma 153-1、13-2、Xi1-1were got in this study.The strain of Xi1-1 was low temperature alkaline lipase,it have prospective development.
引文
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