耐受有机溶剂的脂肪酶产生菌的筛选及其酶的分离纯化的研究
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摘要
脂肪酶(Lipase,EC3.1.1.3)是分解三脂酰甘油的水解酶类,它不仅能将底物三脂酰甘油水解为丙三醇和脂肪酸,还能催化水解反应的逆反应,其催化作用具有精确的区域选择性和立体选择性。它不仅在异相系统(油-水界面)中发生作用,而且有些脂肪酶在有机相中也能起催化作用。近年来,随着非水相酶学的不断深入,脂肪酶的应用已超出了油-水界面上进行水解反应的范围,被广泛应用于酯合成、手性化合物的拆分、化工合成中间体的选择性保护、高聚物的合成、肽合成等方面,具有广阔的应用前景。
近年来对酶在有机介质中催化作用的研究已有大量报道,而采用完整微生物细胞在有机介质中作为生物催化剂的研究却不多见。由于不同菌种来源的脂肪酶的酶学性质和作用机理不同,同时不同菌种所需的营养与培养条件也不同,探讨菌种对不同溶剂的敏感性可为研究在有机溶剂中的催化反应奠定基础。而对酶的各方面的研究要求必须对酶进行纯化。
本论文对耐受有机溶剂的产脂肪酶菌种进行了筛选及鉴定,优化了产酶条件,并对产生的脂肪酶进行了分离纯化和酶学特性的研究。包括以下研究内容:
1.从成都及周边地区的油脂厂、乳品厂、食堂、果园采集土样35份,筛选得到一株耐受有机溶剂的脂肪酶产生菌,命名为L-42,该菌可以在10%的丙酮、石油醚、环己烷、丙三醇中生长良好。根据菌体形态观察、生理生化鉴定和16S rDNA序列分析,该菌株被鉴定为粘质沙雷氏菌(Serratia marcescens)。L-42所产脂肪酶经酯酶同工酶活性染色鉴定有三个同工酶。
2.对筛选得到的L-42菌株产酶发酵条件进行了优化,确定最佳培养基组成成分为:酵母粉1.0%,可溶性淀粉1.0%,橄榄油1.0%,K2HPO40.2%,KH2PO40.1%,MgSO4·7H2O 0.1%,Tween 80 0.1%,pH7.0。最佳摇瓶培养条件为:温度30℃,转速200r/min。优化条件后所产酶活力可达到优化前的2倍。
3.对L-42菌株所产脂肪酶进行了分离纯化研究。菌体发酵液经过丙酮沉淀、DEAE-Sepharose离子交换和Sephadex G-100凝胶过滤等步骤处理,最终将酶纯化了45.57倍,比活为1166.61 U/mg,回收率为20%。纯化的酶经SDS-PAGE分析为单一蛋白染色带。
4.对酶的部分酶学性质的研究,发现其酶活最适pH为9.0,最适温度为35℃,分子量约为50kDa。测试了K~(+)、Ca~(2+)、Co~(2+)、Fe~(2+)、Cu~(2+)、Mn~(2+)、Mg~(2+)、Ni~(+)等九种金属离子对酶活的影响,发现该酶对金属离子均很敏感,Ca~(2+)、Mg~(2+)、K~(+)、Mn~(2+)、Ni~(+)对酶有激活作用,而Sn~(2+)、Fe~(2+)、Zn~(2+)、Co~(2+)对酶活有抑制作用。
Lipases(EC3.1.1.3) are glycerol ester hydrolases, they can catalyze the hydrolysis of triacyglycerols into free fatty acids,partial acylglycerols and glycerol. Its catalytic effect has the regional selective and the precise stereoselective. It is not only in the out-of-phase system(oil-water interface), which play a role, in addition, they also remain active in a variety of organic solvents. In recent years, lipase applications are beyond the oil- water interface on the scope of the hydrolysis reaction, they are widely used in synthesis and separation chiral compounds, chemical synthesis intermediates selective protection, polymer synthesis, peptide synthesis.
Recently, the enzyme catalysis in organic media have been reported numerously, but a full-cells in organic media as a catalyst for biological research is rare. Because different strain sources of the enzyme lipase have different nature and mechanisms. Study the strain of the sensitivity of different solvents lay the foundation for research in organic solvents in the catalytic reaction. On the various aspects of the research, purification of the enzyme is required.
In this paper, the yield lipase strains were screened and identificated; lipase fermentation conditions were optimized; lipase was purificated and researched its basic properties. The main findings are as follows:
1. A strain of bacterium with high lipase activity was isolated form soil sample. It was identified as Serratia marcescens by physiological and biochemical detection and 16S rDNA sequence analyse, named as L-42.The strain was to tolerant to organic solvents and was can able to grow on liquid medium with 10%(v/v) acetone ,petroleum benzene,hexamethylene,glycerol. There are three isozyme EST produced by L-42.
2. Factors affecting the Serratia sp.SL-11 lipase production were investigated in shake flask level. Obtained results showed that the optimum medium composition and cultural condition were(%):yeast 1.0%,soluble starch 1.0%, olive oil 1.0%, K2HPO4 0.2%, KH2PO4 0.1%; MgSO4·7H2O 0.1%, Tween 80 0.1%, and pH was 7.0,30℃, 200 r/min shaking for 48h. Under such conditions the lipase activity in culture supernatant was 2 times high as the original one.
3. The lipase was purified of acetone precipitation, ion-exchange chromatography on DEAE-Sepharose and Sephadex G-100 gel-chromatography. Obtained one lipase sample, and its specific activity was 1166.61 U/mg, purified 45.57-fold, yield was 20.0%.
4. Some characters of the enzyme were also in identified. The optimum temperature of lipase is 35℃and the optimum pH is 9.0.The molecular weight of the purified L-42 lipase is about 50kDa.The lipase was keenness to metal-ion , the enzyme activity was stimulated by Ca~(2+)、Mg~(2+)、K~(+)、Mn~(2+)、Ni~(+),but inhibited by Sn~(2+)、Fe~(2+)、Zn~(2+)、Co~(2+).It indicated that lipase from L-42 would be applied widely as it showed high thermostabilities and stabilities of wide pH range.
近年来对酶在有机介质中催化作用的研究已有大量报道,而采用完整微生物细胞在有机介质中作为生物催化剂的研究却不多见。由于不同菌种来源的脂肪酶的酶学性质和作用机理不同,同时不同菌种所需的营养与培养条件也不同,探讨菌种对不同溶剂的敏感性可为研究在有机溶剂中的催化反应奠定基础。而对酶的各方面的研究要求必须对酶进行纯化。
本论文对耐受有机溶剂的产脂肪酶菌种进行了筛选及鉴定,优化了产酶条件,并对产生的脂肪酶进行了分离纯化和酶学特性的研究。包括以下研究内容:
1.从成都及周边地区的油脂厂、乳品厂、食堂、果园采集土样35份,筛选得到一株耐受有机溶剂的脂肪酶产生菌,命名为L-42,该菌可以在10%的丙酮、石油醚、环己烷、丙三醇中生长良好。根据菌体形态观察、生理生化鉴定和16S rDNA序列分析,该菌株被鉴定为粘质沙雷氏菌(Serratia marcescens)。L-42所产脂肪酶经酯酶同工酶活性染色鉴定有三个同工酶。
2.对筛选得到的L-42菌株产酶发酵条件进行了优化,确定最佳培养基组成成分为:酵母粉1.0%,可溶性淀粉1.0%,橄榄油1.0%,K2HPO40.2%,KH2PO40.1%,MgSO4·7H2O 0.1%,Tween 80 0.1%,pH7.0。最佳摇瓶培养条件为:温度30℃,转速200r/min。优化条件后所产酶活力可达到优化前的2倍。
3.对L-42菌株所产脂肪酶进行了分离纯化研究。菌体发酵液经过丙酮沉淀、DEAE-Sepharose离子交换和Sephadex G-100凝胶过滤等步骤处理,最终将酶纯化了45.57倍,比活为1166.61 U/mg,回收率为20%。纯化的酶经SDS-PAGE分析为单一蛋白染色带。
4.对酶的部分酶学性质的研究,发现其酶活最适pH为9.0,最适温度为35℃,分子量约为50kDa。测试了K~(+)、Ca~(2+)、Co~(2+)、Fe~(2+)、Cu~(2+)、Mn~(2+)、Mg~(2+)、Ni~(+)等九种金属离子对酶活的影响,发现该酶对金属离子均很敏感,Ca~(2+)、Mg~(2+)、K~(+)、Mn~(2+)、Ni~(+)对酶有激活作用,而Sn~(2+)、Fe~(2+)、Zn~(2+)、Co~(2+)对酶活有抑制作用。
Lipases(EC3.1.1.3) are glycerol ester hydrolases, they can catalyze the hydrolysis of triacyglycerols into free fatty acids,partial acylglycerols and glycerol. Its catalytic effect has the regional selective and the precise stereoselective. It is not only in the out-of-phase system(oil-water interface), which play a role, in addition, they also remain active in a variety of organic solvents. In recent years, lipase applications are beyond the oil- water interface on the scope of the hydrolysis reaction, they are widely used in synthesis and separation chiral compounds, chemical synthesis intermediates selective protection, polymer synthesis, peptide synthesis.
Recently, the enzyme catalysis in organic media have been reported numerously, but a full-cells in organic media as a catalyst for biological research is rare. Because different strain sources of the enzyme lipase have different nature and mechanisms. Study the strain of the sensitivity of different solvents lay the foundation for research in organic solvents in the catalytic reaction. On the various aspects of the research, purification of the enzyme is required.
In this paper, the yield lipase strains were screened and identificated; lipase fermentation conditions were optimized; lipase was purificated and researched its basic properties. The main findings are as follows:
1. A strain of bacterium with high lipase activity was isolated form soil sample. It was identified as Serratia marcescens by physiological and biochemical detection and 16S rDNA sequence analyse, named as L-42.The strain was to tolerant to organic solvents and was can able to grow on liquid medium with 10%(v/v) acetone ,petroleum benzene,hexamethylene,glycerol. There are three isozyme EST produced by L-42.
2. Factors affecting the Serratia sp.SL-11 lipase production were investigated in shake flask level. Obtained results showed that the optimum medium composition and cultural condition were(%):yeast 1.0%,soluble starch 1.0%, olive oil 1.0%, K2HPO4 0.2%, KH2PO4 0.1%; MgSO4·7H2O 0.1%, Tween 80 0.1%, and pH was 7.0,30℃, 200 r/min shaking for 48h. Under such conditions the lipase activity in culture supernatant was 2 times high as the original one.
3. The lipase was purified of acetone precipitation, ion-exchange chromatography on DEAE-Sepharose and Sephadex G-100 gel-chromatography. Obtained one lipase sample, and its specific activity was 1166.61 U/mg, purified 45.57-fold, yield was 20.0%.
4. Some characters of the enzyme were also in identified. The optimum temperature of lipase is 35℃and the optimum pH is 9.0.The molecular weight of the purified L-42 lipase is about 50kDa.The lipase was keenness to metal-ion , the enzyme activity was stimulated by Ca~(2+)、Mg~(2+)、K~(+)、Mn~(2+)、Ni~(+),but inhibited by Sn~(2+)、Fe~(2+)、Zn~(2+)、Co~(2+).It indicated that lipase from L-42 would be applied widely as it showed high thermostabilities and stabilities of wide pH range.
引文
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