脂肪酶产生菌的筛选、鉴定及脱墨的初步研究
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摘要
生物酶脱墨是当今国内外废纸回用研究的热点之一。已有的研究报道证明脂肪酶在生物脱墨过程中具有重要的作用。在脱墨过程中,脂肪酶主要是以分解油墨与纤维表面的连接料的方式释放出油墨粒子,对纤维强度和纸浆得率的影响较小,因此在生物脱墨的过程中有较大的应用潜力。
为了能筛选到对油墨连接料有专一性降解作用的脂肪酶产生菌,本研究从印刷厂车间、排水渠内采集被油墨污染较严重的土样和水样,分离筛选到一株具有较高脂肪酶活性、能利用矿物油为唯一碳源,并分解油墨连接料的菌株LP502。经菌体形态、菌落特征观察,生理生化测定,16S rRNA基因以及gyrB基因全序列分析,初步鉴定该菌为乙酸钙不动杆菌(Acinitobacter calcoaceticus);通过酯酶特异性活性染色研究表明,该酶对脂肪酶底物有专一性催化作用,由两个同功酶构成;对该酶发酵条件进行了优化,最适培养基为:K2HPO4 0.1%,(NH4)2SO4 0.1%,蛋白胨3%,MgSO4.7H2O 0.1%,矿物油0.5%,蔗糖1%,牛肉膏1%;最佳培养条件为:起始pH7.0,20 mL装液量,30℃,发酵时间72 h;酶活力达到25000U/L。利用该酶对废旧新闻纸进行生物脱墨的试验研究,并经过浮选、抄片,制成的再生纸白度值为224.9513,与未经脱墨的再生纸相比,二者白度值相差42.5315,具有统计学意义。该结果表明Acinitobacter calcoaceticus LP502所产脂肪酶能够有效地脱除油墨颗粒。
构建了LP502的基因组文库,为进一步从该菌中克隆编码脂肪酶的基因奠定了基础。
Biological deinking is one of the focuses of wastepaper recycle.Many studies and reports prove that lipase have important effect in the biological de-inking process. Lipase can decompose vehicle material of printing ink to release ink particle from firber. It can increase production efficiency and fiber intensity. So lipase may have promising application in wastepaper de-inking reproduction.
In this study, 176 bacterial strains were isolated from the soil polluted by printing ink by method of enrichmental cultivating with mineral oil as its unique carbon source. One of the bacterial strains named LP502 with high enzyme activity was isolated. According to the strain morphology,colony character, physiological and biochemical experiments,it should belong to Acinetobacter. According to 16S rDNA gene and gyrB gene of LP502 sequences,it ccould be identified as Acinetobacter calcoaceticus.The specail lipase enzyme active dyeing proved that the enzyme could specially hydrolaze lipase substrate. Fermentation process was optimized. The optimized cultural medium of fermentation for lipase production was studied. The optimized cultural medium was constituted with: K2HPO4 0.1%,(NH4)2SO4 0.1%,peptone 3%, MgSO4.7H2O 0.1%,mineral oil 0.5%,sucrose 1%,beef extract 1%, The optimal intivital pH was 7.0,volume of substrate was 20mL,best temperature was 30℃.Under the optimized condition,Enzyme activity reached at 25000U/L afer fermentation for 72h. The enzyme was used for the old newsprinting paper de-inking. After flotating and sheeting, the brightness of reproduced paper was to 224.9513, which was 42.5315 higher than old newspaper without biological deinking. The results suggested that lipase of Acinitobacter calcoaceticus LP502 could make ink particle released from firber in the process of de-inking.
A genomic DNA library of the strain LP502 has been constructed, which set a solid foundation for cloning of the lipase gene.
为了能筛选到对油墨连接料有专一性降解作用的脂肪酶产生菌,本研究从印刷厂车间、排水渠内采集被油墨污染较严重的土样和水样,分离筛选到一株具有较高脂肪酶活性、能利用矿物油为唯一碳源,并分解油墨连接料的菌株LP502。经菌体形态、菌落特征观察,生理生化测定,16S rRNA基因以及gyrB基因全序列分析,初步鉴定该菌为乙酸钙不动杆菌(Acinitobacter calcoaceticus);通过酯酶特异性活性染色研究表明,该酶对脂肪酶底物有专一性催化作用,由两个同功酶构成;对该酶发酵条件进行了优化,最适培养基为:K2HPO4 0.1%,(NH4)2SO4 0.1%,蛋白胨3%,MgSO4.7H2O 0.1%,矿物油0.5%,蔗糖1%,牛肉膏1%;最佳培养条件为:起始pH7.0,20 mL装液量,30℃,发酵时间72 h;酶活力达到25000U/L。利用该酶对废旧新闻纸进行生物脱墨的试验研究,并经过浮选、抄片,制成的再生纸白度值为224.9513,与未经脱墨的再生纸相比,二者白度值相差42.5315,具有统计学意义。该结果表明Acinitobacter calcoaceticus LP502所产脂肪酶能够有效地脱除油墨颗粒。
构建了LP502的基因组文库,为进一步从该菌中克隆编码脂肪酶的基因奠定了基础。
Biological deinking is one of the focuses of wastepaper recycle.Many studies and reports prove that lipase have important effect in the biological de-inking process. Lipase can decompose vehicle material of printing ink to release ink particle from firber. It can increase production efficiency and fiber intensity. So lipase may have promising application in wastepaper de-inking reproduction.
In this study, 176 bacterial strains were isolated from the soil polluted by printing ink by method of enrichmental cultivating with mineral oil as its unique carbon source. One of the bacterial strains named LP502 with high enzyme activity was isolated. According to the strain morphology,colony character, physiological and biochemical experiments,it should belong to Acinetobacter. According to 16S rDNA gene and gyrB gene of LP502 sequences,it ccould be identified as Acinetobacter calcoaceticus.The specail lipase enzyme active dyeing proved that the enzyme could specially hydrolaze lipase substrate. Fermentation process was optimized. The optimized cultural medium of fermentation for lipase production was studied. The optimized cultural medium was constituted with: K2HPO4 0.1%,(NH4)2SO4 0.1%,peptone 3%, MgSO4.7H2O 0.1%,mineral oil 0.5%,sucrose 1%,beef extract 1%, The optimal intivital pH was 7.0,volume of substrate was 20mL,best temperature was 30℃.Under the optimized condition,Enzyme activity reached at 25000U/L afer fermentation for 72h. The enzyme was used for the old newsprinting paper de-inking. After flotating and sheeting, the brightness of reproduced paper was to 224.9513, which was 42.5315 higher than old newspaper without biological deinking. The results suggested that lipase of Acinitobacter calcoaceticus LP502 could make ink particle released from firber in the process of de-inking.
A genomic DNA library of the strain LP502 has been constructed, which set a solid foundation for cloning of the lipase gene.
引文
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