菲律宾马岭山森林保护区树冠蕨类植物酵母胞外脂肪酶的筛选、鉴定和优化

F. Elegado, Charisse Leanne B. Legaspi, Joseph Martin Paet, Florabelle Querubin, Jarel Elgin Tolentino, J. Vilela, A. Paguio, J. Maloles, J. Zarate
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引用次数: 4

摘要

脂肪酶(三酰基甘油酰基水解酶,E.C. 3.1.1.3)是广泛应用于工业应用的酶。本研究旨在筛选从菲律宾Makiling森林保护区(MFR)的树冠蕨类植物中分离的脂肪酶产酵母菌,并优化条件,使酶的批量生产和活性最大化。在144株分离菌株中,B1-7在固体培养基(EIA 7.6)和液体培养基(0.082 U/mL-min)中脂肪酶活性最高。经ITS引物分子鉴定和显微观察,该分离物为GRAS安全微生物黄隐球菌(Cryptococcus flavescens)。响应面法(Box-Behnken Design)结果表明,在碳氮比为5.0、pH为6.0、诱导剂(Tween 20)含量为0.5%的条件下,脂肪酶活性为0.66 U/mL-min,生物量为4 g/L。C:N-%诱导剂相互作用和诱导剂浓度对脂肪酶活性有显著影响。经72h补料分批发酵试验,脂肪酶活性比优化结果降低了10倍,且脂肪酶活性与生物量呈负相关(r=-0.405),表明脂肪酶活性与生物量可利用性不相关。最后,总糖浓度保持不变,表明生物利用脂肪酶的降解产物作为碳源。综上所述,MFR中的C. flavescens可用于大规模脂肪酶生产,但建议对其他参数进行研究和优化。脂肪酶(三酰基甘油酰基水解酶,E.C. 3.1.1.3)是广泛应用于工业应用的酶。本研究旨在筛选从菲律宾Makiling森林保护区(MFR)的树冠蕨类植物中分离的脂肪酶产酵母菌,并优化条件,使酶的批量生产和活性最大化。在144株分离菌株中,B1-7在固体培养基(EIA 7.6)和液体培养基(0.082 U/mL-min)中脂肪酶活性最高。经ITS引物分子鉴定和显微观察,该分离物为GRAS安全微生物黄隐球菌(Cryptococcus flavescens)。响应面法(Box-Behnken Design)结果表明,在碳氮比为5.0、pH为6.0、诱导剂(Tween 20)含量为0.5%的条件下,脂肪酶活性为0.66 U/mL-min,生物量为4 g/L。C:N-%诱导剂相互作用和诱导剂浓度对脂肪酶活性有显著影响。经72h补料分批发酵后…
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Screening, identification and optimization of extracellular lipase production of yeast (Cryptococcus flavescens) isolated from a tree canopy fern in the Mount Makiling Forest Reserve, Philippines
Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are enzymes vastly used in industrial applications. The current study aims to screen lipase-producing yeasts isolated from a tree canopy fern from the Makiling Forest Reserve (MFR), Philippines and to optimize conditions that can maximize the mass production and activity of the enzyme. From the 144 isolates, B1-7 showed the highest lipase activity in both solid (EIA 7.6) and liquid selection media (0.082 U/mL-min). Molecular identification using Internally Transcribed Spacer (ITS) primers and microscopic observation revealed that the isolate was Cryptococcus flavescens, a generally regarded as safe (GRAS) microorganism. Response Surface Method (Box-Behnken Design) showed that the maximum lipase activity (0.66 U/mL-min) and a biomass of 4 g/L were achieved at 5.0 Carbon:Nitrogen ratio, pH 6.0 and 0.5% inducer (Tween 20). Also, C:N-% inducer interaction and inducer concentration significantly affected lipase activity. After a 72h fed-batch fermentation experiment, lipase activity was ten-fold lower than the optimization results and a negative correlation (r=-0.405) between lipase activity and biomass suggested the non-dependence of lipase activity to biomass availability. Lastly, total sugar concentration remained constant implying that the organism used the degradative products of lipase as its carbon source. In conclusion, C. flavescens from MFR can be utilized for mass lipase production, but it was recommended that other parameters be examined and optimized.Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are enzymes vastly used in industrial applications. The current study aims to screen lipase-producing yeasts isolated from a tree canopy fern from the Makiling Forest Reserve (MFR), Philippines and to optimize conditions that can maximize the mass production and activity of the enzyme. From the 144 isolates, B1-7 showed the highest lipase activity in both solid (EIA 7.6) and liquid selection media (0.082 U/mL-min). Molecular identification using Internally Transcribed Spacer (ITS) primers and microscopic observation revealed that the isolate was Cryptococcus flavescens, a generally regarded as safe (GRAS) microorganism. Response Surface Method (Box-Behnken Design) showed that the maximum lipase activity (0.66 U/mL-min) and a biomass of 4 g/L were achieved at 5.0 Carbon:Nitrogen ratio, pH 6.0 and 0.5% inducer (Tween 20). Also, C:N-% inducer interaction and inducer concentration significantly affected lipase activity. After a 72h fed-batch fermentation ...
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