College of Liquor and Food Engineering, Guizhou University, Guiyang, 550025, China.
Key Laboratory of Agricultural and Animal Products Storation & Processing of Guizhou Province, Guizhou University, Guiyang, 550025, China.
Appl Microbiol Biotechnol. 2021 Mar;105(5):1925-1941. doi: 10.1007/s00253-021-11160-x. Epub 2021 Feb 9.
The sn-1,3 extracellular lipase from Aspergillus niger GZUF36 (EXANL1) has important potential applications. The cross-linked enzyme aggregate (CLEA) of purified EXANL1 (CLEA-EXANL1) achieved optimum activity recovery (148.5 ± 0.9%), immobilization yield (100 ± 0%), and recovered activity (99.7 ± 0.6%) with 80% tert-butanol as the precipitant, glutaraldehyde (GA) concentration of 30 mM, GA treatment time of 1.5 h, and centrifugal speed of 6000×g. The effect of CLEA strategy on the characterization of EXANL1 was evaluated in this work. CLEA-EXANL1 exhibited a broader optimum pH range (4-6) compared with free EXANL1 (6.5). CLEA-EXANL1 presented optimum activity at 40 °C, which was 5 °C higher than that of free EXANL1. CLEA strategy decreased the maximum reaction rate and increased the Michaelis-Menten constant of EXANL1 when olive oil emulsion was used as a substrate. Moreover, after 30 days, free EXANL1 lost more than 80.0% of its activity, whereas CLEA-EXANL1 retained more than 90.0% of its activity. CLEA strategy improved the tolerance of EXANL1 in polar organic solvents. Fourier transform infrared spectroscopy results showed that the CLEA technique increased the contents of β-sheets and β-turns in EXANL1 and reduced those of α-helixes and irregular crimps. CLEA strategy caused no change in the sn-1,3 selectivity of EXANL1. Therefore, EXANL1 in the form of CLEA is a valuable catalyst in the synthesis of 1,3-diacylglycerol. KEY POINTS: • Cross-linked enzyme aggregate (CLEA) strategy broadened the optimum pH range of sn-1,3 extracellular lipase from Aspergillus niger GZUF36 (EXANL1). • CLEA strategy improved the tolerance of EXANL1 in polar organic solvents. • CLEA strategy caused no change in the positional selectivity of EXANL1.
黑曲霉 GZUF36 来源的 sn-1,3 胞外脂肪酶（EXANL1）具有重要的潜在应用。用 80%叔丁醇作为沉淀剂、戊二醛（GA）浓度为 30mM、GA 处理时间 1.5h、离心速度 6000×g 可使纯化的 EXANL1 的交联酶聚集体（CLEA）达到最佳的酶活回收率（148.5±0.9%）、固定化产率（100%）和酶活回收率（99.7±0.6%）。本工作评价了 CLEA 策略对 EXANL1 特性的影响。与游离 EXANL1（6.5）相比，CLEA-EXANL1 具有更宽的最适 pH 范围（4-6）。CLEA-EXANL1 在 40°C 时表现出最佳活性，比游离 EXANL1 高 5°C。当橄榄油乳液用作底物时，CLEA 策略降低了 EXANL1 的最大反应速率并增加了米氏常数。此外，30 天后，游离 EXANL1 失去了超过 80.0%的活性，而 CLEA-EXANL1 保留了超过 90.0%的活性。CLEA 策略提高了 EXANL1 在极性有机溶剂中的耐受性。傅里叶变换红外光谱结果表明，CLEA 技术增加了 EXANL1 中β-折叠和β-转角的含量，降低了α-螺旋和不规则卷曲的含量。CLEA 策略没有改变 EXANL1 的 sn-1,3 选择性。因此，以 CLEA 形式存在的 EXANL1 是合成 1,3-二酰基甘油的有价值的催化剂。关键点： • 交联酶聚集体（CLEA）策略拓宽了黑曲霉 GZUF36 来源的 sn-1,3 胞外脂肪酶（EXANL1）的最适 pH 范围。 • CLEA 策略提高了 EXANL1 在极性有机溶剂中的耐受性。 • CLEA 策略没有改变 EXANL1 的位置选择性。
