Abstract

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.

中文翻译：

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交联酶聚集策略对黑曲霉GZUF36 sn-1,3细胞外脂肪酶特性的影响

摘要

黑曲霉GZUF36（EXANL1）的sn-1,3细胞外脂肪酶具有重要的潜在应用。纯化的EXANL1（CLEA-EXANL1）的交联酶聚集体（CLEA）达到了最佳活性回收率（148.5±0.9％），固定化率（100±0％）和回收活性（99.7±0.6％）（80％叔） -丁醇作为沉淀剂，戊二醛（GA）浓度为30 mM，GA处理时间为1.5 h，离心速度为6000× g。在这项工作中评估了CLEA策略对EXANL1表征的影响。与游离EXANL1（6.5）相比，CLEA-EXANL1具有更宽的最佳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。当使用橄榄油乳液作为底物时，CLEA策略降低了EXANL1的最大反应速率并提高了Michaelis-Menten常数。此外，在30天后，游离EXANL1丧失了超过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-转折的含量，减少了α-螺旋和不规则卷曲的含量。CLEA策略不会改变EXANL1的sn-​​1,3选择性。因此，CLEA形式的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）策略扩大了黑曲霉GZUF36（EXANL1）的sn-1,3细胞外脂肪酶的最佳pH范围。

•CLEA策略提高了EXANL1在极性有机溶剂中的耐受性。

•CLEA策略不会改变EXANL1的位置选择性。