Abstract
A cell surface displayed system in Pichia pastoris GS115 was developed by using GCW61, a glycosylphosphatidylinositol-modified cell wall protein from P. pastoris, as the anchor protein. Thermomyces lanuginosus lipase (TLL) was successfully displayed on the P. pastoris cell wall by fusing GCW61 gene with TLL2 gene (NCBI Accession: O59952) that was optimized with codon bias and synthesized. Cell surface displayed TLL2 was confirmed by the immunofluorescence microscopy. Flask fermentation was performed for 144 h with lipase activity up to 1964.76 U/g. Enzymatic properties of cell surface displayed TLL2 were also investigated. Displayed TLL2 occurred the maximum activity at pH 9 and 55°C and demonstrated characteristics of wide thermal adaptability and alkaline pH resistance. The optimum substrate was p-nitrophenyl hexanoate. Bivalent metal ions Ca2+, Mn2+, and Zn2+ had the activation effect on displayed TLL2, while Cu2+, Fe2+, Fe3+, K+, Li+, Na+, and Co2+ ions had the inhibitory effect on it. Since cell surface displayed TLL2 required less purification steps compared with free enzyme and showed high enzyme activities, it would be able to be further applied in various potential applications.
Highlights
Lipases (Triacylglyceroyl hydrolase, EC 3.1.1.3) are one of the most extensive used enzymes in the biocatalyst, which can catalyze a wide range of biological transformations, including hydrolysis, esterification, interesterification, alcoholysis, acidolysis, and ammonolysis (Jaeger and Eggert, 2002; Yamada et al, 2016)
As an promising industrial biocatalyst, Thermomyces lanuginosu lipase (TLL) is able to be used in many different fields, e.g., detergents, cosmetics, modification of oils and fats, production and recycling of biodiesel, organic chemistry, etc. (Prathumpai et al, 2004; Fernandez-Lafuente, 2010; Huang et al, 2012; Li et al, 2014)
The results showed that hydrolytic activities of C4 (p-nitrophenyl butyrate), C6, and C8 (p-nitrophenyl octanoate) were higher than other p-nitrophenyl esters above C10, which suggested that cell surface displayed TLL2 preferred to catalyze the short and medium length chain of aliphatic acid ester
Summary
Lipases (Triacylglyceroyl hydrolase, EC 3.1.1.3) are one of the most extensive used enzymes in the biocatalyst, which can catalyze a wide range of biological transformations, including hydrolysis, esterification, interesterification, alcoholysis, acidolysis, and ammonolysis (Jaeger and Eggert, 2002; Yamada et al, 2016). Lipases exhibit a wide specificity, recognizing very different substrates. This permits them to be as catalysts for very different reactions in a wide range of applications, including detergent, food, medicine, fine chemicals, biodiesel, biosensors, and biodegradation (Rodrigues and Fernandez-Lafuente, 2010a,b). TLL was immobilized on silicate and commercialized with the name of Lipozyme TLIM in 1994 as a detergent additive by Novozymes Co. Nowadays, as an promising industrial biocatalyst, TLL is able to be used in many different fields, e.g., detergents, cosmetics, modification of oils and fats, production and recycling of biodiesel, organic chemistry, etc. Limited by the complicated separation and purification steps, the high cost or price seemed to be the bottleneck for the industrial production
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