Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags

Abstract

1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The Rhizopus oryzae lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.

Enhancing the Thermostability and Catalytic Activity of the Lipase from Rhizopus oryzae via Introducing N-Glycosylation

“…Our results indicated that enhancing the thermostability and catalytic activity of ROL through N-glycosylation was equally effective. 32 This approach can be efficiently combined with other non-N-glycosylation modification strategies, thereby developing high-performance biocatalysts.…”

Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system

“…Importantly, we have with this work demonstrated that a pro region is required for efficient secretion and strain stability, and that both pro-ROL or pro-Lip2 could be used for ROL production. In contrast, the pro-ROL region was not used for the expression of ROL in Pichia pastoris by Chow and Nguyen (Chow and Nguyen 2022 ), who used the Saccharomyces cerevisiae prepro region of the alpha factor. Using the strong methanol-inducible AOX1 promoter for ROL production, they could produce wild-type and thermostable variants of ROL at 40 to 120 mg/L in P. pastoris .…”

Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization

“…Amino acid sequences for the selected proteins were taken directly from these reports (Supplementary Data 2). As RoL-WT could not be expressed in E. coli, a previously described mutant which had previous successful E. coli expression (RoL10X) was used 108 . The Ser-His-Asp (SHD) hydrolase group of enzymes was selected from our in-house PET hydrolase library, including two previously reported thermostabilized variants, LCC-ICCG and HotPETase 21,58 .…”