Alteration of Chain-Length Selectivity and Thermostability of
            <i>Rhizopus oryzae</i>
            Lipase via Virtual Saturation Mutagenesis Coupled with Disulfide Bond Design

Huang, Jinsha; Dai, Shuhan; Chen, Xiying; Xu, Li; Yan, Jinyong; Yang, Min; Yan, Yunjun

doi:10.1128/aem.01878-22

Cited by 16 publications

(17 citation statements)

References 66 publications

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“…8 a). A new apolar interaction detected after the mutation further improved the catalytic activity of lipase by influencing substrate binding and product release [ 27 , 64 ]. Thus, a certain degree of increase in the hydrophobic interaction of the substrate binding channel could improve the catalytic efficiency of lipase toward hydrophobic substrates.…”

Section: Resultsmentioning

confidence: 99%

“…A positive value indicates that the mutations decrease the stability of the enzyme. The code for Rosetta Cartesian_ddg is described in our previous research [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

“…The gene sequences of FuncLib designs were amplified by numerous overlap extension PCRs and inserted into the Eco RI/ Not I-digested (Takara, Beijing, China) vector pGAP815α via homologous recombination [ 27 ]. Thereafter, the Sal I-linearized (Takara, Beijing, China) plasmids were integrated into the genome of P. pastoris strain GS115 [ 67 ].…”

Section: Methodsmentioning

confidence: 99%

“…The T m values of single mutation A287P and A265P were, respectively, increased by 2.13 and 0.69 °C. In our previous study [ 27 ], Rosetta Cartesian_ddg was utilized to evaluate the impact of mutation on the stability of Rhizopus oryzae lipase. The virtual saturated mutant library was reduced from 41 to 17, considerably improve the screening efficiency.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production

Huang,

Xie,

Zheng

et al. 2024

Biotechnol Biofuels

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Background Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement. Results In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. Thermomyces lanuginosus lipase (TLL) with high activity and thermostability was obtained using the Pichia pastoris expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt–TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, $$T_{50}^{15}$$ T 50 15 (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt–TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt–TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil. Conclusions An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.

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Section: Resultsmentioning

confidence: 99%

“…A positive value indicates that the mutations decrease the stability of the enzyme. The code for Rosetta Cartesian_ddg is described in our previous research [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production

Huang,

Xie,

Zheng

et al. 2024

Biotechnol Biofuels

Self Cite

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“…After 12 h, the mutant demonstrated a notable hydrolysis rate of 97.2%, outperforming the WT, which achieved only 78.7%. This disparity underscores the positive impact of increased thermostability on catalytic efficiency (Huang et al 2023 ). Additionally, techniques such as B-factor analysis based on structural biology, have been widely employed to enhance the thermal stability of enzymes (Sun et al 2019 ).…”

Section: Enhancing Functional Lipid Synthesis Via Protein Engineeringmentioning

confidence: 99%

Enzyme engineering for functional lipids synthesis: recent advance and perspective

Guan,

Hou,

Yang

et al. 2024

Bioresour. Bioprocess.

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Functional lipids, primarily derived through the modification of natural lipids by various processes, are widely acknowledged for their potential to impart health benefits. In contrast to chemical methods for lipid modification, enzymatic catalysis offers distinct advantages, including high selectivity, mild operating conditions, and reduced byproduct formation. Nevertheless, enzymes face challenges in industrial applications, such as low activity, stability, and undesired selectivity. To address these challenges, protein engineering techniques have been implemented to enhance enzyme performance in functional lipid synthesis. This article aims to review recent advances in protein engineering, encompassing approaches from directed evolution to rational design, with the goal of improving the properties of lipid-modifying enzymes. Furthermore, the article explores the future prospects and challenges associated with enzyme-catalyzed functional lipid synthesis.

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Tuning Thermostability and Catalytic Efficiency of Aflatoxin-Degrading Enzyme by Error-prone PCR

Jia

Tian

Yang

et al. 2023

Appl Microbiol Biotechnol

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Alteration of Chain-Length Selectivity and Thermostability of Rhizopus oryzae Lipase via Virtual Saturation Mutagenesis Coupled with Disulfide Bond Design

Abstract: Rhizopus oryzae lipase (ROL) is very attractive in biotechnology and industry as a safe and environmentally friendly biocatalyst. Functional expression of ROL in Escherichia coli facilitates effective high-throughput screening for positive variants.

Cited by 16 publications

References 66 publications

In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production

In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production

Enzyme engineering for functional lipids synthesis: recent advance and perspective

Tuning Thermostability and Catalytic Efficiency of Aflatoxin-Degrading Enzyme by Error-prone PCR

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