Improvement of the thermostability and enzymatic activity of cholesterol oxidase by site-directed mutagenesis

Sun, Yan; Yang, Hai-Ling; Wu, Wei

doi:10.1007/s10529-011-0669-6

Cited by 12 publications

(6 citation statements)

References 13 publications

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“…The LAB strains in this study were stored in Man–Rogosa–Sharpe (MRS) broth with 15% (v/v) glycerol at −70 °C and cultured in 50 mL liquid MRS for 48 h at 37 °C without shaking, as described previously (Wang et al ., ).…”

Section: Methodsmentioning

confidence: 97%

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A new high phenyl lactic acid-yieldingLactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene

Zhang

Shi

et al. 2014

FEMS Microbiol Lett

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Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1) ) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124.

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Section: Methodsmentioning

confidence: 97%

“…Several studies have reported that enzymatic activity could be improved significantly by site-directed mutagenesis of specific amino acid residues surrounding the active centre or binding site (Kang et al, 2007;Sun et al, 2011;Zhang et al, 2013).…”

Section: Comparison Of the Ability To Produce Pla And The Antifungal mentioning

confidence: 99%

A new high phenyl lactic acid-yieldingLactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene

Zhang

Shi

et al. 2014

FEMS Microbiol Lett

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“…Thus, thermal stability modification for lichenase as well as other industrial enzymes, coupled with isolation of thermostable lichenases, has become a research focus during the past few decades [ 5 , 6 ]. Indeed, conventional methods represented by directed evolution, and rational design have contributed to remarkable successes in improving the thermal stability of enzymes [ 7 – 10 ]. Nonetheless, inferior success frequencies for rational design, as well as time consuming in nature, have limited a broad utilization of these methods [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced thermal stability of lichenase from Bacillus subtilis 168 by SpyTag/SpyCatcher-mediated spontaneous cyclization

Wang

et al. 2016

Biotechnol Biofuels

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BackgroundSpyTag is a peptide that can form an irreversible covalent linkage to its 12 kDa partner SpyCatcher via a spontaneous isopeptide bond. Herein, we fused SpyTag at the N-terminal of lichenase and SpyCatcher at C-terminal so that the termini of lichenase were locked together by the covalent interaction between the partners. In addition, an elastin-like polypeptides tag was subsequently attached to the C-terminus of SpyCatcher, thereby facilitating the non-chromatographic purification of cyclized lichenase.ResultsThe study showed that the optimum temperature of the cyclized lichenase was about 5 °C higher in comparison to its linear counterpart. Moreover, nearly 80 % of the cyclized lichenase activities were retained after 100 °C exposure, whereas the linear form lost almost all of its activities. Therefore, the cyclized variant displayed a significantly higher thermal stability as temperature elevated and was resistant to hyperthermal denaturation. Besides, the Km value of the cyclized lichenase (7.58 ± 0.92 mg/mL) was approximately 1.7-fold lower than that of the linear one (12.96 ± 1.93 mg/mL), indicating a higher affinity with substrates.ConclusionsThis new SpyTag/SpyCatcher cyclization strategy is deemed as a generalized reference for enhancing enzyme stability and can be effectively customized to the cyclization of various enzymes, hence a tremendous potential for successful application in the biocatalytic conversion of biomass to produce fuels and chemicals.

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“…To further improve the thermal stability of COX, site direct mutagenesis was applied to the Brevibacterium sp. grown at 50˚C for 2 h. It was observed that three amino acid residues (Q153E, F128L and S143H) located near the FAD-binding site of the enzyme were responsible for an increase in specific activity by 11.6% and 47% of two mutated sites Q153E and F128L, respectively [111]. Further studies on this bacterium showed that 0.15% (w/v) cholesterol emulsified with 0.05% Tween-80 induced the highest amount of COX production in comparison to use of other detergents [112].…”

Section: Stability and Activity Of Coxmentioning

confidence: 99%

Cholesterol Oxidase and Its Applications

Kumari¹,

Kanwar²

2012

AiM

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Cholesterol oxidase (COX), a bi-functional FAD-containing microbial enzyme belongs to the family oxidoreductases. COX catalyses the oxidation of cholesterol into 4-cholesten-3-one. In recent time, cholesterol oxidase has received great attention due to its wider use in clinical (determination of serum cholesterol) laboratories practice and in the biocatalysis for the production of a number of steroids. COX has been shown to possess potent insecticidal activity, besides its use to track cell cholesterol. Moreover, COX is also implicated in the manifestation of some of the diseases of bacterial (tuberculosis), viral (HIV) and non-viral prion origin (Alzheimer's). These applications and disease mechanisms have promoted the need of screening, isolation and characterization of newer microbes from diverse habitats as a source of COX to learn more about its structural and functional aspects. In this review, we discuss microbial sources of COX, its structure and important biochemical properties besides its broad range of biological functions and applications.

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Improvement of the thermostability and enzymatic activity of cholesterol oxidase by site-directed mutagenesis

Cited by 12 publications

References 13 publications

A new high phenyl lactic acid-yieldingLactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene

A new high phenyl lactic acid-yieldingLactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene

Enhanced thermal stability of lichenase from Bacillus subtilis 168 by SpyTag/SpyCatcher-mediated spontaneous cyclization

Cholesterol Oxidase and Its Applications

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