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Effective Expression of the Serratia marcescens Phospholipase A1 Gene in Escherichia coli BL21(DE3), Enzyme Characterization, and Crude Rapeseed Oil Degumming via a Free Enzyme Approach.

ABSTRACT: Crude oil degumming by phospholipid removal is crucial to guarantee oil quality. Phospholipase degumming could produce green vegetable oil by reducing energy consumption and protecting the environment. To develop a novel phospholipase for oil degumming, we cloned the Serratia marcescens outer membrane phospholipase A gene (OM-PLA1) and expressed its 33 KDa protein in engineered Escherichia coli BL21(DE3). OM-PLA1 activity reached 18.9 U mL-1 with the induction of 0.6 mM isopropyl ?-D-1-thiogalactopyranoside for 4 h. The optimum temperature and pH were 50°C and 7.5, respectively. Mg2+, Ca2+, Co2+, and Mn2+ at 0.1 mM L-1 significantly increased OM-PLA1 activity. The kinetic equations of OM-PLA1 and Lecitase Ultra were y = 13.7x+0.74 (Km = 18.53 mM, Vmax = 1.35 mM min-1) and y = 24.42x+0.58 (Km = 42.1 mM, Vmax = 1.72 mM min-1), respectively. The phosphorus content decreased from 22.6 to 9.3 mg kg-1 with the addition of 15 units of free recombinant OM-PLA1 into 150 g of crude rapeseed oil. OM-PLA1 has the close degumming efficiency with Lecitase Ultra. The S. marcescens outer membrane phospholipase gene (OM-PLA1) possessed higher substrate affinity and catalytic efficiency than Lecitase Ultra. This study provides an alternative approach to achieve crude vegetable oil degumming with enzymatic technology.

SUBMITTER: Yang P

PROVIDER: S-EPMC6811509 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Effective Expression of the Serratia marcescens Phospholipase A1 Gene in Escherichia coli BL21(DE3), Enzyme Characterization, and Crude Rapeseed Oil Degumming via a Free Enzyme Approach.

Yang Peizhou P Wu Yun Y Jiang Suwei S Zheng Zhi Z Hou Zhigang Z Mu Dongdong D Xiao Wei W Jiang Shaotong S Yang Yung-Hun YH

Frontiers in bioengineering and biotechnology 20191017

Crude oil degumming by phospholipid removal is crucial to guarantee oil quality. Phospholipase degumming could produce green vegetable oil by reducing energy consumption and protecting the environment. To develop a novel phospholipase for oil degumming, we cloned the Serratia marcescens outer membrane phospholipase A gene (OM-PLA1) and expressed its 33 KDa protein in engineered Escherichia coli BL21(DE3). OM-PLA1 activity reached 18.9 U mL-1 with the induction of ...[more]

PMID: 31681748

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Project description:BACKGROUND:Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3). RESULTS:We show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body. CONCLUSIONS:As strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate.

Project description:IntroductionThe Serratia marcescens enzymes (SMEs) are chromosomally encoded Ambler Class A carbapenem-hydrolysing β-lactamases, which distinctively express resistance to carbapenems while remaining susceptible to extended-spectrum cephalosporins. Global reports of SMEs are infrequent. Here we describe the isolation of an SME-2-producing S. marcescens from the sputum of a patient who was hospitalized at Christchurch Hospital, New Zealand.MethodsAn immunosuppressed asthmatic patient who presented with shortness of breath and hypoxia grew S. marcescens from a sputum culture. Antimicrobial susceptibilities were determined by Phoenix, with MICs of meropenem and imipenem determined by Liofilchem® MIC gradient strips and interpreted according to EUCAST breakpoints. Investigation for carbapenemase was performed using Carba NP, modified CIM (mCIM) and GeneXpert® Carba-R. WGS was performed using the Illumina DNA Prep library kit and sequenced using MiSeq.ResultsThe isolate showed an unusual susceptibility profile, including high-level resistance to meropenem and imipenem, while remaining susceptible to extended-spectrum cephalosporins. The Carba NP and mCIM were positive and WGS demonstrated the presence of a blaSME-2 gene located on the chromosome within the SmarGI1-1 genomic island. In addition, a blaSRT-like class C β-lactamase, aac(6')-Ic aminoglycoside-modifying enzyme and various multidrug efflux mechanisms were found. Phylogenetic core-genome analysis indicated no matching genome with RefSeq database strains.ConclusionsS. marcescens is an opportunistic pathogen of concern, harbouring a variety of intrinsic resistance mechanisms, including the potential for stable AmpC hyperproduction. Globally, SME-type carbapenemases have been infrequently reported; however, isolates carrying this mechanism could have limited treated options, having implications for patient management. To the best of our knowledge this is the first report of SME in New Zealand.

Dataset Information

Effective Expression of the Serratia marcescens Phospholipase A1 Gene in Escherichia coli BL21(DE3), Enzyme Characterization, and Crude Rapeseed Oil Degumming via a Free Enzyme Approach.

Publications

Effective Expression of the <i>Serratia marcescens</i> Phospholipase A1 Gene in <i>Escherichia coli</i> BL21(DE3), Enzyme Characterization, and Crude Rapeseed Oil Degumming via a Free Enzyme Approach.

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