High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements

Zhang, Junjiao; Kang, Zhen; Ling, Zhenmin; Cao, Wenlong; Liu, Long; Wang, Miao; Du, Guocheng; Chen, Jian

doi:10.1016/j.biortech.2013.07.129

Cited by 54 publications

(23 citation statements)

References 35 publications

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“…[17], the extracellular Pel production achieved in the present study exhibited an 8.54-fold increase, and the secretory capability increased from 69.3 to 89.2%. Pels have also been expressed in other expression systems, such as B. subtilis [18,19] and Pichia pastoris [20,21], among which the highest yield reported previously was 2,138 U mL −1 in B. subtilis after 44-h cultivation in a 7.5-L fermentor [22]. This is 1.15-fold lower than the production reported in this study.…”

Section: Discussionmentioning

confidence: 49%

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Wang

Zhou

et al. 2014

BMC Biotechnol

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BackgroundBiotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli.ResultsA Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL−1, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg−1 on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca2+ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber.ConclusionsThis work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production.

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Section: Discussionmentioning

confidence: 49%

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Wang

Zhou

et al. 2014

BMC Biotechnol

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“…Heterologous expression is an effective strategy to improve the target protein production, and many strategies have been carried out to improve the protein yield [3, 9, 29–31]. It has been previously shown that SPP can play an important role during protein secretion [32], However, prior to the current study, no research has been reported on the improvement of protein secretion by overexpression of SPP.…”

Section: Discussionmentioning

confidence: 99%

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

Cai

Wang

et al. 2017

Microb Cell Fact

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BackgroundSignal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases.ResultsIn this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins.ConclusionsOur results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0688-7) contains supplementary material, which is available to authorized users.

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“…The resulting 1557-bp DNA fragment was purified, digested with EcoRI and BamHI, and subsequently ligated into each of the seven pMA0911 derivatives that harbor the signal peptides oppA, bpr, yvgO, amyX, wapA, yclQ, and nprE to generate the corresponding plasmids pMA0911-1, pMA0911-2, pMA0911-3, pMA0911-4, pMA0911-5, pMA0911-6, and pMA0911-7 (Brockmeier et al 2006) (Table 1). Each recombinant plasmid was transformed into the host strain B. subtilis WB600 to obtain the recombinant strains WB600-1, WB600-2, WB600-3, WB600-4, WB600-5, WB600-6, and WB600-7, respectively (Zhang et al 2013). The vector pMA0911 without insert was transformed into B. subtilis WB600 and used as the control.…”

Section: Construction and Expression Of Recombinant Plasmids In B Sumentioning

confidence: 99%

Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater

Zhang

Song

et al. 2015

Environ Sci Pollut Res

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Fungal laccases are typically unstable at high pH and temperature conditions, which limit their application in the decolorization of textile wastewater. By contrast, the highly stable bacterial laccases can function within a wider pH range and at high temperatures, thus have significant potential in treatment for textile wastewater. In our previous work, a thermo-alkali-stable CotA-laccase gene was cloned from Bacillus pumilus W3 and overexpressed in Escherichia coli. In this study, the robust CotA-laccase achieved efficient secretory expression in Bacillus subtilis WB600 by screening a suitable signal peptide. A maximum CotA-laccase yield of 373.1 U/mL was obtained at optimum culture conditions in a 3-L fermentor. Furthermore, the decolorization and detoxification of textile industry effluent by the purified recombinant CotA-laccase in the presence and absence of redox mediators were investigated. Among the potential mediators that enhanced effluent decolorization, acetosyringone (ACS) was the most effective. The toxicity of the CotA-laccase-ACS-treated effluent was greatly reduced compared with that of the crude effluent. To the best of our knowledge, this study is the first to report on the heterologous expression of CotA-laccase in B. subtilis. The recombinant strain B. subtilis WB600-5 has a great potential in the industrial production of this bacterial enzyme, and the CotA-laccase-ACS system is a promising candidate for the biological treatment of industrial textile effluents.

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High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements

Cited by 54 publications

References 35 publications

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater

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