Cloning and Expression of Aspergillus tamarii FS132 Lipase Gene in Pichia pastoris

Shi, Bihong; Zeng, Lingfeng; Song, Haolei; Qiaoqin, Shi; Songgang, Wu

doi:10.3390/ijms11062373

Cited by 10 publications

(3 citation statements)

References 31 publications

(35 reference statements)

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“…This increased the sensitivity of the assay and detection of the pathogen was possible when as low as 10 pg of P. granati DNA was present. Many other researchers have used nested PCR to increase the sensitivity of the reaction for the detection of pathogens1924373839404142.…”

Section: Discussionmentioning

confidence: 99%

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

Yang

Hameed

Zhang

et al. 2017

Sci Rep

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Pilidiella granati, a causal agent of twig blight and crown rot of pomegranate, is an emerging threat that may cause severe risk to the pomegranate industry in the future. Development of a rapid assay for the timely and accurate detection of P. granati will be helpful in the active surveillance and management of the disease caused by this pathogen. In this study, a nested PCR method was established for the detection of P. granati. Comparative analysis of genetic diversity within 5.8S rDNA internal transcribed spacer (ITS) sequences of P. granati and 21 other selected fungal species was performed to design species-specific primers (S1 and S2). This primer pair successfully amplified a 450 bp product exclusively from the genomic DNA of P. granati. The developed method can detect 10 pg genomic DNA of the pathogen in about 6 h. This technique was successfully applied to detect the natural infection of P. granati in the pomegranate fruit. The designed protocol is rapid and precise with a high degree of sensitivity.

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Section: Discussionmentioning

confidence: 99%

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

Yang

Hameed

Zhang

et al. 2017

Sci Rep

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“…In that study, the lipase activity was measured with p-NPP in pH 6 buffer for 30 min, whereas in this study, it was measured with p-NPL in pH 7 buffer for 10 min. Production of crude recombinant A. melanogenum lipase expressed in P. pastoris was lower than productions of recombinant Candida thermophila lipase (33 U/mL) 21) and recombinant Aspergillus tamarii FS132 lipase (20 U/mL) 22) when p-NPP and tributyrin were used as substrates, respectively. The low production level of recombinant A. melanogenum lipase might attribute to the small amount of wet cell used for induction.…”

Section: Cloning Of Lipase Gene From a Melanogenummentioning

confidence: 84%

Cloning, expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

Wongwatanapaiboon

Klinbunga

Ruangchainikom

et al. 2016

Bioscience, Biotechnology, and Biochemistry

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cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35-37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg, Mn, Li, Ca, Ni, CHAPS, DTT, and EDTA and inhibited by Hg, Ag, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.

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“…High-level expression of enzymes is regarded as a prerequisite for commercial biotechnological production. To date, several microbial lipase genes have been cloned and expressed in P. pastoris [Luo et al, 2006;Shi et al, 2010;Yang et al, 2009].…”

Section: Introductionmentioning

confidence: 99%

Molecular Cloning and Heterologous Expression of a True Lipase in <b><i>Pichia pastoris</i></b> Isolated via a Metagenomic Approach

Zheng

Liu

et al. 2012

Microb Physiol

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Lipases are important enzymes for various biotechnological applications. By using functional expression screening, lipZ03, a novel lipase gene, was isolated from a soil-derived metagenomic library. The gene was supposed to encode a protein of 617 amino acids with a C-terminal targeting signal region and four potential N-linked glycosylation sites. The protein sequence shared a conserved GXSXG motif (X represents any amino acid residue) with other microbial lipases. Gene lipZ03 was expressed in Pichia pastoris and the molecular weight was estimated to be approximately 65 kDa by electrophoresis. The optimum reaction temperature and pH value for LipZ03 was 50°C and 9.0, respectively. The enzyme was highly stable in the temperature range of 40–60°C and under alkaline conditions (pH 8–10). Lipolytic activity was significantly enhanced by Ca²⁺ and Mg²⁺ ions, but dramatically inhibited by Cu²⁺, Ni²⁺ and Hg²⁺ ions and EDTA. The purified enzyme preferentially hydrolyzed relatively long-chain triacylglycerols and was a true lipase rather than an esterase. Using a multi-stepwise methanol supply, the purified LipZ03 achieved a conversion yield of biodiesel production up to 74% after 36 h. Some interesting characteristics described here showed that the recombinant lipase may have potential to be a useful enzyme in industrial applications.

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Cloning and Expression of Aspergillus tamarii FS132 Lipase Gene in Pichia pastoris

Cited by 10 publications

References 31 publications

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

Cloning, expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

Molecular Cloning and Heterologous Expression of a True Lipase in <b><i>Pichia pastoris</i></b> Isolated via a Metagenomic Approach

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