Error-prone PCR of a fungal xylanase for improvement of its alkaline and thermal stability

Stephens, D. E.; Singh, Surendra; Permaul, Kugen

doi:10.1111/j.1574-6968.2009.01519.x

Cited by 51 publications

(25 citation statements)

References 35 publications

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“…This result showed that the subtle changes of amino acids did not make drastic changes for biochemical parameters of xylanase, even though xylanase activity has been improved by gene modification. This is consistent with the previous report, in which it was found that gene mutation did not change the optimal temperature and thermal stability of original genetics, compared with the native xylanase (ZHOU et al, 2008;CHEN et al, 2018); however, the other reports indicated that gene modification improved pH range and thermal stability of xylanase (CHEN et al, 2001;STEPHENS et al, 2009). Maybe the different modified points in xylanase gene will have different effects on biochemical parameters of xylanase.…”

Section: The Mutant Xylanase Gene Expression In P Pastoris and Xylansupporting

confidence: 82%

“…A transparent zone will be formed around the positive colony if the colony is able to secrete xylanase for degrading xylan to cause the red color disappeared. The previous researches have obtained some good mutant genes by error-prone PCR technique for alkali resistance and high stability of xylanase (CHEN et al, 2001;STEPHENS et al, 2009), in agreement with this study.…”

Section: Error-prone Pcr and Base Modification Of Xylanase Genesupporting

confidence: 78%

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Xylanase gene mutation by error-prone pcr and expression in Pichia pastoris

Li¹,

Huang²,

Chang³

et al. 2018

Biosci. J.

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Xylanase can hydrolyze xylan for reducing its anti-nutritional impact and improving nutrient availability, so obtaining suitable xylanase to degrade xylan is essential. Error-prone PCR and gene transformation were used in this study to obtain the ideal xylanase for degrading xylan effectively. The result showed that one mutant xylanase gene with high xylanase expression was obtained. After the mutant xylanase gene was connected with pGAPZαA and transformed into Pichia pastoris (P. pastoris), the recombinant P. pastoris with mutant gene was found to produce higher xylanase activity (0.1480 U/mL) than that with the native xylanase gene (0.1360 U/mL) after 12 h incubation (p<0.05). The optimal temperature and pH of xylanase expressed by native and mutant genes were the same, i.e. 40°C and 5.50 (p<0.05). In addition, adding 0.2% Tween 80 during recombinant P. pastoris incubation could significantly increase xylanase yield by about 30-35% (p<0.05). The mutant xylanase could significantly increase xylose yield from wheat meal more than the native xylanase (p<0.05).

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Section: The Mutant Xylanase Gene Expression In P Pastoris and Xylansupporting

confidence: 82%

Section: Error-prone Pcr and Base Modification Of Xylanase Genesupporting

confidence: 78%

Xylanase gene mutation by error-prone pcr and expression in Pichia pastoris

Li¹,

Huang²,

Chang³

et al. 2018

Biosci. J.

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show abstract

“…The locations of glycosylated residues Asn37 and Asn88 in the structure of XynCDBFV were indicated in the left panel. b The enzyme activities of the wild-type and deglycosylated mutants were determined at the indicated temperatures, and the relative activity of each sample was presented as a percentage of the value of wild type at 55°C reactivity at higher temperature and alkaline conditions to meet the demands of bio-bleaching (Paes and O'Donohue 2006;Song et al 2012;Stephens et al 2009;Pokhrela et al 2013;Umemoto et al 2009). Notwithstanding, the needs for enzymes with higher reactivity at milder conditions remain.…”

Section: Discussionmentioning

confidence: 99%

Improving the catalytic performance of a GH11 xylanase by rational protein engineering

Cheng

Chen

Huang

et al. 2015

Appl Microbiol Biotechnol

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XynCDBFV from Neocallimastix patriciarum is among the most effective xylanases and holds great potentials in a wide variety of industrial applications. In the present study, several active site residues were modified referring to the instrumental information of the complex structure of XynCDBFV and xylooligosaccharides. Among the 12 single active site mutants, W125F and F163W show increased activity comparing to the wild-type protein. The double mutant W125F/F163W was then generated which displayed nearly 20 % increase in the enzyme activity. Although W125F/F163W showed 5 °C reduction in the optimal temperature, it still preserves similar thermostability and is more active than the wild-type enzyme at temperatures lower than 60 °C. These properties make the double mutant a suitable candidate for commercial applications that involve lower operating temperatures. Furthermore, we investigated the effect of N-glycosylation on the thermostability of XynCDBFV when expressed in the yeast strain Pichia pastoris for industrial use. Two potential glycosylation sites (Asn-37 and Asn-88) were examined, and their roles in enzyme performance were validated. We found that the N-glycosylations of XynCDBFV are related to both catalytic activity and heat stability, with Asn-37 motif playing a dominant role. Collectively, the enzymatic properties of XynCDBFV were improved by molecular engineering, and the influences of N-glycosylations on the enzyme have been clearly elucidated herein.

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“…PCR amplicons of the desired length were purified using ethanol precipitation and sequenced with an ABIPRISM DNA Sequencer. All chromatograms were viewed using Chromas LITE version 2.1.1 (Technelysium Pty Ltd) (Stephens et al 2009). The similarity between generated and published sequences was determined using NCBI reference sequences with BLAST.…”

Section: Methodsmentioning

confidence: 99%

First report of the molecular detection of Ancylostoma caninum in Lahore, Pakistan: the threat from pets

Rehman¹,

Akhtar²,

Akbar³

et al. 2017

Vet. Med. - Czech

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ABSTRACT:The molecular prevalence of Ancylostoma caninum was determined in stray and pet dogs in Lahore, Pakistan from July 2014 to August 2015. A total of 500 dog faecal samples were first evaluated using a sedimentation technique and further through a PCR assay targeting the ITS-2 region of the A. caninum genome. Overall, 130 (26%) samples were positive for hookworm infestation by microscopic examination. Only microscopically positive samples were processed for PCR and 89 (17.6%) were positive for A. caninum. Sequence analysis of amplicons showed 100% homology with A. caninum and the genotypes clustered in one clade with Brazilian A. caninum hookworms. There was a significantly (P < 0.05) increased prevalence in male dogs younger than six months old. Labrador retrievers had higher A. caninum incidence compared to German shepherds and other breeds. To the best of our knowledge, this is the first report of the molecular prevalence of A. caninum in dogs in Pakistan. The novel results of the present study allow us to conclude that A. caninum is more prevalent in pet dogs, especially puppies, and this can be a potential threat for humans that come into contact with such animals. Therefore, the routine monitoring of pets, especially the more susceptible breeds, is essential for disease control.

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Error-prone PCR of a fungal xylanase for improvement of its alkaline and thermal stability

Cited by 51 publications

References 35 publications

Xylanase gene mutation by error-prone pcr and expression in Pichia pastoris

Xylanase gene mutation by error-prone pcr and expression in Pichia pastoris

Improving the catalytic performance of a GH11 xylanase by rational protein engineering

First report of the molecular detection of Ancylostoma caninum in Lahore, Pakistan: the threat from pets

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