Cloning and expression of a xylanase xyn<scp>B</scp> from Aspergillus niger<scp>IA</scp>‐001 in Pichia pastoris

Fang, Wei; Gao, He; Cao, Yunhe; Shan, Anshan

doi:10.1002/jobm.201300078

Cited by 17 publications

(13 citation statements)

References 42 publications

(49 reference statements)

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“…4a). This result is similar to the xylanases from Aspergillus terreus BCC129 at pH 4.0-10.0 [24], from Aspergillus Niger IA-001at pH 2.2-10.0 [25], from Thermobifida halotolerans YIM 90462 T at pH 4.0-10.0 [26], from Streptomyces sp. CS428 at pH 4.0-13.5 [27] and Streptomyces sp.…”

Section: Enzymatic Characterization Studies Of Xynbsupporting

confidence: 79%

Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71 / Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu

Nie

Zhang

Shan

et al. 2015

Turkish Journal of Biochemistry

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Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu Abstract: Objective: To clone a full-length cDNA sequence of xynB, encoding endo-1,4-β-xylanase of Aspergillus niger C71 and express in Escherichia coli BL21(DE3).Methods: The xynB was cloned using rapid amplification of cDNA ends (RACE) methods. The sequenced DNA was compared with the available sequences from GenBank using the BLASTX program, and the phylogenetic tree of the xylanases was then constructed using MEGA version 5.0. The amino acid sequences of XYNB were submitted to the ESyPred3D Web Server 1.0 for Homology modeling. The XYNB was purified by MonoQ an ion exchange chromatography and analyzed by SDS-PAGE.Results: The results showed that xynB is 678 bp in length, and encodes a 18 amino acid signal peptide as well as a 22 amino acid mature peptide with a calculated molecular weight of 24.127 kDa. Phylogenetic analysis of xynB, three-dimensional structure and overlap analysis of XYNB demonstrated that XYNB has a β-jelly-roll architecture, which is more conversation in the catalytic domain of GH11 xylanases, belonging to the family 11 of glycosyl hydrolases. A maximum enzyme activity of 62,242.33 U•ml -1 was obtained from XYNB. The XYNB with MW of 41 kDa demonstrated a broad pH stability from pH 3.0 to pH 12. 0.5 mM of Fe 2+ could greatly enhance activity of XYNB to 567.02%. The mainly hydrolysis products of beechwood xylan were xylobiose. The hydrolysis products of XYNB effecting on beechwood xylan were analysed by HPLC.Conclusion: The xynB had been successfully expressed in Escherichia coli BL21, with high enzyme activity and a broad pH stability, and it would be a very good application prospect as the feed enzyme.

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Section: Enzymatic Characterization Studies Of Xynbsupporting

confidence: 79%

Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71 / Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu

Nie

Zhang

Shan

et al. 2015

Turkish Journal of Biochemistry

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“…Pichia pastoris GS115 with the vector pPICZa carrying xylanase‐encoding gene‐(XynB‐opt) 2 was preserved in our laboratory . Briefly, the XynB gene encoding a mature xylanase of 225 amino was subcloned into the pPICZαA vector under the control of the alcohol oxidase I (AOX1) promoter and transformed into P. pastoris GS115 .…”

Section: Methodsmentioning

confidence: 99%

The optimization of fermentation conditions for Pichia pastoris GS115 producing recombinant xylanase

Sun

Yan

Zhan

et al. 2020

Engineering in Life Sciences

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Xylanase is a member of an important family of enzymes that has been used in many biotechnological processes. However, the overall cost of enzyme production has been the main problem in the industrial application of enzymes. To obtain maximum xylanase production, statistical approaches based on the Plackett-Burman design and response surface methodology were employed. The results of the statistical analyses demonstrated that the optimal conditions for increased xylanase production were the following: inoculum size, 3.8%; maize meal, 4.5%; histidine, 0.6%; methanol, 1%; culture volume, 20%; bean pulp, 30 g L −1 ; and Tween-80, 0.8%; and pH 5.0. Verification of the optimization demonstrated that 3273 U mL −1 xylanase was observed under the optimal conditions in shake flask experiments. SDS-PAGE results showed that the size of xylanase protein was about 23 kDa. The results showed that the xylanase produced by fermentation came from Aspergillus Niger by MALDI-TOF-MS. The optimized medium resulted in 2.1-and 1.4-fold higher the activity of xylanase compared with the unoptimized medium (the main nutrients are maize meal and bean pulp) and laboratory medium (the main nutrients are yeast extract and peptone), respectively.The optimization of fermentation conditions is an effective means to reduce production cost and improve xylanase activity. K E Y W O R D Smethodology optimization, Plackett-Burman, xylanase Abbreviations: PBS, phosphate-buffered saline; RSM, response surface method.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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“…The purified protein had a molecular mass of 24 kDa with an optimal pH of 5 and an optimal temperature of 50 °C. The specific activity, K m and V max over beechwood xylan were 1916 U mg −1 , 4.4 mg L −1 , and 1429 U mg L −1 , respectively …”

Section: Production Of Cellulases and Hemicellulasesmentioning

confidence: 96%

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

Juturu

2017

ChemBioChem

159

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Pichia pastoris is a well‐known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent‐free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site‐specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α‐factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co‐expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high‐quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol‐II, and hyaluronic acid. The cell‐surface display of enzymes has obviously increased enzyme stability, and high‐level intracellular expression of acyl‐CoA and ethanol O‐acyltransferase, lipase and d‐amino acid oxidase has opened up applications in whole‐cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High‐level expression of various food‐grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening.

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Cloning and expression of a xylanase xynB from Aspergillus nigerIA‐001 in Pichia pastoris

Cited by 17 publications

References 42 publications

Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71 / Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu

Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71 / Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu

The optimization of fermentation conditions for Pichia pastoris GS115 producing recombinant xylanase

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

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