Optimization of the Trichoderma reesei endo-1,4-beta-xylanase production by recombinant Pichia pastoris

He, Jun; Chen, Shuai; Yu, Bing; Zhang, Keying

doi:10.1016/j.bej.2010.06.003

Cited by 10 publications

(2 citation statements)

References 35 publications

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“…To date, many recombinant xylanases from different microorganisms have been successfully expressed in P. pastoris , but the expression yields of each protein are highly dependent on the individual enzyme (Table ). For example, the GH11 Xyn2 from T. reesei Rut C‐30 yielded 261 U mL −1 for 96 h cultivation in P. pastoris , but 105 U mL −1 with XynB from Aspergillus sulphureus . As for the GH11 XynB from A. niger IA‐001, a maximized activity of 15 158 U mL −1 was achieved after 120 h of shaking .…”

Section: Resultsmentioning

confidence: 99%

“…However, the existing enzymes are always limited by their high production costs and low catalytic capability, which has posed challenges to developing an economical and feasible hydrolysis process . To advance the utilization of recombinant xylanase, extensive research on further optimization of recombinant DNA technology for large‐scale expression of the enzyme in an heterologous host system and development of more efficient enzymes are necessary …”

Section: Introductionmentioning

confidence: 99%

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Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system

Long

Zhang

Ren

et al. 2019

J of Chemical Tech & Biotech

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BACKGROUND: Previous work has shown that GH10 endo-xylanase presented better synergistic cooperation with cellulase to achieve effective hydrolysis. However, most currently reported commercial xylanolytic enzymes from Aspergillus niger belong to GH11 and there have been few studies regarding GH10 xylanase. In addition, to increase overall expression of heterologous endo-xylanase in Pichia pastoris, a double-plasmid co-expression method, including the construction of recombinant P. pastoris using one expression vector followed by inserting another vector which targeted to different locus, was introduced. RESULTS: A GH10 xylanase gene from A. niger BE-2 (XynC) was optimized and constitutively expressed in P. pastoris GS115.Compared with the conventional single-plasmid method, it appeared that the double-plasmid strategy could considerably increase the XynC yield by ∼33%. To further improve enzyme production, cultivation conditions were optimized at shake-flask level and then scaled up to a 5-L bioreactor. Through high cell-density fermentation, the expression level of GH10 XynC reached 1650 U mL −1 . The XynC showed maximum activity at 55 ∘ C and pH 5.0, and exhibited an excellent stability over a wide range of pH from 4.5 to 7.0. The kinetic parameters K m and V max values for beechwood xylan were 3.5 mg mL −1 and 2327 U mg −1 , respectively. CONCLUSION: The double-plasmid co-expression strategy introduced herein could greatly improve the XynC yield in P. pastoris. The GH10 xylanase from A. niger BE-2 shared similar reaction conditions with the existing enzymatic hydrolysis, which might be a good candidate to assist cellulases for industrial application. Biochemical properties of the xylanase XynCIn order to avoid or minimize interference in the assay by media components, XynC expressed from P. pastoris-D was purified wileyonlinelibrary.com/jctb a Shake-flask level. b Fermentor level. J Chem Technol Biotechnol 2020; 95: 535-543

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system

Long

Zhang

Ren

et al. 2019

J of Chemical Tech & Biotech

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Cloning and Expression of a Novel Xylanase Gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris

Zhang

et al. 2012

Appl Biochem Biotechnol

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A full-length complementary DNA (cDNA) of Auxyn11D, a gene that encodes a novel endo-β-1,4-D-xylanase of Aspergillus usamii E001 (abbreviated to AuXyn11D), was obtained using 3'- and 5'-rapid amplification of cDNA ends (RACE) methods. The cDNA sequence is 855 bp in length, containing 5'- and 3'-untranslated regions and a 696-bp open reading frame (ORF) that encodes a 32-aa signal peptide and a 199-aa mature peptide (namely AuXyn11D). Multiple homology alignment of amino acid sequences verified that AuXyn11D belongs to glycoside hydrolase family 11. Moreover, a mature peptide-encoding cDNA fragment of Auxyn11D was cloned and expressed in Pichia pastoris GS115. One P. pastoris transformant expressing the highest recombinant AuXyn11D (reAuXyn11D) activity of 15.0 U/mL, labeled as P. pastoris GSAuXyn4-16, was chosen by shake flask test. SDS-PAGE assay demonstrated that the reAuXyn11D, a glycosylated protein with an apparent molecular mass of 32.0 kDa, was secreted into the medium. The purified reAuXyn11D displayed the highest activity at pH 4.5 and 55 °C. It was stable at a pH range of 3.5-6.5 and at a temperature of 50 °C or below. Its activity was not significantly affected by most of metal ions tested and EDTA, but increased by Ca(2+) and inhibited by Mn(2+). The K(m) and V(max) of the reAuXyn11D towards birchwood xylan were 6.32 mg/mL and 391.6 U/mg, respectively.

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Extractive Fermentation of Xylanase from Aspergillus tamarii URM 4634 in a Bioreactor

Silva

Queiroz

Nascimento

et al. 2014

Appl Biochem Biotechnol

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Of the many reported applications for xylanase, its use as a food supplement has played an important role for monogastric animals, because it can improve the utilisation of nutrients. The aim of this work was to produce xylanase by extractive fermentation in an aqueous two-phase system using Aspergillus tamarii URM 4634, increasing the scale of production in a bioreactor, partially characterising the xylanase and evaluating its influence on monogastric digestion in vitro. Through extractive fermentation in a bioreactor, xylanase was obtained with an activity of 331.4 U mL(-1) and 72% yield. The xylanase was stable under variable pH and temperature conditions, and it was optimally active at pH 3.6 and 90 °C. Xylanase activity potentiated the simulation of complete monogastric digestion by 6%, and only Mg2+ inhibited its activity. This process provides a system for efficient xylanase production by A. tamarii URM 4634 that has great potential for industrial use.

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Optimization of the Trichoderma reesei endo-1,4-beta-xylanase production by recombinant Pichia pastoris

Cited by 10 publications

References 35 publications

Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system

Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system

Cloning and Expression of a Novel Xylanase Gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris

Extractive Fermentation of Xylanase from Aspergillus tamarii URM 4634 in a Bioreactor

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