Expression and characterization of two glucuronoyl esterases from Thielavia terrestris and their application in enzymatic hydrolysis of corn bran

Tang, Jiao; Long, Liangkun; Cao, Ya; Ding, Shaojun

doi:10.1007/s00253-019-09662-w

Cited by 18 publications

(9 citation statements)

References 45 publications

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“…The thermophilic fungus T. terrestris and the mesophilic fungus E. parvum are important sources for production of thermotolerant enzymes (Garcia-Huante et al 2017 ; Long et al 2016 ; Tang et al 2019 ). In the present study, two ABF (TtABF51A and EpABF62C) encoding genes were obtained from the two fungi by artificially synthesis or RT-PCR method.…”

Section: Discussionmentioning

confidence: 99%

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

Long

Sun

Lin

et al. 2020

Appl Microbiol Biotechnol

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Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-l-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-l-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone. Key points • Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability. • The thermal stability of GH62 family EpABF62C was dependent on calcium. • Buffer pH affects the thermal stability of the two ABFs. • Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

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Section: Discussionmentioning

confidence: 99%

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

Long

Sun

Lin

et al. 2020

Appl Microbiol Biotechnol

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“…To harness the catalytic ability of GE, understand its specific role, and determine optimum dosages, pure enzyme preparations are required. Notably, the recent increase in publications which evaluate application of GE, show an urgent need for GE production [7–10, 12].…”

Section: Introductionmentioning

confidence: 99%

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

et al. 2019

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Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489–2780 mg L−1), and were secreted at moderate to high percentages of the total extracellular protein (51–94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5–6-fold, reaching enzyme activity levels (1020–202 U L−1) that could allow their direct application.

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“…Glucuronoyl esterase (GE) is a recently defined carbohydrate esterase that has been isolated from several microorganisms [1][2][3][4][5][6]. GE has potential for production of biofuels and biomaterials in lignocellulose biorefineries [7][8][9][10][11][12]. GE cleaves alkali-labile bonds from the lignincarbohydrate complexes at acidic pH [12].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Conacher

García-Aparicio

Coetzee

et al. 2019

Preprint

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Objective: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L -1 ), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U.L -1 ) that could allow their direct application.

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Expression and characterization of two glucuronoyl esterases from Thielavia terrestris and their application in enzymatic hydrolysis of corn bran

Cited by 18 publications

References 45 publications

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

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