Abstract
BackgroundXylanase degrades xylan into monomers of various sizes by catalyzing the endohydrolysis of the 1,4-β-D-xylosidic linkage randomly, possessing potential in wide industrial applications. Most of xylanases are susceptible to be inactive when suffering high temperature and high alkaline process. Therefore, it is necessary to develop a high amount of effective thermoalkaliphilic xylanases. This study aims to enhance thermoalkaliphilic xylanase production in Pichia pastoris through fermentation parameters optimization and novel efficient fed-batch strategy in high cell-density fermentation.ResultsRecombinant xylanase activity increased 12.2%, 7.4%, 12.0% and 9.9% by supplementing the Pichia pastoris culture with 20 g/L wheat bran, 5 mg/L L-histidine, 10 mg/L L-tryptophan and 10 mg/L L-methionine in shake flasks, respectively. Investigation of nutritional fermentation parameters, non-nutritional fermentation parameters and feeding strategies in 1 L bioreactor and 1 L shake flask revealed that glycerol and methanol feeding strategies were the critical factors for high cell density and xylanase activity. In 50 L bioreactor, a novel glycerol feeding strategy and a four-stage methanol feeding strategy with a stepwise increase in feeding rate were developed to enhance recombinant xylanase production. In the initial 72 h of methanol induction, the linear dependence of xylanase activity on methanol intake was observed (R2 = 0.9726). The maximum xylanase activity was predicted to be 591.2 U/mL, while the actual maximum xylanase activity was 560.7 U/mL, which was 7.05 times of that in shake flask. Recombinant xylanase retained 82.5% of its initial activity after pre-incubation at 80 °C for 50 min (pH 8.0), and it exhibited excellent stability in the broad temperature (60–80 °C) and pH (pH 8.0–11.0) ranges.ConclusionsEfficient glycerol and methanol fed-batch strategies resulting in desired cell density and xylanase activity should be applied in other P. pastoris fermentation for other recombinant proteins production. Recombinant xylanases with high pH- and thermal-stability showed potential in various industrial applications.
Highlights
Xylanase degrades xylan into monomers of various sizes by catalyzing the endohydrolysis of the 1,4-β-Dxylosidic linkage randomly, possessing potential in wide industrial applications
Strains and reagents To obtain the secreted thermoalkaliphilic xylanase, a P. pastoris strain X-33 transformed with pPICZɑ-A vector bearing the construct xynA of Thermobifida fusca YX with a C-terminal histidine tag locates at the downstream of alcohol oxidase 1 (AOX1) promoter was used, and the xylanase was optimally active at 80 °C and pH 8.0 [25]
Yeast extract and peptone were obtained from OXOID (Basingstoke, Hampshire, UK), yeast nitrogen base (YNB) without amino acids was purchased from BD (Sparks, MD, USA), biotin and phenylmethylsulfonyl fluoride (PMSF) were obtained from Amresco (Solon, OH, USA)
Summary
Xylanase degrades xylan into monomers of various sizes by catalyzing the endohydrolysis of the 1,4-β-Dxylosidic linkage randomly, possessing potential in wide industrial applications. Most of xylanases are susceptible to be inactive when suffering high temperature and high alkaline process. It is necessary to develop a high amount of effective thermoalkaliphilic xylanases. Xylanase (β-1,4-Endoxylanases, EC 3.2.1.8) degrades xylan into monomers by catalyzing the endohydrolysis of the 1,4-βD-xylosidic linkage between molecules of xylose in the main chain randomly [2]. Endoxylanases play an important role in bioconversion of lignocellulose in feed, food, biofuel, pulp and paper industry and are widely used as raw materials in lots of industrial processes [2, 4]. The development of a high amount of effective thermoalkaliphilic xylanases is highly desirable
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