Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis

Abstract

BackgroundXylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking.ResultsThe xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread.ConclusionsXylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory properties of bread. Therefore, the expression yield of recombinant XYNZG can be further improved through engineered strain containing high copy numbers of the XYNZG, and optimized fermentation condition, making bread-baking application possible.

Functional diversity and properties of multiple xylanases from Penicillium oxalicum GZ-2

“…Therefore, multiple xylanases with diverse biochemical properties, structures, specific activities, and substrate specificities are required to increase the efficiency and extent of xylan hydrolysis 11 12 . Currently, hundreds of xylanases have been widely reported and characterized 3 8 13 14 15 16 17 , but few studies have focused on the multiplicity of functions and eco-physiological properties of these enzymes. Particularly, there are few reports on the occurrence of multiple xylanases from Penicillium .…”

Thermostable xylanase from thermophilic fungi: Biochemical properties and industrial applications

“…Similarly, the potential of xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis was studied. They obtained improvement in sensorial characteristics, volume, texture, and handling time (6.5 to 6.0 min) (Zhan et al, 2014). The purified xylanase from Remersonia thermophila CBS 540.59 (Rtl) also showed increased loaf volume by 8%, softness (19.6%) and decreased in 20.4% hardening of the bread after four days of storage compared to the control (McPhillips et al, 2014).…”

Tunnel engineering to accelerate product release for better biomass-degrading abilities in lignocellulolytic enzymes

“…The xylanase activity was evaluated by measuring the amount of reducing sugar released in the enzymatic hydrolysis by a modified DNS method [39]. The reaction mixtures containing 500-μL 1% (w/v) xylan in 20-mM glycine–NaOH buffer (pH 9.0) and appropriately diluted enzymes were incubated at 70 °C for 10 min, and stopped by adding 700-μL DNS reagent, followed by boiling for 10 min and determination of the absorption at 540 nm.…”