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

Abstract

Background: Xylanase 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.

Results: The 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.

Conclusions: Xylanase 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.

Figure 1

The map for the expression plasmid pKLAC2- xynZG in K. lactis . xynZG (the target gene encoding XYNZG), P_LAC4-PBI (LAC4 Promoter), α-MF (α-mating factor secretion leader sequence), Ap^R (Ampicillin resistance gene), amdS (encoding acetamidase to break down acetamide for use as a nitrogen source), ori (origin of E. coli), TT_LAC4 (LAC4 transcription terminator).

Figure 2

The transformants of activity screening. The transformants were cultured on the YPD plates containing 1% RBB xylan. The plates were photographed after 12 h incubation at 28°C. Negative control is K. lactis transformant harboring pKLAC2.

Figure 3

SDS-PAGE and zymogram analysis of XYNZG. Lane 1, zymogram analysis of the fermentation supernatant. Lane 2, the fermentation supernatant. Lane 3, the protein marker.

Figure 4

The fermentation of xylanase XYNZG in different media. a: The activity changes of XYNZG in YPL, YLP, YLU and YLPU medium during 96 h fermentation. The samples were taken and measured every 24 h. b: The comparison of biomass and activities at 72 h culture. The experiments were performed in triplicate.

Figure 5

Mass spectrometry analysis of xylan hydrolysate.

Figure 6

Pictorial showing the effects of various concentrations of XYNZG on the volume of wheat bread. a: Photograph of bread and cut bread. b: The relative specific volume of bread. Data represent mean±standard deviation of three replicates. The activity of XYNZG was 50 U/ml.