Background: In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeastPichia pastoriscan neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes.Results: The Xyn2 gene, which encodes the mainTrichoderma reeseiRut C-30 endo-β-1, 4-xylanase was cloned into the pPICZαA vector and expressed inPichia pastoris. The selectedP. pastorisstrains produced as 4,350 nkat/ml β-xylanase under the control of the methanol inducible alcohol oxidase 1 (AOX1) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60°C and it was active over a broad range of pH (3.0–8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50°C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparentKmand kcatvalues were 2.1 mg/ml and 219.2 S-1, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity.Conclusion: TheP. pastorisexpression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore,P pastorisis a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.
Intra-uterine growth-retarded (IUGR) neonates have shown an impairment of postnatal intestinal development and function. We hypothesised that the immune function of IUGR neonates might be affected by increased nutrient intake (NI) during the suckling period. Therefore, we investigated the effects of high NI (HNI) on the growth performance, intestinal morphology and immunological response of IUGR and normal-birth weight (NBW) piglets. A total of twelve pairs of IUGR and NBW piglets (7 d old) were randomly assigned to two different nutrient-level formula milk groups. After 21 d of rearing, growth performance, the composition of peripheral leucocytes, serum cytokines and intestinal innate immune-related genes involved in the Toll-like receptor (TLR)-4–myeloid differentiation factor 88–NF-κB pathway were determined. The results indicated that IUGR decreased the average daily DM intake (ADMI) and the average daily growth (ADG). However, the ADMI and ADG were increased by HNI, irrespective of body weight. Likewise, serum cytokines (TNF-α and IL-1β) and ileal gene expressions (TLR-4, TLR-9, TRAF-6 and IL-1β) were lower in IUGR piglets, whereas HNI significantly increased blood lymphocyte percentage and serum IL-10 concentrations, but decreased neutrophil percentage, serum IL-1β concentrations and ileal gene expressions (NF-κB and IL-1β). Furthermore, IUGR piglets with HNI exhibited lower serum concentrations of TNF-α and IL-1β than NBW piglets, and these alterations in the immune traits of IUGR piglets receiving HNI were accompanied by decreasing ileal gene expressions of TLR-4, TLR-9, NF-κB and IL-1β that are related to innate immunity. In conclusion, the present findings suggest that increased NI during the suckling period impaired the immune function of neonatal piglets with IUGR.