Improved Production of Xylanase in Pichia pastoris and Its Application in Xylose Production From Xylan

Xylanases with high specific activity has been focused with great interest as a useful enzyme in biomass utilization. The production of recombinant GH11 xylanase (MYCTH_56237) from Myceliophthora thermophila has been improved through N-terminal signal peptide engineering in P. pastoris. The production of newly recombinant xylanase (termed Mtxyn11C) was improved from 442.53 to 490.7 U/mL, through a replacement of α-factor signal peptide with the native xylanase signal peptide segment (MVSVKAVLLLGAAGTTLA) in P. pastoris. Scaling up of Mtxyn11C production in a 7.5 L fermentor was improved to the maximal production rate of 2503 U/mL. In this study, the degradation efficiency of Mtxyn11C was further examined. Analysis of the hydrolytic mode of action towards the birchwood xylan (BWX) revealed that Mtxyn11C was clearly more effective than commercial xylanase and degrades xylan into xylooligosaccharides (xylobiose, xylotriose, xylotetraose). More importantly, Mtxyn11C in combination with a single multifunctional xylanolytic enzyme, improved the hydrolysis of BWX into single xylose by 40%. Altogether, this study provided strategies for improved production of xylanase together with rapid conversion of xylose from BWX, which provides sustainable, cost-effective and environmental friendly approaches to produce xylose/XOSs for biomass energy or biofuels production.

Effects of Enzyme Loading, Incubation Time and Incubation Temperature on Sawdust Hydrolysis by Locally Produced Bacterial Xylanase

The present study aims to investigate the enzymatic hydrolysis conditions of bacterial xylanase on alternative cheaper substrate which is hardwood sawdust (SD) in order to produce reducing sugars (xylose). The bacterial xylanase was produced and secreted from the Bacillus sp. The wood industry in Malaysia has become a major source of foreign exchange across the globe for developing the countries. Therefore, more wood residues (sawdust) are produced during the logging and processing of wood. Most of the sawdust will be disposed into the landfills. In actual, the sawdust can be utilised into more valuable products such as in producing reducing sugars. Thus, previous researches have studied on xylose production from wooden sawdust using commercial xylanases, but only few with the bacterial xylanase. Therefore, a study on the best conditions of enzymatic hydrolysis in producing xylose from sawdust using bacterial xylanase is essential. Prior to the enzymatic hydrolysis, the hardwood sawdust was pre-treated by autoclave at 121°C for 20 min in order to breakdown the lignin linkage and obtain the hemicellulosic xylan (delignification). The enzymatic hydrolysis conditions such as enzyme loading, incubation time and incubation temperature were experimented by One-Factor-At-Time (OFAT) method. Based on the experiment, the fifth cycle pre-treated autoclaved sawdust showed 5.5-fold higher than the untreated sawdust. The best enzymatic hydrolysis conditions for xylose production were enzyme loading of 1.4%, incubation time of 30 min, and incubation temperature of 56.9 °C. These conditions also succeeded in producing 2.5-fold higher xylose than the one without the enzymatic hydrolysis.

Xylose Release From Sunflower Stalk by Coupling Autohydrolysis and Enzymatic Post-Hydrolysis

The purpose of this study was to obtain xylose-based fermentation media from autohydrolysis liquors of sunflower stalk by using commercial xylanase formulation. Xylose is generally produced from xylan by diluted acid hydrolysis that causes the formation of some unwanted compounds during the process. As an alternative to dilute acid hydrolysis method, enzymatic hydrolysis of xylan can provide more specific hydrolysis under moderate conditions and does not cause the formation of undesirable compounds. In this study, xylose production carried out with Trichoderma longibrachiatum xylanase on solubilized xylan form of sunflower stalk, which was hydrothermally pretreated for 1 hour at 160ºC. The effects of substrate concentration and enzyme activity were investigated for the production of xylose. To obtain a high xylose yield and selectivity, the optimization study was conducted by the response surface methodology. The optimum substrate concentration and enzyme activity were found as 60 mg ds/mL CAL and 234 U/mL, respectively. Under the optimum condition, xylose yield and selectivity were found to be 69.5% and 8.2 g/g, respectively. This study showed that xylose could be produce with a high yield without requiring a neutralization process and corrosive chemical reagent apart from water.

Xylitol Production From Sugarcane Bagasse Through Ultrasound-Assisted Alkaline Pretreatment and Enzymatic Hydrolysis Followed by Fermentation

This study focused on the optimization of xylitol production from sugarcane bagasse by using response surface methodology (RSM). Xylitol was produced through a series of processes, firstly, optimization of ultrasound assisted mild alkaline pretreatment for the xylan extraction from sugarcane bagasse followed by enzymatic hydrolysis of xylan to xylose by enzyme β-1,4-xylanase and finally microbial fermentation of xylose to xylitol using yeast (Candida guilliermondii), bacteria (Corynebacterium glutamicum) and their mixed culture for different time periods (0-96 h). Maximum xylan recovery of 12.059% (w/w) was observed at pretreatment; 0.73 M NaOH, 1:38.55 solid to liquid ratio and 34.77 min ultrasonication. The enzyme concentration of 400 U/g xylan at 48 h of incubation showed the highest xylose production (81.51 mg/g bagasse). Yeast (Candida guilliermondii) resulted in the highest xylitol yield (Yp/s= 0.43 g/g) after 72 h. This bioprocess route can contribute as a suitable alternative for chemical methods of xylitol production.

Comprehensive study on the effects of process parameters of alkaline thermal pretreatment followed by thermomechanical extrusion in sugar liberation from Eucalyptus grandis wood

A combination of alkaline thermal pretreatment followed by thermomechanical extrusion was studied as a novel sequential pretreatment process for an effective breakdown of the lignocellulosic structure of Eucalyptus grandis wood (EW). The first step was studied by analysing the influence of two factors: the NaOH-to-dry biomass ratio or NaOH loading (NaOH/DM) and the liquid-to-solid ratio (L/S). Optimization of these two parameters provided good results in terms of enzymatic hydrolysis at 5% (w w−1) solids loading, obtaining a total sugar concentration of 24.9 g L−1 and a total sugar production of 36.9 g 100 g−1 raw EW after pretreating the biomass at 20% NaOH/DM and L/S = 1/1. The second step of extrusion, when followed by a final washing step, provided a significant increase in glucose and xylose production when working at 10% NaOH/DM. For a soda loading of 20%, there was a clear improvement in sugars conversion yield after extrusion and washing: 71% for glucan conversion and 89% for xylan.