Date Published: March 21, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Youzhi Miao, Yanqiong Kong, Pan Li, Guangqi Li, Dongyang Liu, Qirong Shen, Ruifu Zhang.
Xylanase with a high thermostability will satisfy the needs of raising the temperature of hydrolysis to improve the rheology of the broth in industry of biomass conversion. In this study, a xylanase gene (xyn10A), predicted to encode a hydrolase domain of GH10, a linker region and a CBM1 domain, was cloned from a superior lignocellulose degrading strain Aspergillus fumigatus Z5 and successfully expressed in Pichia pastoris X33. Xyn10A has a specific xylanase activity of 34.4 U mg−1, and is optimally active at 90 °C and pH 6.0. Xyn10A shows quite stable at pHs ranging from 3.0 to 11.0, and keeps over 40% of xylanase activity after incubation at 70 °C for 1 h. Removal of CBM1 domain has a slight negative effect on its thermostability, but the further cleavage of linker region significantly decreased its stability at high temperature. The transfer of CBM1 and linker region to another GH10 xylanase can help to increase the thermostability. In addition, hydrolase domains between the two Xyn10A proteins naturally formed a dimer structure, which became more thermostable after removing the CBM1 or/and linker region. This thermostable Xyn10A is a suitable candidate for the highly efficient fungal enzyme cocktails for biomass conversion.
Xylanases have been used in many industrial processes as the additives to improve the quality of baked goods and animal feeds as well as to bleach kraft pulp. In biomass conversion, xylanases also play a crucial role to synergistically deconstruct lignocellulose with cellulases and release soluble sugars from polysaccharide of xylan (Inoue et al. 2015). Besides the pre-treatment of biomass and the design of efficient enzyme cocktails, the whole industrial process also need high thermostable enzymes, which have many benefits of increased specific activity, stability, prevention of growth of contaminants, and increased mass transfer rate due to lower fluid viscosity at high substrate concentrations at high temperature, to save the cost (Kubicek and Kubicek 2016). To this end, the searching of high thermostable xylanases is valuable.
Enzymatic hydrolysis of hemicellulose is a complex process, in which multi-enzymes are required. Endo-β-1,4-xylanase plays the most important role for its function of breaking xylan backbone, and therefore be considered as a key enzyme for biomass conversion in industry. In microbes, fungi are excellent degraders of lignocellulosic biomass, such as Neurospora crassa (Phillips et al. 2011), a model organism for the study of biomass degradation mechanisms, and Trichoderma reesei (Martinez et al. 2008), being widely used in industry to produce enzymes for lignocellulosic biomass degradation. These well-studied filamentous fungi, however, can only grow normally below 30 °C, which may cause that their secreted xylanases usually have an optimal reaction temperature about 50 °C (Polizeli et al. 2005). Obviously, large amount of xylanases will be needed in biomass conversion, and the property of high thermostability could significantly save the cost for the longer working life and higher enzyme activity. Until now, thermostable xylanases have been mainly discovered in bacteria, such as Thermotoga thermarum (Shi et al. 2013), Geobacillus thermoleovorans (Sharma et al. 2007), Acidothermus cellulolyticus, Enterobacter sp. and Clostridium sp. (Bhalla et al. 2013), having the optimal temperatures ranged from 75 to 100 °C. However, the application of bacterial-derived enzymes as complements to fungal enzyme cocktails is severely limited by the still unsolved difficulty of expressing them in filamentous fungi. A. fumigatus Z5 was previously isolated from the compost heaps of crop straws and its genomic sequence was determined (Miao et al. 2015a). The genes encoding Xyn10A, Xyn10B and Xyn10C (three different xylanases) were identified in Z5’s genome. In the same species, such as A. awamori, A. fischeri, A. kawachii, A. nidulans, A. oryzae, A. niger and A. terreus xylanases were also expressed and purified, but only have the low optimal temperatures ranging from 40 to 60 °C (Polizeli et al. 2005). The protein of Xyn10A had several similar homologs (> 70%) when blasted against the NCBI database (http://www.ncbi.nlm.nih.gov), however, it was firstly reported in this study as a high thermostable GH10 xylanase with an optimal temperature of 90 °C in Aspergillus species. It will be suitable for adding this thermostable GH10 xylanase into enzyme cocktails by expressing it in an efficient protein expression system of A. niger (Wanka et al. 2016) or other industrial strains like T. reesei. If being considered, the potential of this enzyme still could be increased, such as enhancing the stability at high temperatures (> 80 °C), or improving the xylanase activity at low temperatures to make this enzyme work efficiently in a wide range of temperatures.