Abstract
Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.
Highlights
Carbohydrate degradation involves a series of hydrolases, especially xylanase (EC3.2.1.8) [1]
Several xylanases have been extracted from thermophilic microorganisms, their expression levels and enzymatic activities are insufficient for industrial use [5]
(Figure 1a), and CDBFV, C2-CDBFV, and CDBFV-C2 (Figure 1b) constructs appeared on the electrophoresis gel as single bands with estimated molecular masses close to 750 bp (CDBFV-1, C2L, LC2, and CDBFV) and 1500 bp (C2-CDBFV and CDBFV-C2) (Figure 1c), which are close to the theoretical values
Summary
Carbohydrate degradation involves a series of hydrolases, especially xylanase (EC3.2.1.8) [1]. The application value of xylanases in industrial processes depend on their thermal stability and activity. In some procedures, such as production drying, feed pelleting, maltification, etc., xylanases with excellent thermal stability to adapt high-temperature environments are highly demanded [3]. Most natural xylanases belong to mesophilic enzymes [4]. Several xylanases have been extracted from thermophilic microorganisms, their expression levels and enzymatic activities are insufficient for industrial use [5]. Many projects have been undertaken to develop xylanases with improved thermostability and activity to create novel enzymes withstanding harsh conditions [3,6,7,8]
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