Isolation of highly thermostable β-xylosidases from a hot spring soil microbial community using a metagenomic approach

Abstract

The DNA extracted from a high-temperature environment in which micro-organisms are living will be a good source for the isolation of thermostable enzymes. Using a metagenomic approach, we aimed to isolate thermostable β-xylosidases that will be exploited for biofuel production from lignocellulosic biomass. DNA samples obtained from the soil near a spout of a hot spring (70°C, pH7.2) were subjected to sequencing, which generated a total of 84.2 Gbp with 967,925 contigs of >500 bp in length. Similarity search for β-xylosidase in the contigs revealed the presence of 168 candidate sequences, each of which may have arisen from more than one gene. Individual genes were amplified by PCR using sequence-specific primers. The resultant DNA fragments were cloned and introduced into Escherichia coli BL21 Star(DE3). Consequently, 269 proteins were successfully expressed in the E. coli cells and then examined for β-xylosidase activity. A total of 82 proteins exhibited β-xylosidase activity at 50°C, six of which retained the activity even at 90°C. Out of the six, three proteins were originated from a single candidate sequence, AR19M-311. An amino acid sequence comparison suggested the amino acid residues that appeared to be crucial for thermal stability of the enzymes.

Xylanolytic Extremozymes Retrieved From Environmental Metagenomes: Characteristics, Genetic Engineering, and Applications

“…Existing β-xylosidases are associated with several limitations, such as poor efficiency, low thermostability, salt sensitivity, and by-product inhibition (Bao et al, 2012;Anand et al, 2013). Therefore, efforts have been made to discover novel candidates of β-xylosidases using metagenomic approaches, with which little success has been achieved (Wagschal et al, 2009;Jordan et al, 2016;Cheng et al, 2017;Sato et al, 2017;Liu et al, 2018;Rohman et al, 2019; Table 2). Of the available metagenomic β-xylosidases, fewer than 20 have been extensively characterized to date.…”

“…Thermostable β-xylosidases are in high demand in industries to overcome microbial contamination and reduce the viscosity of the reaction mixture, which improves the overall reaction efficiency manifold (Haki and Rakshit, 2003). The β-xylosidases (AR19M-311-2, AR19M-311-11, and AR19M311-21) reported by Sato et al (2017) are highly thermostable having optimum activity at 90 • C with fair stability at 70 • C for 1 h. It corroborates the characteristics of thermostable β-xylosidases of Thermoanaerobacter ethanolicus, Thermotoga maritima, and Thermotoga thermarum having the temperature optima at and above 90 • C (Shao and Wiegel, 1992;Shi W. et al, 2013). In another report, four thermostable β-xylosidases have been reported from a compostsoil metagenome that show optimum activity at 60-75 • C (Wang et al, 2015).…”

“…Direct sequencing of the randomly picked clones from a metagenomic library is a straightforward approach to decode the hidden information in the sequences. This approach has also been employed in generating significant information for hundreds of xylanolytic enzyme-encoding genes ( Sato et al, 2017 ; Ariaeenejad et al, 2019 ). The direct sequencing of a porcupine metagenome uncovered four genes having β-glucosidase, α-L-arabinofuranosidase, β-xylosidase, and endo-1,4-β-xylanase activities ( Thornbury et al, 2019 ).…”

MCIC: Automated Identification of Cellulases From Metagenomic Data and Characterization Based on Temperature and pH Dependence

“…However, the process of mining for novel enzymes solely through functional screening techniques and without the utilization of in silico methods can be laborious and time-consuming. For instance, to isolate highly thermostable beta-xylosidases from hot spring soil microbiota, the researchers had to express, examine, and screen 269 candidate proteins based on their xylosidase activity (Sato et al, 2017). Doubtlessly, powerful in silico approaches could effectively facilitate this process and reduce experimental expenses and time consumption.…”

Effects of nitrogen fertilization and bioenergy crop species on central tendency and spatial heterogeneity of soil glycosidase activities

“…In general, AG and BG are the most important glycosidases in soils, and their hydrolysis products are source of energy for soil microorganisms. AG acts on the α-d-glucoside bonds present in maltose 14 ; BG catalyzes the hydrolysis of β-d-glucopyranoside and is involved in the saccharification of cellulose 11,15 ; BX cleaves the β-1,4-linkage of xylan from the non-reducing terminus to release d-xylose and can be used for bioenergy production [16][17][18] ; CBH is known to hydrolyze the ends of the cellulose chain and to processively produce glucose or cellobiose as the end product 19 .…”