Cellulase, including endoglucanase (endo-1,4-D-glueanase, EC3.2.1.4), exoglucanase (exo-1,4-D- glucanase, EC3.2.1.91) and glucosidase (1,4-D-glueosidase, EC3.2.1.21), is a type of enzymes that can hydrolyze the β-1,4-glycosidic bond between cellulose molecules (Lin et al. 2016). Endocellulase mainly catalyzes degradation of the amorphous region in cellulose, providing the starting site for further catalysis of exocellulase, so it plays an important role in efficient degradation of cellulose. Cellulase is distributed in many families, and the endoglucanases belonging to the glycoside hydrolase family 5 (GH5) have high similarity in both amino acid sequence and two strict conserved catalytic residues of glutamic acid (Yuan et al. 2019; Matsuyama et al. 1999). The two residues are considered as catalytic proton donors and active site nucleophiles, respectively. They can maintain the structure of the substrate isocarbon of the enzyme (Valérie et al. 1995). Cellulase is widely used in many fields, such as biofuel production, papermaking, textiles, food processing, brewing, and extraction of active ingredients of traditional Chinese medicine (Garg et al. 2016; Chia et al. 2016; Samkelo et al. 2020). Endocellulase has attracted attention because of its catalytic efficiency. So far, although various cellulase has been isolated from bacteria, fungi (Okada 1976), plants and higher animals, thermostable cellulase with excellent properties still needs to be discovered (Morteza et al. 2020; Zheng et al. 2018).
Ferula asafoetida is a rare medicinal plant resource distributed in Xinjiang in China. The distribution area of Ferula asafoetida in Shihezi on the southern edge of Junggar Basin experiences much dramatic temperature changes (e.g. long time sunshine, large difference of daily temperature) and high content of salt-alkali. Therefore, microbes and enzymes in the soils of this area are characteristic of salt alkali resistance and high thermal stability. Hence, the soils here are a good sample for screening new enzymes with industrial application potential. However, most of the microorganisms in the environment cannot be cultured with the existing techniques or be used for biotechnology or basic research (Amann et al. 1995; Schmitz et al. 2004). For example, only 1% or even less of prokaryotes in the soils are readily cultivatable (Griffiths et al. 1996). Metagenomic libraries containing DNA extracted directly from environmental samples provide genomic sequences, and phylogenetic and functional information (Jo et al. 2002). Thus, metagenomics, the genomic analysis of collective genomes in an assemblage of organisms, is suitable for screening new functional biocatalysts and molecules with industrial application potential from soils (Handelsman et al. 2004). Many new enzymes have been discovered by using metagenomics, such as lipases (Tirawongsaroj et al. 2008; Bayer et al. 2009), amylases (Yun et al. 2004), and cellulase (Nimchua et al. 2012; Ko et al. 2013; Amitha et al. 2013). To our knowledge, no study has reported any highly thermostable cellulase that is found through metagenomic screening from the soils of Ferula asafoetida distribution area.
Burning fossil fuels can cause environmental pollution (Bao et al. 2011). Cellulose is the most abundant component of lignocellulose in the biosphere, and the cheapest renewable and natural organic substance (Liu et al. 2011). Cellulosic ethanol is the best alternative to fossil fuels (Raj et al. 2018; Costa et al. 2018). However, many intermolecular, intramolecular hydrogen bonds and other intermolecular interactions result in the long-lasting chain conformation and tight chain filling of cellulose, which cannot be dissolved by ordinary solvents (Medronho et al. 2012). Moreover, the hydrolysis of cellulose by chemical methods to produce cellulosic ethanol is complicated and expensive (Salinas et al. 2011). The utilization of lignocellulosic biomass lacks effective low-cost means (Lynd et al. 2002).
Water hyacinth (Eichhornia crassipes) has abundant utilizable lignocellulosic biomass (Ismail et al. 1995). It can tolerate seasonal changes in flow rate, water level, pH, nutrient availability, temperature, and toxic substances (Asrofi et al. 2018; Sumrith and Dangtungee 2019). Water hyacinth can multiply quickly at suitable temperature (between 10 and 35°C) in a nutrition balanced environment (Mayo and Hanai 2017). The infestation of water hyacinth affects water transport and ecological balance, and causes secondary pollution of water bodies. These limitations are great challenges to the ecosystem. To solve these problems and make reasonable use of water hyacinth resources, some researchers have studied enzymatic degradation of water hyacinth. The reactions of 170 U/g cellulase from Trichoderma reesei with alkali-treated water hyacinth for 36 h (Ganguly et al. 2013), 30 FPU/g cellulase from Aspergillus fumigatus with acid-treated water hyacinth for 24 h (Das et al. 2013, 2016), and 10 FPU/g cellulase from T. atroviride with acid-treated water hyacinth for 70 h (Rajesh et al., 2014) lead to the maximum sugar yields of 299.13, 425.6 and 380.97 mg/g respectively. In this work, after 121.25 U/g Cel1029 (the enzyme loading of 1 g of water hyacinth is 121.25 U) reacts with acid-treated water hyacinth for 22 h, the reducing sugar yield is 430.39 mg/g (sugar yield per gram of water hyacinth).
We metagenomically screened a novel type of cellulase through from the soils of Ferula asafoetida distribution area. The cellulase shows excellent thermal stability, tolerance against metal ions, organic solvents, and salt solutions, and high ability of degrading water hyacinth, which together prove its great potential for industrial application.