The growth of societal demand has exacerbated the global shortage of resources and energy, especially the depletion of oil resources, making it urgent to seek alternative energy sources. Plant biomass in nature is the largest renewable resource in existence and has great potential for conversion into biofuel ethanol and other valuable chemicals. The first generation of industrially produced biofuel ethanol uses starch-based feedstock such as food crops, which has the problem of competing with people and animals for food and is scale limiting and unsustainable in the long run. The second generation of lignocellulose-based biofuel ethanol preparation technology has become the key to achieving large-scale oil substitution. Because of the complex structure of lignocellulose, its utilization rate is low and most of it is burned, which not only causes waste of resources, but also causes serious environmental problems. Therefore, achieving efficient conversion of lignocellulose to bioethanol can not only alleviate the energy crisis, but also avoid environmental pollution.

Even after pretreatment of plant biomass some crystalline cellulose remains [1]. The degradation of crystalline cellulose is inefficient using the available enzymes, and thus its conversion is slow and expensive [2], [3], [4], [5]. However, the lytic polysaccharide monooxygenases (LPMOs) can cleave the glycosidic bonds of crystalline polysaccharides by oxidation [6]. LPMOs are copper-dependent enzymes with a highly conserved active center, generally consisting of two histidines, one tyrosine and one copper ion [7]. The side chains of two histidines and the nitrogen atom at the amino terminus of the first histidine are linked to form a T-shaped structure known as the His-brace motif [8], [9]. LPMOs can effectively disrupt and loosen the intrinsic structure of crystalline polysaccharides, providing more accessible sites for hydrolases [10]. Therefore, the synergistic action of LPMO and cellulase can accelerate the decomposition of cellulose and make the conversion of plant biomass more economical [11], [12], [13], [14], [15].

In industrial applications, enzyme activity and thermostability are significant enzyme parameters, which determine the enzyme’s economic feasibility. However, the enzymatic activity and thermostability of LPMOs are suboptimal in the context of industrial applications. In order to meet the industrial demand, the prevalent protein engineering methods are often used to improve enzymatic performance [16], [17]. Rational design based on computer-aided methods has been widely used to identify the key amino acids and conduct directed evolution. For example, the oxidation capacity of LPMO to cellulose was improved by mutating the residues bound to cellulose [18], [19], [20], [21]. The efficiency of cellulose degradation by LPMO was improved by rational design [22], [23]. In addition, the enzymatic activity of LPMO could also be improved by adding the cellulose-binding module region [24], [25], [26]. It can be seen that there have been many studies on improving LPMO enzyme activity by rational design. Also, a few researchers have improved the thermal stability of LPMO by amino acid mutation [27], [28], [29]. For example, the L90V/D131S/M134L/A141W variant of AfAA9B from Aspergillus fumigatus was obtained based on the computer calculations, which had a 7 °C improvement of Tm compared to AfAA9B wild type [27]. However, there are relatively few research on improving the thermal stability of LPMOs, which dramatically affects their industrial application.

In previous studies, a LPMO (GenBank: AKO82493.1) from Myceliophthora thermophila C1 was studied [30], [31]. It was the first LPMO discovered to have catalytic activity for both cellulose and xylan, which is important for the conversion of lignocellulose [30]. In our previous work, the LPMO was named as MtC1LPMO and was cloned and expressed [31]. Since it is derived from thermophilic fungi, its thermal stability was explored. It was found that the optimum temperature of MtC1LPMO was 85 °C, and it had good stability at 40–60 °C. But its enzyme activity dramatically decreased after incubation at 70 °C [31]. Since the thermostability is an important parameter for the enzyme in industrial applications, we further improved thermal stability of MtC1LPMO by constructing disulfide bonds to enhance its economic feasibility. Firstly, the structural changes of MtC1LPMO at different temperatures were explored by molecular dynamics (MD) simulations, and eight pairs of amino acids were selected to mutate to cysteine by combining the results of Disulfide by Design (DBD), Multi agent stability prediction upon point mutations (Maestro) and Bridge disulfide (BridgeD) websites. Then, the enzymatic properties of eight mutants were determined after their expression and purification. Mutants with improved thermal stability were obtained and their degradability of different biomasses was analyzed. Finally, the mechanism of the increased thermal stability of the mutant was investigated by MD simulations.