**1. Introduction**

The use of enzymes in industrial processes is a multi-billion-dollar market. One of the first enzymes discovered in 1833 was diastase, an enzyme able to hydrolyze starch [1]. Nowadays, amylases, also able to hydrolyze starch, constitute up to 25% of the market for enzymes and have virtually replaced chemical methods for degrading starch in the industrial sector (reviewed in [2]). Amylases are

the most important class of enzymes for degrading starch and can be subdivided into three subclasses: α-, β-, and gluco-amylases based on their reaction specificity and product profiles. α-amylases degrade the α- 1,4 linkage between adjacent glucose units and are extensively used for example in bioethanol production or in washing powder and detergents [3] (and reviewed in [4]). One of the most widely used α-amylases is that from *Bacillus licheniformis*, known under the tradename "Termamyl". Microbial amylases are generally used in detergent applications and other industrial processes, including bioethanol production, with new amylases, in particular those from hyperthermophilic organisms, offering further improvement in the production process (reviewed in [5]).

α-amylases belong to glycoside hydrolase family 13 (GH13) in the CAZy database classification [6]. They have a (β/α)8 barrel domain harboring the active site, a subdomain which includes the canonical calcium binding site inserted between the third β-strand and the third α-helix and a C-terminal β-sandwich domain, thought to be important for the interaction with raw starch (reviewed in [7]) [8,9]. Amylases follow a retaining mechanism with an aspartate as nucleophile and one glutamate as general acid/base [10,11]. Up to ten consecutive sugar subsites forming the active site cleft have been identified in bacterial amylases [12].

To date, recombinant fungal amylases have been isolated from mesophilic hosts such as *Aspergillus oryzae* and are of particular interest to the food industry as they match the temperature and pH range used in typical applications in the baking process, where they are active in the dough but inactivated during baking. Due to the widespread use of fungal enzymes for the production of food and food ingredients (such as citric acid), they are classified as GRAS (generally recognized as safe) organisms by organizations including the FDA (US Food and Drug Administration) [13].

Up till now, fungal enzymes with a higher pH-tolerance and thermostability have not been reported. Here, we describe the structure and function of three novel α-amylases from *Cordyceps farinosa* (CfAM), *Rhizomucor pusillus* (RpAM) and *Thamnidium elegans* (TeAM) with a higher stability and pH-tolerance with the potential to act as novel biocatalysts for various industrial processes. The sequence of all three enzymes groups them in the GH13 sub-family 1 along with, for example, the amylase from *Aspergillus oryzae* (also known as TAKA amylase). However, unlike other fungal amylases, the enzymes in this study have been shown to have a broad pH profile with an optimum around pH 5 while retaining activity at pH 8. Furthermore, their more open crevice leads to the production of longer oligomers compared to TAKA amylase.

The native RpAM and TeAM have a four-domain fold with a carbohydrate binding domain (CBM20) at the C-terminus and a short serine-rich linker in between, while native CfAM lacks this CBM20 domain. In this study, only the core of the amylases including the A, B and C domains was cloned and expressed. In addition, crystallization of *Cordyceps farinosa* amylase again demonstrates the power of the microseed matrix screening technique [14].

#### **2. Results**

#### *2.1. Biochemical Characterization*

The pH, temperature and product profiles were characterized for all three amylases. Of great desire are amylases with a broader pH-tolerance compared to TAKA amylase. Our analysis showed that all three amylases have a pH optimum around 5. Whereas TeAM has no significant activity above pH 7, RpAM and CfAM retain significant activity at pH 7 extending up to a pH of 9 (Figure 1a). In particular, CfAM shows the highest pH tolerance, retaining 70% of its activity at pH 8. RpAM and TeAM both show a pronounced shoulder, suggesting the involvement of more titratable residues in the substrate recognition and catalysis process. The temperature profiles reveal that RpAM and CfAM also have a considerably higher thermotolerance compared to TAKA and TeAM (Figure 1b). In particular, RpAM retains full activity even at 80 ◦C, making it an attractive enzyme for industrial high temperature starch saccharification processes. Compared to TAKA amylase, all three amylases

show a tendency to produce higher amounts of oligomers with a degree of polymerization (dp) of three, with trace amounts of oligomers with a dp of up to seven for TeAM (Figure 1c).

**Figure 1.** Biochemical characterization of RpAM, CfAM. TeAM and TAKA. (**a**) pH-profile of all three amylases in comparison with TAKA amylase; (**b**) temperature profile of all three amylases in comparison with TAKA amylase; (**c**) product profile of all three amylases and the abundance of oligomers with a degree of polymerization (dp) of 1 to 7 after hydrolysis of starch.
