Starch is one of the most abundant polysaccharide in nature and serves as a substrate for the production of numerous valuable products. The use of amylolytic enzymes such as α-amylase, glucoamylase and pullulanase in starch processing is considered economically advantageous [1].

β-Amylase (α-1,4-glucan maltohydrolase, EC 3.2.1.2) is an exoenzyme that hydrolyzes α-1,4-glucosidic bonds from the non-reducing ends of starch molecules, releasing maltose. β-Amylases are widely used in the food industry, particularly in brewing and in the saccharification of liquefied starch to produce maltose syrup [2]. Maltose and maltose syrups are widely used in the food and pharmaceutical industries due to their colourlessness, low viscosity and mild sweetness.

β-Amylases have been predominantly identified in plant sources such as barley and soybean, as well as in microorganisms, mainly Bacillus species [3], [4] and Clostridium thermosulphurogenes. At present, industrial applications primarily rely on plant-derived β-amylase preparations obtained through extraction. However, the production of plant-based enzymes poses several challenges, including vulnerability to crop failure, reliance on grain availability, limited storage stability, and high production costs. Moreover, unlike microbial β-amylases, plant enzymes are generally unable to hydrolyze raw starch [5], further limiting their applicability.

In contrast, microbial production of β-amylases is not dependent on climatic conditions and allows for consistent enzyme supply. Additionally, microbial β-amylases can be optimized through recombinant DNA technology and directed evolution. Although bacterial β-amylases are actively investigated, only a few have been commercialized due to limitations in yield, thermostability, and food safety [6]. There is still a need for bacterial β-amylases with enhanced properties, such as higher specific activity and broader pH stability profiles [7].

For industrial applications, β-amylases should be active at temperatures ranging from 55 to 62 °C and at pH 5.6–6.0, as these conditions are typical for barley mashing in brewing and for maltose syrup production [5], [8], [9], [10]. It is known that the wild-type β-amylase from Priestia flexa (formerly Bacillus flexus) strain APC 9451 is active under these technological conditions [11]. In our laboratory, two wild-type strains of P. flexa from the BRC VKPM collection were previously investigated. However, their β-amylase productivity was very low, not exceeding 30 U•mL−1 when cultured in flasks in TB medium.

Wild-type strains typically produce low levels of β-amylase, rendering them unsuitable for industrial applications. For example, the wild-type strain Paenibacillus chitinolyticus CKS1 produced only 0.82 U•mL−1 of β-amylase [12], while the native P. flexa strain yielded 121 U•mL−1 after 36 h of cultivation in complete nutrient medium [13]. Therefore, enhancing enzyme production through appropriate genetic manipulation is a high priority.

Recombinant DNA technology can substantially increase enzyme yield: β-amylase from C. thermosulfurogenes, heterologously expressed in Escherichia coli, showed activity of 215 U•mL−1 [14], and recombinant β-amylase from Bacillus aryabhattai, produced in Bacillus subtilis strain TEV1030, reached 1590 U•mL−1 in Terrific Broth (TB) medium [4]. Thus, the highest productivity was achieved in the Bacillus expression system. However, the high concentrations of peptone (12 g•L−1) and yeast extract (24 g•L−1) in TB medium significantly increase production costs, making it less viable for industrial-scale applications. Therefore, alternative expression systems are needed for cost-effective and efficient β-amylase production [15].

Komagataella phaffii (formerly Pichia pastoris) is a well-established platform for producing a wide range of industrial recombinant proteins [16]. These methylotrophic yeasts are regarded as biosafe, capable of high-density growth on minimal media, and secrete few endogenous proteins, thereby facilitating high-yield production of heterologous proteins at low cost. Over 70 commercial products, including phytase, mannanase, xylanase, and lipase, have been successfully produced using K. phaffii [16]. While K. phaffii is not typically known for naturally producing β-amylases, it can be engineered to express and secrete them as a host for recombinant protein production. It is known that the yield of previously unmanufacturable commercial enzymes can be increased through the engineering of recombinant producer strains [4]. However, to date, β-amylase production in K. phaffii has not been reported. Therefore, the possibility of producing microbial β-amylases using the K. phaffii expression system is of considerable interest.

In the present study, genes encoding β-amylases from P. flexa strains previously investigated in our laboratory were heterologously expressed in K. phaffii to evaluate the possibility of obtaining yeast recombinant strains with increased production of the target enzyme. The biochemical properties of the recombinant enzymes were characterized, and sequence analysis was performed to identify amino acid substitutions associated with changes in enzyme properties. A mutant recombinant β-amylase with increased specific activity and improved kinetic parameters was obtained. A high level of recombinant β-amylase production was achieved in fed-batch fermentation using K. phaffii cells.