L-asparaginase (L-ASNase EC 3.5.1.1) is a crucial enzyme that plays a complementary role in the pharmaceuticals and food industry [1]. L-ASNase can specifically catalyze the conversion of free L-Asn into aspartic acid and ammonia. Thus, L-ASNase can inhibit tumor cell growth by exhaustion of the circulating L-Asn in the blood [2,3]. Therefore, L-ASNase has been widely employed as an essential anticancer drug for treating acute lymphoblastic leukemia (ALL) and other related cancer worldwide [4,5]. Besides being an important therapeutic drug, L-ASNase gained wide attention as a promising AA mitigating agent in fried and baked foods [6,7].

AA is formed via the Maillard reaction that occurred between the amide group of free L-Asn and the carbonyl group of reducing sugar at a temperature above 120 °C [8,9]. In recent studies, L-Asn was identified as a primary AA precursor in potatoes and wheat [7,10,11]. Also, it is the most abundant free amino acid in plants, constituting approximately one-third of the total amino acid pool [12]. In addition, non-Asn routes, including acrolein, acrylic acid, Amadori products, and decarboxylated Schiff base, also led to the formation of AA in processed foods [13,14]. According to the International Agency for Research on Cancer (IARC), 2002, AA is classified as a group-2A potential human carcinogen [15]. AA-associated neurotoxicity, genotoxicity, and mutagenicity have been confirmed by various studies [16,17]. AA intake of 5 mg/kg/day triggered neurobehavioral disorder in rats within six months [18]. AA concentration of 2.5 mM induced BV2 microglial cytotoxicity in mice [19]. In addition, the cytotoxic, genotoxic, and carcinogenic effect was observed after AA exposure of 0.5, 1.0, and 5 mM, which could induce DNA strand breaks, cell transformation, and anchorage-independent growth of BEAS-2B human lung cells. Therefore, AA detection in food may pose a potential threat to human health through dietary exposure and raised concerns for public health to mitigate AA.

The agronomical and technological approaches such as compromising Asn and reducing sugar concentration, lowering the pH of the food matrix, using additives, reducing excessive frying conditions, and blanching before frying and baking have been introduced to inhibit AA [20,21]. The pH-lowering agents in a concentration of 0.02 % are responsible for 0.5 unit pH reduction of the food matrix [22]. However, due to acidification and sour taste, these methods are ineffective as they negatively impact the food's taste, color, and aroma [23,24]. Additionally, various additives, including citric acid, tartaric acid, L-lactic acid, acetic acid, and free amino acid, block the nucleophilic addition of L-Asn with a carbonyl compound, which prevents the formation of the Schiff base, a primary intermediate in the Maillard reaction and AA formation [8,25]. Because Maillard reaction is also responsible for the taste and the sensory properties of the food, possibly affecting the food quality. In addition, blanching is a commonly used approach for minimizing AA content, resulting in a 20–78 % reduction in acrylamide formation [21]. However, a significant loss of soluble nutrients adversely affects the texture quality of vacuum-packaged potato strips [23,24]. Therefore, food products without AA content while preserving sensorial properties are challenging for the food industry.

L-ASNase mitigates AA by depleting the free L-Asn level without affecting the sensory properties of the final product. The Acrylway® and PreventASe™ are the only commercially available L-ASNase from fungi Aspergillus oryzae and Aspergillus niger, respectively, for AA reduction in the food industry [26,27]. The bacterial L-ASNase is categorized into type I and type II, based on their cellular localization and affinity towards the substrate [28]. The type I L-ASNase exhibited low substrate affinity and was constitutively expressed intracellularly in the cell. However, the type II L-ASNase is known as periplasmic or membrane-bound enzymes with relatively higher substrate affinity [28,29,32]. The various bacterial L-ASNase from Bacillus megaterium H-1 [30], Paenibacillus barengoltzii [31], Acinetobacter soli [32], and Pseudomonas sp. PCH182 [7] has been investigated for AA mitigation. It is known that type I L-ASNase has a lower substrate affinity than type II. Hence, fewer studies were performed in the past using type I L-ASNase to reduce AA formation in foods.

The Himalayan region is largely free from large-scale anthropogenic disturbances and is an underexplored reservoir of bioresources [33]. Hence, microbes inhabiting these niches acquire unique functions with novel properties such as high substrate specificity, pH, and thermostability [[34], [35], [36]]. The Pangi-Chamba Himalayan (PCH) region in the Western Himalayas is a good source, offering tremendous possibilities for identifying novel and robust biomolecules with commercial applications in therapeutics for the treatment of ALL, bioplastic synthesis, laccase production, and lignin depolymerization [[33], [34], [35], [36], [37], [38]]. Previously, our group explored the diversity of L-ASNase-producing bacterial isolates from PCH niches of Himalayan regions [33]. The study revealed new bacterial sp., including Rahnella sp., for L-ASNase production. Interestingly, Pseudomonas sp. PCH182 and Rahnella sp. PCH162 demonstrates the potential for L-ASNase with low-glutaminase (L-GLNase).

Here, in the present study, two L-ASNase genes (Type I) from Pseudomonas sp. PCH182 and Rahnella sp. PCH162 was cloned, expressed, purified, and analysed for its biochemical properties. Additionally, the role of L-Asn and its effects on AA formation was investigated to develop a sustainable and effective method for AA mitigation.