The seaweeds are well known for industrially beneficial compounds production such as agar, carrageenan, alginate, fucoidan, pigments, etc [1]. However, research on bacteria associated with seaweed remains limited. [2]. The bacterial communities associated with seaweeds are either epibionts or endobionts and produce plant growth-promoting substances, quorum-sensing signalling molecules, bioactive compounds, and other effective molecules that are responsible for normal morphology, development, and growth of seaweeds [3], [4]. Most novel pharmaceutical compounds have been found in marine environments [5], [6]. Natural products derived from bacteria associated with seaweeds have shown promising results in combating antibiotic resistance [7]. The Okha Coast is situated in the north-western part of the Saurashtra region, Gujarat, India, which is the central point of the Gulf of Kutch and the Arabian Sea. The intertidal region of the Okha coastline is 0.7–0.9 km long, consisting of stones and sand [8], [9]. The annual climatic conditions of Okha seawater were reported as 20–33.6˚C, pH 8.0–8.37, dissolved oxygen 6.4–8.2 ppm, and salinity 26.89–45.44 ppt [10]. Till today, diverse floral and faunal species have been reported from the coastal areas of Gujarat, India. However, only limited research has focused on the microbial diversity of coastal Gujarat, especially from the Saurashtra region. Despite the ecological and industrial importance of seaweed-associated bacteria, there is a significant lack of studies on marine actinobacteria associated with seaweeds from the Saurashtra coastline, particularly the Okha region. Moreover, no existing studies report the co-production of multiple industrial enzymes from such isolates or their application using agricultural waste substrates [4], [11], [12]. Therefore, the Okha coast from the Saurashtra region was selected in this study to investigate marine actinobacteria associated with seaweed.

The actinobacteria can survive and grow in harsh environments, renowned for their versatile bioactive metabolites production [13], [14]. Therefore, deriving enzymes from marine actinobacteria improves enzyme functionality under alkaline and saline conditions, which are required enzymatic characteristics for the detergent and food industries [15], [16]. For optimal enzyme production, it is significant to optimize medium conditions as they strongly affect cell growth rate and enzyme secretion [16]. To systematically enhance enzyme production, a statistical optimization approach was employed to identify and fine-tune the key physicochemical parameters influencing enzyme yield. This strategy enabled the determination of optimal conditions for stable co-production of both amylase and protease using a marine actinobacterial strain [17], [18].

In this globalized world, millions of tons of agricultural waste are produced annually to fulfil the basic demands of the growing population. Agricultural waste mainly includes crop straw and livestock manure [19]. Consequently, utilizing such materials for valuable products facilitates recycling, mitigates pollution, and offers agricultural waste as a viable alternative substrate for enzyme production [16]. The extracellular protease and amylase are the most significant enzymes from a commercial perspective, used mainly in food, leather, dairy, brewing, textile, and pharmaceutical industries [16]. Protease accounts for about 60 % of the total enzyme market worldwide [20], whereas amylase accounts for about 30 % [7]. Gujarat state is linked with agriculture and allied activities. About 51.8 % of employment in Gujarat is based on agricultural activities. The largest cash crop production in Gujarat includes cotton, groundnut, tobacco, and cumin, whereas cotton, sugarcane, sorghum, millet, wheat, groundnut, and castor are the major agricultural harvests of Kathiawar, Gujarat, India (https://www.agrifarming.in). Agricultural wastes, especially livestock manure and crop straw, are rich sources of protein, carbohydrates, fats, and minerals. Therefore, the use of such agricultural waste for the production of large value-added products enhances the quality of products and minimizes production costs [19].

In recent years, immobilization techniques have emerged as an effective approach for enhancing enzyme properties, including stability and reusability [21]. The support matrix used for immobilization plays a pivotal role in the stability of the enzyme. For intense entrapment, using sodium alginate is wildly used method for immobilization of protease produced by B. brevis [22] and amylase produced by B. subtilis [23]. Further addition of cross-linker, chitosan, and nanoparticles such as TiO2 and SiO2 was reported earlier, enhancing enzyme immobilization [24] [25]. Therefore, in the current study, immobilization of crude amylase and protease was achieved by the entrapment method using a combination of sodium alginate, zinc oxide nanoparticles, hydroxyapatite, and glutaraldehyde. The significance of this study includes the simultaneous production of more than one enzyme using standard chemicals and agricultural waste by haloalkaliphilic actinobacteria and the immobilization of crude enzymes that can be beneficial for various industrial sectors.

The objective of this study was to isolate haloalkaliphilic actinobacteria from seaweed Sarconema sp. collected from the Okha coastal region and assess the biocatalytic potential of the identified strain, Streptomyces sp. OSs-1, which secretes protease, amylase, and L-asparaginase. The study specifically aimed to achieve co-production of protease and amylase using a single strain and a single medium, evaluate their co-production using agricultural waste as an alternative substrate, and further enhance their industrial applicability through immobilization of the crude enzymes.