In multifarious industrial sectors like food, feed, pharmaceutical and textiles, enzyme-based bioprocessing is increasingly being adopted as an efficient, economical and environment-friendly alternative to conventional methods [2], [3]. This is reflected by the steadily increasing demand (USD $6.95 billion in 2022 and USD $9.1 billion by 2026; CAGR 6.4%) for industrial enzymes world-wide [marketsandmarkets.com, 4].

In nature, multifunctional proteins like sub-cellular reaction chambers on cell surfaces, scaffold-organized proteins and cellulosomes occur as a single polypeptide chain wherein numerous enzymes are brought together in close proximity to produce a final product [5], [6]. Many investigators have tried to mimic these naturally existing multi-functional proteins by developing chimeras bestowed with a wide range of desired functions using modern biology tools [5], [7], [8], [9]. The heterogeneous, complex and recalcitrant nature of plant-derived biomass warrants a coordinated action and interaction between multiple CAZymes (www.cazy.org) for its effective bioprocessing and transformation into versatile biochemicals and biofuels [5], [10]. To this end, cocktails of single enzymes [native or genetically engineered] have been used but have met with limited commercial success due to poor techno-economics [6]. Chimeric enzymes having multi-functionality in a single polypeptide offer a plethora of benefits like synergistic action, higher reaction rates and easier production, besides better structural and biochemical (temperature and pH profile) properties over single enzymes or their cocktails, and thus can be a plausible low-cost alternative from industrial biotechnology perspective [6], [7].

The carbohydrate binding module (CBM) (Mw 20–40 kDa) is an auxiliary domain with autonomous folding and comprises of 30–200 amino acids. They are classified into 88 families based on their amino acid sequence (http://www.cazy.org/Carbohydrate-Binding-Modules.html). Fusion of DNA sequences encoding catalytic domains with CBMs can facilitate the hydrolysis of insoluble substrates due to improved recognition of the substrate, increased local concentration of enzyme, higher enzyme activity and processivity besides modifying the interfacial properties of the substrate [11]. As part of the multifunctional proteins, CBMs by adhering to a substrate have not only improved the activity of associated domains/enzyme but also the activity of enzymes for substrates that are in close proximity [8]. Such versatile features of CBMs have led to novel protein engineering applications like designing glycosyl hydrolase-CBM fusions for enhanced stability and catalysis [8].

Traditionally, oil from oleaginous material (oil-bearing fruits and seeds) is produced either by mechanical extraction (hydraulic pressing, expeller pressing) or organic solvent extraction or their suitable combinations [12]. However, these methods suffer from several disadvantages like high capital investment and operational costs, high carbon footprint and lack of eco-friendly nature. On the other hand, aqueous extraction (AE) of oil is a promising, low-cost, less energy-intensive and environmentally-safe method wherein water (devoid of any chemical refining, bleaching and deodorizing) is used as the extraction medium and the co-products (meal, fiber and protein) are non-toxic and can be readily used. AE based methods have been used to extract oil from mature coconut kernel (Cocos nucifera L.) but have shown low oil yield [12]. As hemicellulose, cellulose, arabinogalactans, mannans, galactomannans, and pectin represent the major complex glycans in the coconut cell wall, usage of plant cell wall degrading enzymes-assisted aqueous extraction has been suggested as a possible strategy to increase the oil yield [13]. However, the high cost associated with extraction involving a single enzyme or their cocktail remains a major bottleneck.

In our previous study [1], two chimeric enzymes [bi-chimera: GH26 endo-mannanase (ManB-1601) and GH11 endo-xylanase (XynB) (ManB-XynB); tri-chimera: GH26 endo-mannanase (ManB-1601), GH11 endo-xylanase (XynB) and GH5 cellulase (CelB) (ManB-XynB-CelB)] connected by flexible peptide linkers (5–15 amino acids long) were generated and used for pectin extraction from fruit peels. The present study demonstrates that CBM3 [derived from Bacillus subtilis CAM21 CelB (GenBank Accession No: ON646199) [14] improves the temperature optima, pH and kinetic stability, substrate affinity, catalytic efficiency and structural properties of single and bi- and tri-chimeric enzymes. Furthermore, for the first time, we demonstrate that the fusion of CBM to CAZymes helps in improving the oil yield from mature coconut kernel during aqueous enzyme-assisted extraction.