Systemic Concocting of Cross‐Linked Enzyme Aggregates of Candida antarctica Lipase B (Novozyme 435) for the Biomanufacturing of Rhamnolipids

Abstract

In the present study, Candida antarctica lipase B was immobilized on amine‐functionalized silica microspheres as cross‐linked enzyme aggregates (CLEA) and utilized for the biomanufacturing of rhamnolipids (RL). Lipase CLEA synthesized under optimized conditions of 2.0:1.0 by volume of silica microsphere/enzyme concentration, a 1.0:2.5 (v/v) ratio of enzyme/2‐propanol, 7 mM glutaraldehyde concentration, when incubated at pH 9.0 and 40 °C, for a cross‐linking time of 30 min were observed to exhibit superior biocatalytic properties and a maximum enzyme load of 770 U g−1. Lipase CLEA exhibited enhanced pH stability in acidic and alkaline media and increased temperature resistance as compared to free lipase. Both free and CLEA lipases were used to synthesize RL in different solvent systems. After 12 h, from initiation of the esterification, the degree of esterification (molar conversion yield) reached 46% and 71% in the batch mode. 1H and 13C nuclear magnetic resonance (NMR) and high‐performance liquid chromatographic (HPLC) analysis confirm RL production by CLEA lipase. The CLEA showed greater confrontation to enzyme‐mediated bioprocess approach as compared to its soluble counterpart and exhibited excellent RL production and catalytic activity even after its tenth successive reuse.

Amino‐functionalised mesoporous silica microspheres for immobilisation of Candida antarctica lipase B – application towards greener production of 2,5‐furandicarboxylic acid

“…Water molecules in trace amount are important in lipase‐mediated reaction, as it maintains the three‐dimensional structural integrity and the biocatalytic activity of the enzyme. However, an excess amount of water molecules is deleterious to the activity of lipases [21]. Hence, in order to draw a comparison between the synthesis of the two esters using free and covalently immobilised CaLB in aqueous and hexane systems, the equivalent amount of acid consumed for 20 h, with periodic estimation of percentage ester formation, was studied: The volume of the base used corresponded to an equivalent amount of acid in the reaction mixture, which is indicative of the amount of acid being used for ester synthesis, and pointed to the percentage ester formation.…”

“…The reaction mixture was then incubated for 0.5–24 h, followed by washing three times to obtain the desired covalently bound CaLB. The activity of the free and immobilised CaLB was measured according to the procedure previously established by our group [21] and the absorbance was measured at 410 nm ( ɛ max 15,000 M −1 cm −1 ). One unit (U) of lipase activity was defined as the amount of enzyme needed to produce 1 μmol of pNP from pNPP at pH 8.0 and 40°C in 1 min.…”

“…Water molecules in trace amount are important in lipase-mediated reaction, as it maintains the three-dimensional structural integrity and the biocatalytic activity of the enzyme. However, an excess amount of water molecules is deleterious to the activity of lipases [21]. Hence, in order to draw a comparison between the synthesis of the two esters using free and covalently immobilised CaLB in aqueous and hexane systems, the equivalent amount of acid consumed for 20 h, with periodic estimation of percentage ester formation, was studied:…”

“…Thus, the present work is aimed at synthesising the biopolymer precursor, FDCA, using CaLB covalently immobilised on the commercially available mesoporous silica. The mesoporous silica microspheres used for immobilisation are complex but have structured inorganic framework, high surface area and particle size, which made the immobilisation process more efficient [21]. This study developed an operationally stable CaLB system with enhanced biocatalytic activity, and, subsequently, a one-pot biocatalytic toolbox was established for the synthesis of alkyl esters, which were employed as acyl donors in tandem with hydrogen peroxide in the biocatalytic conversion of 2,5-diformylfuran (DFF) to FDCA.…”

Magnetically assisted commercially attractive chemo-enzymatic route for the production of 5-hydroxymethylfurfural from inulin

“…The reusability of immobilized inulinase (2.5 U/mg) was evaluated by performing repeated batch runs of inulin hydrolysis at 50°C and it was observed that 10 cycles of operation could be achieved with the immobilized inulinase. In the first cycle of inulin hydrolysis, the fructose release was 34 g/L, which gradually declined to 23.6 g/L at the fifth cycle, which could be due to the leaching out of the enzyme due to continuous washing [54]. Further, at the tenth cycle, the fructose released subsequently dropped to 9.2 g/ L, probably due to the decrease in thermal stability of the immobilized inulinase after prolonged incubation at 50°C [54] (Fig.…”