5In present study, we have synthesized lipase-nano-bio-conjugates via immobilization of various lipases on multiwall carbon nano-tube (MCNT), in order to construct an efficient and recyclable biocatalytic system. In screening study lipase Pseudomonas fluorescens (PFL) acted as an efficient biocatalyst (lipase-nano-bio-conjugates) which showed higher retention of lipase activity and protein loading. Consequently immobilization support:lipase (MCNT:PFL) composition was screened in which MCNT:PFL (2:1) was worked out as robust biocatalyst composition which showed higher activity retention and protein loading. This nano-bio-conjugate was then 10 characterized in details with physical and biochemical techniques using SEM, TEM, FTIR, Km, Vmax, catalytic efficiency and (%) water content analysis. This developed biocatalyst was further used for practical biocatalytic applications such as O-acylation reactions. Various reaction parameters were optimized in details like reactant molar ratio (2:3.5), solvent, MCNT:PFL biocatalyst amount (36 mg), temperature (50 o C) etc. The developed biocatalytic protocol was then extended to synthesize several (twenty-two) industrially important acylated moieties with an excellent yield, these products are well characterized by 1 HNMR, 13 CNMR and GCMS analysis. Moreover in 15 present study, we have reviewed potential industrial applications of various synthesized compounds. Also, we have studied thermodynamic aspect which demonstrated more feasibility of use of immobilized MCNT:PFL lipase over free lipase. Interestingly, immobilized MCNT:PFL lipase showed 2.3 folds higher catalytic activity than free PFL. Besides this, biocatalyst was efficiently recycled upto five cycles. Thus the present protocol demonstrated, (i) synthesis of nano-bio-conjugates as a bio-catalyst, (ii) detail physical-biochemical characterization of nano-bio-conjugates, (iii) optimization of biocatalytic protocol (iv) practical biocatalytic 20 applications along with mechanistic study (v) thermodynamic feasibility study and (vi) recyclability study. 65 conjugates as compared to bulk solid materials. This is because of key properties such as higher surface area, low mass transfer resistance, effective enzyme loading, nano-scale dispersion and ease of surface functionalization/ modification. [10][11][12][13][14][15][16][17][18][19] Thus, discovery of nano-scale materials proved countless 70 applications and scope based on their extraordinary properties