| Due to organic solvent tolerance, wide substrate scope, catalytic specificity, and no need to add the expensive cofactors in reactions, lipase plays an important role in biocatalysis. Lipase preparations for industrialization should be reused continuously with stable high activity in a long time in the view of technology and economic. Knoevenagel condensation is a classic C-C bond forming reaction, the product is often an α, β-conjugated enone, as pharmaceutical intermediate. In this work, low cost viscose fibred nonwoven fabric for lipase immobilization was developed and evaluated, combined with hydrophobic treatment. To extend the application of lipases, we also studied lipases catalytic promiscuity under Knoevenagel condensation of aldehydes and ester substrates. In addition, the role of nucleotides and amino acids as pre-enzyme catalysts (functional in non-aggressive solvents at neutral pH) is of fundamental interests for evolutionary biochemistry. Herein we explore the potential of neutralized basic amino acids or RNA/DNA as catalysts. The detained contents are listed as follows:1. Lipase immobilization and its application in esters synthesis:(1) Lipase immobilzation. Nonwoven fabrics of polypropylene (PP), polyethylene terephthalate (PET) and viscose fiber were used as novel and inexpensive support membranes for the immobilization of lipase. The properties of lipase immobilized on nonwoven fabric membranes by six methods, including co-solvent, co-solution, chitosan-coated, direct cross-linking and hydrophobic treatment were comparatively studied. A hydrophobic treatment improved the protein coupled content of silk cotton, viscose and PET surfaces more than threefold. Viscose prepared with the hydrophobic treatment could be reused up to 44 times. The initial rate of esterification remained high even when the amount of added water was increased to 10%. The post-hydrophobic treated immobilized lipase (retained a static water contact angle of 131.8°) can be reused for at least 20 batches, with acid conversion rate improved by at least 10% in each of the first five batches. Nonwoven viscose could be a potential enzyme immobilization matrix for industrial processes.Expand the possibility of various kinds of carriers that can be applied in esterification. We choose two kinds of carriers; with differentiate values in potential industrial application and mechanism study on lipase structure and catalysis. The study shows, the common polyurethane sponge performed similar immobilized lipase properties with polyester viscose fiber nonwoven fabric (5-6mg protein/g carrier,7000U/g carrier). Lipase immobilized from flocculent sedimentation can be reused for at least 15 batches. And amphiphilic polymer coated Fe3O4 nano-composite particles can be adsorbed directly in lipase concentrated solution with 847.2 mg protein/g carrier.(2) Application for esters synthesis with hydrophobic immobilized lipase. The lipase immobilization method in Yarrowia lipolytica fermentation broth in our lab was optimized on broth activity units without pH adjustments. Repeated adsorption could increase the utilization of lipase activity from fermentation broth. The temperature stability of the post-hydrophobic treated immobilized lipase was improved; 45℃ for 4h, lipase activity was still higher than 70%. As fitted by the equation of the ordinary ping-pong mechanism with EtOH inhibition, hydrophobic immobilized lipase reduced the inhibition effect of ethanol. The lipase immobilization method was investigated by the final activity of the lipase, as well as the water that had accumulated and the amount of lipase that had detached from the membrane under stirring. The immobilized lipase membrane could be reused in 10 iterative batch stirred tank reactor processes (a total reaction time of 150 h). The simple enzyme immobilization method can also be applied to the esterification synthesis of other esters, such as vitamin A palmitate and 2-ethylhexyl palmitate.2. Biocatalysis for Knoevenagel condensation:a mechanistic elucidation:(1) Natural basic a-amino acid of L-lysine, L-arginine and L-histidine neutralized to physiological pH as salts were investigated for their ability to catalyze Knoevenagel condensation of benzaldehyde and ethyl cyanoacetate. Compared with their free base forms, although neutralized alkaline amino acid salts reduced the catalytic activity markedly, they were still capable to perform an efficient catalysis at physiological pH as porcine pancreatic lipase (PPL), one of the best enzymes that catalyse Knoevenagel condensation. In agreement with the fact that the three basic amino acids were well neutralized, stronger basic amino acid Arg and Lys showed more obvious variation in NH bend peak from the FTIR spectroscopy study. Study of ethanol/water system and quantitative kinetic analysis suggested that the microenvironment in the vicinity of amino acid salts and protonability/deprotonability of the amine moiety may determine their catalytic activity and mechanism.(2) Mechanistic study on lipase catalytic promiscuity. Lipase-catalyzed Knoevenagel condensation of a ketone/aldehyde (e.g. benzaldehyde 1) and the active hydrogen compound (e.g. ethyl cyanoacetate 2 or malononitrile 3) is often regarded as catalytic promiscuity. The alternative mechanism suggests partial enzymatic hydrolysis of 2, whose products (but not the enzyme) initiate fusion 1+2. We found that three lipases (porcine pancreatic, Mucor javanicus and Yarrowia lipolytica) did not hydrolyze 2; nevertheless they significantly accelerated condensations 1+2 and 1+3 (where 3 is not hydrolyzable). The reaction was, therefore, associated with the promiscuous enzymatic activity, but did not evolve from the conventional esterase function. Conversion was performed mainly within the active site (based on competitive inhibition by caffeic acid). Yet, Ser residue (responsible for esterase activity) was unimportant, because its covalent modification did not affect condensation. The reaction was to some extent promoted by unspecific residues of lipase, as well as albumin and simple proton acceptors (e.g. sodium acetoacetate, sodium methoxide, water). This especially concerned 1+3 condensation, where 3 is a better H+-donor than 2. Spontaneous condensation in water/ethanol surprisingly revealed kinetics with substrate saturation. We explained this depart from linearity by a two-step steady state mechanism including deprotonation of the active hydrogen substrate 3H by polar solvent, followed by direct collision of a temporary complex solvent-H+-3-with 1. Similar mechanism with more sophisticated binding and positioning of substrates was conjectured for the lipases.(3) We demonstrated that (i) salts of RNA/DNA segments were capable to catalyse Knoevenagel condensation at physiological pH 7.0 with efficiency comparable to one of the best enzymes, porcine pancreatic lipase (PPL); and (ii) a broad scope of substrates could be successfully used in this reaction. Velocity of catalysis was positively correlated with the content of GC nucleosides in DNA/RNA; while the 3D-organization in DNA segments largely contributed to the elevated turnover number of catalysis. An insight into the reaction mechanism (based on quantitative analysis of kinetics) elucidated general similarities between DNA, RNA, and PPL in the substrate binding mechanisms. |
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