Conjugation of Candida rugosa lipase with hydrophobic polymer improves esterification activity of vitamin E in nonaqueous solvent

We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide(NIPAM),was grafted onto Candida rugosa lipase(CRL)to synthesize poly(NIPAM)(pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL.The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates(19.5-30.3 L·s^(-1)·mmol^(-1))were at least 7 times higher than that of free CRL(2.36 L·s^(-1)·mmol^(-1))in DMSO.It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media.The synthesis catalyzed by pNIPAM-CRL co njugates was influenced by the length and density of the grafted polymer,water content,solvent polarity and molar ratio of the substrates.In the optimal synthesis,the reaction time was shortened at least 7 times,and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4%and 6.6%at 55℃after the reaction for 1.5 h.The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction,thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length of pNIPAM.At the optimal reaction conditions(55℃and 1.5 h),pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept~70%of its catalytic activity after ten consecutive cycles.The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.

Non-Alcohol Route of Biodiesel Synthesis from Fried Palm Oil using Immobilized Candida rugosa Lipase

Synthesis biodiesel using biocatalyst is an emerging and attracting alternative process to replace the conventional process. However, biocatalyst is easy to be deactivated by alcohol, which is a reactant in biodiesel synthesis reaction. Therefore, it is needed to develop new method to maintain the activity and stability of the biocatalyst during reaction. New method to be developed is by changing the reaction route which is using alcohol to the reaction route which is not using alcohol. Route reaction of non alcohol can be done by changing the alkyl alcohol with alkyl acetate. Both have the same function as alkyl supplier during the reaction. In this research, methyl acetate was reacted with triglyceride from fried palm oil using Candida rugosa lipase in batch reactor. The reactants and products were analyzed using HPLC. The effect of operating factors such as enzyme concentration, substrates ratio, operating temperature and addition of inhibitor using free and immobilized enzyme were investigated. The experimental results showed that 89.6% of triglyceride from fried palm oil was converted to its corresponding methyl esters under the condition of 4% wt lipase based on substrate weight, 1/12 mol rasio of oil/methyl acetate after 50 hours reaction using immobilized lipase. Stability test indicated that the activity of the immobilized biocatalyst was still remained after three reaction cycles.

Synthesis of Vitamin E Succinate from Candida rugosa Lipase in Organic Medium

A screening of commercially available lipases for the synthesis of vitamin E succinate showed that lipase from Candida rugosa presented the highest yield. The synthesis of vitamin E succinate in organic solvents with dif- ferent lgP values ranging from -1.3 to 3.5 was investigated. Of particular interest was that dimethyl sulfoxide （DMSO） with the lowest lgP exhibited the highest yield among all the organic solvents used. It suggests that lgP is incapable of satisfactorily predicting the biocompatibility of organic solvents due to the complexity of enzymatic reaction with hydrophilic and hydrophobic substrates in organic solvent. Effects of different operating conditions, such as molar ratio of substrate, enzyme concentration, reaction temperature, mass transfer, and reaction time were also studied. Under the optimum conditions of 10 g/L enzyme, a stirring rate of 100 r/min, a substrate molar ratio of 5：1 at 55℃ for 18 h, a satisfactory yield（46.95%） was obtained. The developed method has a potential to be used for efficient enzymatic production of vitamin E succinate.

Lipase-catalyzed Kinetic Resolution of Racemic 1-Trimethylsilylethanol in Organic Solvent

The enantioselective esterification of racemic 1-trimethylsilylethanol with acids catalyzed by lipase in organic solvent was successfully performed. The influence of some factors on the reaction was investigated. Among the four lipases explored, Candida rugosa lipase (CRL) showed the highest activity and enantioselectivity. Octanoic acid was the best acyl donor among the eleven acids studied and n-hexane was the most suitable medium for the reaction. The optimum shaking rate and temperature were found to be 150r·min-1 and 20℃ to 30℃, respectively. The enantiomeric excess of the remaining (5)-(-)-1-trimethylsilylethanol was 93% when substrate conversion was 53% upon incubation of the reaction mixture at 30℃, 150r·min-1 for 12 h.

Candida rugosa Lipase-catalyzed Kinetic Resolution of Hydroxyalkanephosphonates

An efficient lipase-catalyzed enantioselective hydrolysis of bu-tyryloxyalkanephosphonates in water-equilibrated diisopropyl ether was developed. The relationship between the substrates' structure and the reactivity, as well as the enantioselectivity of this enzymatic transformation was studied. The catalytic preference of crude Candida rugosa lipase toward such molecules was assigned according to modified Mosher's method and X-ray crystallographic analysis. Optically pure 2-hydroxy-2-arylethanephosphonates, 3-hydroxy-3-phenylpropanephosphon-ate, and 3,3,3-trifluoro-2-hydroxypropanephosphonates were conveniently prepared in this manner.