Carica Papaya Lipase Purification And Its Application

Carica papaya is a soft-stemmed and unbranched tree, as a native to the Central America, the papaya tree has successfully established in many ecological niches in tropical and subtropical climates. In China it has been widely grown in Hainan, Guangdong, Guangxi, Yunnan and Taiwan. All aerial parts of the plant, including the unripe fruits present laticifers of which contained varieties of proteases and lipases, these proteases and lipases complex is called crude Carica papaya enzyme or papain. If incisions are made in those aerial parts, especially in the unripe fruits, an abrupt release of latex (the so-called C. papaya latex) is observed. The latex contains about85%of water,15%of hydrolytic enzymes by weight. The crude enzyme has already been widely used in medicine, food, leather, textile and cosmetic fields. Moreover the widely grown Carica papaya in China supplied the large scale production of the above enzymes with rich source of raw materials, therefore the development of papaya enzymes has important application value. This paper mainly did the following work:In the first part of this paper, as the latex of Carica papaya contains varieties of hydrolytic enzymes including papain, glycyl endopeptidase, chymopapain and caricain and lipases, this limits its use in the field of medicine, chiral compound resolution, cosmetic use. So firstly we intend to separate the important enzymes-contained in the latex:papain and payapa lipase. As papain could be easily dissolved in water, and payapa lipase was tightly integrated with unknown materials which made payapa lipase have poor solubility. So papain and lipase could be separated by centrifugation after redissolving the crude enzyme in pH7.5phosphate buffer solution.Subsequently, we successfully prepared lyophilized Carica payapa lipase(CPL). As stated above the payapa lipase was tightly integrated with unknown materials, and it was traditionally considered as "naturally immobilized" lipase, so it could be separated from other enzymes by washing it with phosphate buffer, after lyophilized15%of CPL by weight was collected. By measuring its water content and the activity, it was found that the lyophilized CPL need2%(w/w) water to maintain its maximum activity and the optimum lyophilization time was8h. Then the enzymatic properties including its activity towards olive emulsification, optimum temperature, pH, thermal stability, organic tolerance, specificity in the esterification of fatty acids with alcohols were systematically studied. The experiment results showed that CPL has good thermal stability, organic solvent tolerance, and very suitable for the application in non aqueous media, for example it still remained more than90%activity after treatment under65℃for6h. CPL could also catalyze the direct esterification of acid and alcohol, and showed high activity towards medium chain length acid and alcohol, the produced esters is important fragrance, so the study of CPL’s specificity in the esterification of fatty acids with alcohols is valuable for theory and practice purpose.For the secondary part of this paper, CPL was used for terpene esters synthesis. It was found that CPL showed the highest activity in n-hexane with vinyl octanoate as the best acyl donor. To obtain high yield of terpene esters, the main reaction parameters were studied and further optimized by response surface methodology. The optimal conditions were:reaction temperature is55℃,50mg CPL, aw=0.32, equimolar ratio of substrates. Under these conditions, yield of more than99%was achieved after8h reaction. Ping-Pong Bi-Bi mechanism with dead end complex of citronellol(when citronellol concentration is>0.6M) was found to fit the initial rate data and the kinetic parameters Km for citronellol(821.7mM) and vinyl octanoate(30.9mM) were obtained by regression analysis.For the third part of this paper, CPL was used as catalysis in the preparition of chiral pharmaceutical intermediates. Secondary alcohols, acids and amino acids are important intermediates for asymmetric synthesis and also the mostly used substrates for lipase catalyzed resolution, in this part by optimizing the reaction temperature, solvent, acyl chain length, molar ratio, the three kind of substrate mentioned above were successfully resolved by CPL. Initially,4-phenyl-2-butanol was chosen as a model alcohol and the effects of prime reaction parameters were investigated on its enzymatic acylation. The effect of the chain length of the vinyl esters as acyl donors was studied. Vinyl hexanoate was found to be the best one among the acyl donors examined with regard to both the conversion(49.5%) and enantioselectivity(e.es99.4%, E>200). As to the temperature effect (30-55℃) on the acylation of4-phenyl-2-butanol with vinyl hexanoate, lowering the temperature resulted in a large decrease in the reaction rate, while temperature did not affect the enantioselectivity. As to the solvent, n-hexane proved to be the best one among the solvents examined in terms of enantioselectivity. Based on the above results, CPL was also successfully used in the enantioselective acylation other benzylcarbinols with vinyl hexanoate as acyl donor.The Carica papaya lipase (CPL) was furthermore explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-profen esters in water-saturated organic solvents. The kinetic analysis in water-saturated n-hexane indicated that both acyl donor and acyl acceptor have profound influences on the lipase activity, E-value, and enantioselectivity. Best enantioselectivity was found for substrate that contained a bulky substituent at the meta-position of2-phenyl moiety of the acyl part. For naprofen, the conversion reached up to49%after30h reaction with excellent enantioselectivity(E=173). The kinetic analysis also showed that the enantioselectivity came from the different reaction rate for the two isomers, not from the difference of the Km for the two isomers, for naprofen the quotient of K2R/K2S was195which approximately equal to the E value173. In conclusion, the different KS for the two isomers resulted in the enantioselectivity.At last efficient synthesis of3-amino-3-phenylpropanoic acid (BPA) enantiomer had been developed via the lipase-catalyzed enantioselective alcoholysis of the corresponding racemic N-protected-BPA-2,2,2-trifluoroethyl esters in an organic solvent. The reaction conditions and substrate structure effect including amino protecting group size, the space between the benzene ring and amino group, the chain length of the ester, the ester type, effect of substituents on the bezene ring were systematically studied. It found that the enantioselectivity and reaction rate were much enhanced by switching the conventional N-Ac-BPA-methyl ester to its2,2,2-trifluoroethyl ester, large size protecting groups showed negative effects on both the enantioselectivity and reaction rate, high conversion(50%) and enantiselectivity (E=230) were achieved when acetyl group used as amino protected group. When methanol was used as acyl acceptor, the reaction proceeded smoothly and enantiospecifically(E>200), only the (S)-product was formed. Benzene ring with electron-withdrawing groups would significantly reduce the reaction rate, for example, compared with electron-donating group H (6.40*10-3mM/h), the S-isomer reaction rate for Cl was sharply felled to6.80*10-4mM/h. By optimization high conversion(50.1%) and enantioselectivity(504) were achieved in n-hexane at50℃after6h reaction. At the end of this part the reaction was scale-up, the obtained S-3-amino-3-phenylpropanoic acid was successfully used as key chiral intermediate in the synthesis of (s)-Dapoxetine and gave high enantiomeric excess>99%.