Biosynthesis Of L-ascorbyl Aromatic Acid Ester And Studies On Microbial Keratinase

Abstractâ… Transforming L-ascorbic acid to its organic acid esters was a simple and efficient way to solve problems caused by its poor liposolubility.In this process,the lipase-catalyzed esterification in organic media was recently involved and intensively studied.However,no data were available on L-ascorbyl aromatic acid esters,although they found wide applications in perfumery,cosmetics and food.The aim of this work was to study the lipase-catalyzed esterification of ascorbyl benzoate in organic media, to optimize this biosynthesis process using a statistic approach,to investigate interactions among variables,and to explore its antioxidant and antimicrobial ability.1.Novozym435 was used in this work.A series of organic solvents with a log P from -1.30 to 2.50 were investigated,in which cyclohexanone(log P=0.96)was found to be the most suitable for this study.The optimum reaction condition in cylcohexanone was at pH 6.0,a_w 0.33,a substrate concentration form 0.06 M to 0.1 M, 65(?)and over 150 rpm.Experiment results also demonstrated that benzoic acid was not an ideal substrate of lipase,which led to low conversion rates,but its limitation could be overcome by excess L-ascorbic acid.2.The first statistical model showed that only substrate concentration,water activity and temperature had significant effect on the reaction.Enzyme concentration affected this process slightly,and effect of reaction time became minor after equilibrium.For the first time,using a partial derivative method,we quantitatively demonstrated how the optimum of variable studied depended on the constant value of fixed variables used in a single factor experiment.The positive correlation existed between substrate concentration and temperature,and water activity and substrate concentration,whereas a negative correlation existed between temperature and water activity.The second statistical model predicted a maximal conversion rate(47.66%)at enzyme concentration 10 g/L,substrate concentration 0.1031 mol/L,a_w 0.4896,48 h and 66.63(?).This prediction was confirmed by further experiments.3.Schaal oven test illustrated that the antioxidant activity of ascorbyl benzoate was comparable to that of ascorbyl palmitate,and minimal inhibitory concentration (MIC)data showed that its antimicrobial activity was weaker than that of benzoic acid. This will not influence the antimicrobial application of this product,since esters as antimicrobial agent also have some advantages such as a wide range of active pH over acids.Abstractâ…¡Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid,strongly cross-linked structural polypeptide "keratin" recalcitrant to the commonly known proteolytic enzymes trypsin,pepsin and papain. They are widely employed in feed,fertilizer,cosmetics and many other industries, especially in transforming keratin waste to reusable protein resources.However,most keratinase microbial producers were not suitable for industry use due their long production cycle and low productivity.In this study,a keratin-degrading bacterium was isolated and identified;then,its extracellular keratinase production was optimized of by response surface methodology;finally the keratinase was purified for study of characterations and applications.1.Strain L99,an efficient keratin-degrading bacterium,was newly isolated from waste water with decayed feathers.On the basis of physiological,morphological,16S rDNA,and cellular fatty acid studies,the new isolate was identified as Chryseobacterium and named as Chryseobacterium miricola L99.Adding sucrose, glucose,or maltose to medium using keratin as sole nutrition could promote keratinase procution,while adding exogenous nitrogen sources had no siganificant effect on keratinase procution.From Mg²⁺,Ca²⁺to Zn²⁺,their ability of promoting keratinase production descended.The optimal medium(g/L)for keratinase production was sucrose 16.6,feather meal 40.0,MgCl₂·6H₂O 1.9,NaH₂PO₄·2H₂O 6.0,K₂HPO₄·6H₂O 1.0.The optimal cultivation conditions were at 25.7(?).,inoculum size 4.12%,34.1h and 200rpm.After optimization,the keratinase activity increased to 221.37 U/ml in broth,which was about four times of the beginning.There was negative effect between cell growth and keratinase production.2.The keratinase of L99 was purified by saturation of ammonium sulfate precipitation,Q-Sepharose Fast Flow and Sephadex G-75.The final relative activity was 18.26 U//μg,and the purification multiple was 10.20.The molecular of the purified keratinase was about 32.5 kDa.K⁺,Ca²⁺and Mg²⁺stimulated the keratinase when their concentration was lower than 2.5mM;Mn²⁺,Co²⁺and Zn²⁺inhibited the keratinase activity.Concentrations of SDS lower than 0.1%(v/v)increased keratinase activity, while concentrations of Tween-80 or Triton -100 lower than 0.2%inhibited the keratinase activity.DTT or sodium sulfite hydrolyzed keratin greatly by itself.They decreased the keratinase activity by modifying substrates.3.To compare activities of different keratinases,a substrate specificity constant, which was only related to the kinds of keratinase and substrate,was introduced. Generally,substrates with high specific constants were more applicable for keratinase research.Substrate specificity constants of hair,wool,keratin azure,feather,azocasein and casein were 7.44,4.06,3.46,3.44,1.06 and 1.0.The km of keratin azure,wool, hair,feather,casein and azocasein were 0.0250,0.0158,0.0572,0.0060,0.0036 and 0.0050;their Maximum reaction rates were 1250(1000·A₅₉₅/ml/h),526 (1000·A₂₈₀/ml/h),1429(1000·A₂₈₀/ml/h),1000(1000·A₂₈₀/ml/h),59.52(μg/ml/min),5000(100·A₄₄₀/ml/h).4.The keratinase exhibited very excellent performance when only a concentration of 80 U/ml.Its ability of shrink-proofing was nearly the same to Savinase 16L and TG. The high degradation efficiency,low costs,and environment-friendly working conditions made L99 and its keratinase as potential tools for keratin treatment.