| Carotenoids are lipophlic isoprenoid pigments occurring in nature, systhesized by all plants and some photosynthetic microorganisms. Based on the variety of carotenoids and the imparity of cleavage positions, many apocarotenoids are produced, which play key roles in organisms, including retinoids, phytohormones and aroma compounds as the best-known examples. These carotenoids-derived aroma compounds with low flavor thresholds are important contributors to the quality of wine. The carotenoids cleavage enzymes belong to a family of enzyme to catalyze carotenoids, and they can convert substrates to the targeting products. The carotenoids cleavage enzymes have been characterized not only from various plants and animals, but also in microorganisms such as fungi, proteobacteria and photosynthetic bacteria. To our best knowledge, the capacity of Staphylococcus sp. has been rarely reported to catalyze carotenoids cleavage. However, in our previous research, Staphylococcus pasteuri TS-82 has been found to degrade carotenoids efficiently. In order to elaborate the enzymatic characterizations and the catalytic mechanism on carotenoids degradation, in this dissertation, a comprehensive research was made based on S.pasteuri TS-82 from some aspects including pathogenicity characteristics, incubation conditions, and the isolation and purification of carotenoid cleavage enzyme. Then the biochemical properties, substrate specificity, and the enzymatic reaction products(non-volatile and volatile compounds) were studied. The obtained results are listed as follows.(1) Pathogenicity characteristics and cultural conditions of S.pasteuri TS-82. The virulence and antibiotic susceptibility tests of TS-82 have been identified. No production of virulence-related enzymes was detected in S.pasteuri TS-82 and it was only resistant to tetracycline, indicating this strain is safety and can be used in food processing. Response surface methodology was employed to optimize medium conponents and culture conditions for the production and activity of carotenoid cleavage enzyme. Results showed the optimum medium: 41g/L sucrose, 1g/L K2HPO4, 0.5g/L Mg SO4·7H2O, 0.01g/L Fe SO4·7H2O, 0.5g/L KCl, 3g/L Na NO3, and 3.7g/L yeast extract, p H adjusted to 7.0. The incubation conditions: 16 h of culture at 36℃ with an inoculums concentration of 2% in liquid medium. The highest carotenoid cleavage enzyme activity was achieved under these conditions.(2) Isolation and purification of the obtained carotenoid cleavage enzyme. The carotenoid cleavage enzyme from S.pasteuri TS-82 was isolated and purified using methods of Mono Q 10/100 GL anion-exchange chromatography, preparative high performance liquid chromatography(PHPLC), and Superdex Peptide 10/300 GL chromatography. The purified enzyme performed β-carotene degrading activity of 125 U per g protein, 466-fold purification and a yield of 2.39%. By the analysis of high performance liquid chromatography(HPLC), the protein was confirmed that it showed a single peak in HPLC analysis with purity of 95%. It had a molecular weight of 655.093 Da identified by LC-ESI/MS.(3) Enzymatic characterization of the purified enzyme. Substrate specificity of the purified enzyme was investigated by using β-apo-8’-carotenal, β-carotene, zeaxanthin, canthaxanthin, astaxanthin and lycopene as substrates. The experiment results indicated that the purified enzyme by chromatographic column had a catalytic ablitily towards β-apo-8’-carotenal, β-carotene, zeaxanthin, canthaxanthin and astaxanthin. However, the degradation of lycopene was not detected. Km and Vmax for the different substrates were determined by plotting the activity data as a function of substrate concentration according to a Lineweaver-Burk plot. The Km values were 15.403, 10.99, 9.846, 11.285 and 10.952μM respectively, indicating that the enzyme showed the highest affinity towards zeaxanthin followed in a decreasing order by astaxanthin, β-carotene, canthaxanthin, and β-apo-8’-carotenal. And the Vmax were 0.922, 0.962, 1.406, 1.089 and 1.044μM/min respectively, indicating that the zeaxanthin enzymatic conversion was fastest followed by canthaxanthin, astaxanthin, β-carotene, and β-apo-8’-carotenal. In enzymatic characterization, the experiment results indicated that the purified enzyme had optimal temperature of 60℃ for degrading C40 carotenoids and 50℃ for degrading β-apo-8’-carotenal, and the temperature under 50℃ for keeping stable activity. The optimum p H value was 3.0 for degrading all substrates. Metal ions Al3+ and Fe3+ were identified as the potent catalyst of the purified enzyme, whereas, Fe2+ was identified as the potent inhibitor. H2O2 increased enzyme activity in degrading β-carotene at levels of 0~16m M. A low concentration of alcohol(4~16%) did not inhibit the activity of the enzyme at all.(4) Determination of non-volatile compounds. The variation of the non-volatile compounds generated from different substrates was measured along with the changes of zymolytic time, p H and temperature by HPLC. The results showed that the production of non-volatile compounds increased as zymolytic time, p H and temperature increased until the maximum production measured, then as zymolytic time, p H and temperature continued to increase, the productions gradually decreased. In addition, the study found that the enzyme catalyzed β-carotene cleaved to produce different apocarotenoids at different p H. Orthogonal arrays design combining quadratic polynomial stepwise regression analysis was applied to optimize the reaction conditions of the purified enzyme cleaving different substrates. The optimal reaction conditions can be concluded as follows: p H 4.5, 15 min at 30℃ for cleaving β-carotene; p H 4.5, 13 min at 30℃ for cleaving zeaxanthin; p H 4.5, 45 min at 33℃ for cleaving β-apo-8’-carotenal; p H 4.0, 41 min at 30℃ for cleaving canthaxanthin; p H 4.5, 7min at 50℃ for cleaving astaxanthin. The non-volatile compounds collected under the optimal conditions were tentatively identified using LC-APCI+/MS. The enzyme converted β-carotene to β-carotene-5,6-epoxide and β-carotene-5,8-epoxide, β-apo-8’-carotenal to apo-8’-carotenal-5,6-epoxide, zeaxanthin to apo-10’-zeaxanthinal-5,6-epoxide and zeaxanthin epoxide, canthaxanthin to apo-10’-canthaxanthinal-5,6-epoxide and canthaxanthin epoxide, astaxanthin to apo-10’-astaxanthinal-5,6-epoxide and astaxanthin epoxide, respectively.(5) Determination of volatile compounds. The volatile products bioconverted by the purified enzyme from five carotenoid substrates were identified using GS-MS. Evidently, β-carotene and β-apo-8’-carotenal were mainly converted to β-ionone, β-ionone-5,6-epoxide, β-cyclocitral and etc by the purified enzyme from S.pasteuri TS-82. Zeaxanthin, canthaxanthin and astaxanthin were cleaved to 3-hydroxy-β-ionone, 3-oxo-β-ionone and α,β-dihydro-β-ionone, respectively. Moreover, it indicates that astaxanthin is an another source for α,β-dihydro-β-ionone.Analysis of the cleavage products obtained from incubation of β-carotene and β-apo-8’-carotenal with purified enzyme from S.pasteuri TS-82 revealed two cleavage sites at the C9-C10/C9’-C10’ and C7-C8/C7’-C8’ double bonds. The identification of the cleavage products indicated that cleavage of xanthophylls catalyzed by the purifed enzyme occured at the C9’-C10’ double bonds. |
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