การผลิตผงเอนไซม์ไพรูเวตดีคาร์บอกซิเลสหลายระดับความบริสุทธิ์จากเซลล์จุลินทรีย์ผลิตเอทานอลที่ใช้ลำไยสดและลำไยอบแห้งล้นตลาดเป็นแหล่งอาหารคาร์บอน

บทคัดย่อ

The production and utilization of bioethanol had attracted worldwide attention as a strategy for decreasing global warming and improving global energy security. PDC was a key enzyme in homoethanol fermentation process. As a side reaction, PDC could ligate TPPbound ‘active acetaldehyde’ to added benzaldehyde resulting in PAC. PAC was a precursor for the commercial production of ephedrine and pseudoephedrine, which were used primarily as bronchial dilators and nasal decongestants. The most economical and popular method for ephedrine and pseudoephedrine synthesis was through biotransformation with benzaldehyde by yeast PDC followed by reductive animation. The ethanol production capability of 50 microbial strains in glucose medium under shaking condition with the medium working volume of 100 ml, 30oC, 200 rpm for 24 h. In order to compare the capability of these microorganisms, the production levels of ethanol, PAC and dried biomass, carboligase activity of PDC, as well as remnant cells viability after cultivation were determined. The highest carboligase activities (0.39 ? 0.06 U/ml) and PAC production (19.83?3.36 mM) was observed for C. tropicalis TISTR 5350. The most interesting candidates were C. tropicalis TISTR 5350, 5306, and 5144 after taking into account the production levels of dried biomass concentration, ethanol concentration, PAC concentration, PDC activity, and remnant cells viability with corresponding scores of 70.02, 58.03, and 56.21 ? 3.48% respectively. The propagation of seed inocula for Candida spp. and Saccharomyces spp. (five strains of each), namely, C. tropicalis TISTR 5306, C. tropicalis TISTR 5144, C. fennica TISTR 5618, C. lusitaniae TISTR 5156, C. tropicalis TISTR 5350, S. ellipsoideus TISTR 5199, S. ellipsoideus TISTR 5194, S. cerevisiae TISTR 5606, S. cerevisiae TISTR 5339, and S. cureanes UNSW 709800 were performed using expired commercial dried longan extract at 30oC, 200 rpm shaking speed for 48 h. C. tropicalis TISTR5306 and S. cerevisiae TISTR5606 could produce the highest ethanol concentration levels of 44.83 ? 1.26 g/L and 45.58 ? 1.12 g/L, respectively with the similar level of ethanol yield of 0.39 g ethanol/g sugars consumed. The corresponding PAC concentration and PDC activity levels for C. tropicalis TISTR5306 and S. cerevisiae TISTR5606 were 1.22 and 2.11 as well as 0.10 and 0.23 U/ml, respectively after 48 h. The kinetics of batch fermentation processes for ethanol production using C. tropicalis TISTR 5350, 5306, and 5144, which were at the top of the list in the first section, were examined in the second section. The maximum ethanol production rates from C. tropicalis TISTR 5350, 5306, and 5144 were 0.84 ? 0.56, 0.66 ? 0.25, and 0.27 ? 0.21 g/l/h, respectively. The ethanol yield from C. tropicali TISTR 5350 and 5144 also approached the theoretical yields of 0.49 and 0.50, respectively. The highest dried biomass concentration of 15.14 ? 0.33 g/l was found from C. tropicalis TISTR 5350 at 36th h cultivation period. The trend of increasing dried biomass concentration from this strain occurred simultaneously with ethanol formation and sugar utilization. An enzymatic process based on extracted PDC overcomes the problem of byproduct benzyl alcohol production. Four methods of cells disruption which included application of glass beads, freezing/thawing, silver nanoparticles, and combination of all three methods were investigated in the third section to choose the best method for partial isolation of PDC from C. tropicalis TISTR 5350. The maximum level of PDC activity (0.53 ? 0.05 U/ml and 0.17 ? 0.02 U/mg protein) from 15 ?g/ml of silver nanoparticle concentration level was obtained and compared with the other cells disruption methods. The result showed that the highest specific PDC activity was obtained from glass bead method with the corresponding PDC activity of 1.04 ? 0.11 U/ml and 0.27 ? 0.06 U/mg protein. Glass bead method should thus be selected as the method of choice to preserved enzyme activity and could be simply applied when the specialized equipment was unavailable. The PDC from C. tropicalis TISTR 5350 released from glass bead disruption method was prepared for partial purification in comparison with ammonium sulphate and acetone precipitation techniques. Total PDC activity and percentage recovery (yield) of precipitated PDC enzyme based on 50% (v/v) cold acetone were significantly higher (1.13?0.02 U/ml and 98.27%, respectively) than any other concentration levels of acetone used and the precipitation obtained using the 40 to 60% ammonium sulphate saturation (0.75 ? 0.08 U/mg protein). Freeze drying of the precipitation of PDC enzyme by 50% (v/v) cold acetone showed the highest enzyme activity of 1.55 ? 0.07 U/mg protein. The result from this section indicated that the recovery of PDC activity using the 50% (v/v) cold acetone was the most effective strategy for partially purifying PDC and stabilizing the enzyme in a freeze dried powder form. The last investigation to detect benzaldehyde loss and the inhibitory effect of phosphate concentrations on PAC production were conducted in a single-phase enzymatic PAC production from partially purified enzyme and whole cells of C. tropicalis TISTR 5350. The highest PAC production (95.80 ? 0.10 mM) and yield (0.96 ? 0.01 based on benzaldehyde and 0.80 ? 0.01 based on pyruvate) were achieved in a system using 100 mM benzaldehyde, 120 mM pyruvate, and 250 mM phosphate buffer with partially purified PDC from C. tropicalis TISTR 5350 at 6.39 ? 0.01 U/ml.

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