The Preparation Of Protein-Coated Micro-Crystals And Its Application In Biodiesel Production

Enzymatic biodiesel production technology has the advantages of mild reactionconditions, less methanol/ethanol consumption and non-pollution to environmentcompared to chemical technology. Nevertheless, the expensive price, inability to reuse andpoor stability of free lipases result in high cost of enzymatic biodiesel production. Hence,the research and application of new biocatalysts and catalytic processes have become a hotspot. In this research, Pseudomonas cepicia lipase (PS lipase) was immobilized throughthe effective method of protein-coated micro-crystals (PCMC) to obtain PS-PCMCs withimproved characteristics, and the application of PS-PCMCs in biodiesel production wasalso investigated. The main work is as following:1. Through the optimization of single factorial test and response surface methodology,the optimal preparation conditions of PS-PCMCs were:62.3mg of free PS lipase wasfully dissolved in200μL of phosphate buffer (50mM, pH7.0) to obtain the lipasesolution.403μL of saturated K2SO4solution was then added to the lipase solution. Thiscombined mixture was added drop-wise to the stirring tube (150rpm), which contained6.271mL of acetone. Then it was immediately centrifuged at4800×g for5min to removethe supernatant. The obtained precipitate was washed with2mL of acetone, then collectedand vacuum-dried before use. This PS-PCMCs was utilized to catalyze transesterificationof545mg of soybean oil and146μL of ethanol at40oC and200rpm for12h. Therelative biodiesel yield of PS-PCMCs was91.27%(87.22%of average relative proteinloading), while free PS lipase only gave48.72%conversion under the same conditions.2. The optimal reaction temperature of PS-PCMCs and free PS lipase were60oC and50oC, and the corresponding conversions were99.83%and56.64%, respectively. Afterbeing incubated at different temperatures (from25oC to70oC) for1h, PS-PCMCs and PSlipase were used to catalyzed biodiesel production at their optimal reaction temperaturesto investigate thermostability. The conversions of PS-PCMCs were in the range of92.52-99.64%. However, the catalytic ability of free PS lipase dramatically declined as theincubation temperature increased. The results indicated the enhanced thermostability ofPS-PCMCs. In addition, PCMC-PS demonstrated much higher activity than free PS lipase after incubation in different organic solvents for3h. PS-PCMCs showed67.71%conversion, but free PS lipase was almost completely inactivated after incubation inmethanol. After being incubated in tetrahydrofuran, t-butanol, hexane, heptane andisooctane, the conversions of PS-PCMCs were still in the range of92.84-97.41%, whichshowed the satisfied organic solvents stability of the immobilized enzyme. Whencatalyzed by PS-PCMCs, the relative biodiesel yields of most of the seven tested oils wereabove83%. Moreover, stillingia oil of high acid value (17.80mg-KOH/g-oil) gave91.08%conversion without any optimization. This demonstrated the extensive substrateapplicability of PS-PCMCs and also the promising future of its industrialization. Scanningelectron microscopy (SEM) detection showed significant morphological distinctionsamong PS-PCMCs, K2SO4crystals, and free PS lipase, which revealed the process ofPS-PCMCs formation. The results of circular dichroism (CD) anlysis showed that thealpha-helix content of PS-PCMCs was lower than that of free PS lipase, but in contrast tothe random coil content. This conformational transition was probably one of the reasonsfor the salient improvement in catalytic activity of PS-PCMCs.3. The optimized PS-PCMCs was applied to catalyze stillingia oil and ethanol toproduce biodiesel. The optimal reaction system contained588mg of stillingia oil and20%(on the basis of oil weight) of PS-PCMCs. The ethanol/oil molar ratio was4:1and thehexane/oil volume ratio was1:1with no water. The optimal reaction conditions were55oC and200rpm for12h, and the relative biodiesel yields were98.50%. Additionally, theoperational stability of PS-PCMCs was also investigated. For the PS-PCMCs with notreatment after each batch reaction, the catalytic ability dramatically decreased. The datashowed that the acetone treatment could slow down the activity decrease of PS-PCMCs ina certain level. It still exhibited69.92%conversion after8batch reactions. With thetreatment of hexane, PS-PCMCs showed82.29%conversion after8batch reactions,indicating the favorable operational stability.