Screening Of High Lipase Production Strain For Ester Synthesis And Study Of The Enzyme Production And Fermentation Control

Lipase catalyzed esterification/transesterification in nonaqueous system has been used in many esters synthesis, and is also the most intresting field in lipase catalyzed reactions during the past 20 years. Most of the research works in this field focus on the organic synthesis by a known lipase or on the mechanism of the catalytic process involving. Because not all of the lipases could catalyze ester syshtesis reaction in nonaqueous system. It makes few lipases have perfect catalytic ability in nonaqueous system. Therefore, isolation and development of novel lipase with high esters synthesis ability become very important. Hower, to our knowledge, few researchers focus on screening of lipase production strain based on the ester synthesis ability, and also few researchers focus on regulation and production this kind of lipase.In this paper, lipase with synthetic activity was concerned. So, a fast screening and activity determination method for lipase synthetic activity in organic solvent has been built up firstly, and then the strain improvment for R. chinensis Y92 by combination of N+ implantation and nitrosoguanidine (NTG) mutation was carried out. A high producing strain R. chinensis Y92-M was selected base on the new screen method. The optimization of culture conditions were also studied for lipase production with synthetic activity, and the main effect factors on the lipase production was investigated detailedly. Base on the analysis of intracellular lipases biosynthesis, relationship between the lipase synthetic activity and hydrolytic activity was revealed. The morphology changes in submerged fermentation for R. chinensis Y92-M and their effect on production of lipase with synthetic activity were investigated. In order to validate the whole cell lipase ester synthesis ability, ethyl esters and biodisel synthesis by this lipase were performed in organic solvent or in solvent free system. The detailed contents of this dissertation follow:(1) A fast screening and activity determination method for lipase synthetic activity in organic solvent has been built up instead of the traditional olive oil plate in order to screen a strain which can produce the lipase with higher synthetic activity, The principle of this method was based on lipase catalyzed transesterification reactions between p-nitrophenyl esters and ethanol. Liberation of pNP was detected using a common spectrophotometer after extracting aliquots of sample from the reaction medium with 1 mL 0.1 mol/L NaOH solution. Compare with the similar method described by other researchers, the substrate used in new method was less expensive and commercially obtainable, and the measurement time for the new method could be largely shortened. To validate the effectiveness of the methodology, different common commercial lipases were selected and different detection and assasy methods were performed. The result indicated that the new method could distinguish various lipases with different synthetic activities, and the result correlated well with other methods. The kinetics curve of the transesterification reaction between pNPP and ethanol catalyzed by Amano PS-C with different concentrations monitored by the new method confirmed the possibility of monitoring the transesterification by the proposed colorimetric method. (2) Two cycles of N+ implantation combined with nitrosoguanidine treatment were used to improve the lipase production of R. chinensis Y92. A mutant strain R. chinensis Y92-M was selected using the new screen method as primary screen and ethylcaprylate synthesis ability detected by GC as secondary screen. The production of whole cell lipase with synthetic activity by mutant strain Y92-M was remarkably improved from 200 U to 400 U per gram dry mycelium.(3) The cultivation conditions and medium composition for whole cell lipase production with synthetic activity by R. chinensis Y92-M were studied and optimized. Taguchi design was employed to initial optimize the cultivation conditions and medium composition, to screen the important factors, and to confirm the level of each factors. Eight factors were selected for this study, namely maltose, olive oil, peptone, K2HPO4, agitation speed, inoculum level, fermentation volume and pH. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25×108 spores/L, olive oil 2.37% (w/v), fermentation volume 21 mL/250mL flask, peptone 4.06% (w/v), agitation speed 200r/min , maltose 0.5 %(w/v), and K2HPO4 0.3% (w/v), pH adjust to 5.5, a maximal contour (Y = 14349.2) could be predicted. The validation experiments were carried out under the optimum conditions, maximum activity yield for the whole-cell lipase obtained experimentally was found to be 13875 U/L, which 120% improved compare the result of 6300 U/L (non-optimized conditions). From the fermentation process for the R. chinensis Y92 and Y92-M, synthetic activity for the whole cell lipase was found different for two strains, but nearly no different in hydrolytic activity for the whole cell lipase. This result indicated the necessary to study the lipase production base on the synthetic activity.(4) To investigate the relationship between the two catalytic characteristics (hydrolytic and synthetic activity) for the whole lipase production, different amount and kinds of fatty acid and corresponding esters were added to the medium. The hydrolytic activity of the lipase was not induced by lipids efficaciously and could be detected regardless of whether substrate-related compounds were present. But the synthetic activity of the lipase was remarkably improved by the fatty acid and corresponding esters which C>18, the maxium activity was 17 times higher than medium without lipids.(5) The analysis of protein for the intracellular enzymes indicated that intracellular lipases have high hydrolytic activity, but nearly have no synthetic activity. However, the membrane-bound lipases exhibit both high synthetic activity and hydrolytic activity. The regulations of membrane bound lipase production by R. chinensis Y92 might have two ways: regulation of enzyme quantity and regulation of enzyme activity. The regulation of enzyme quantity was confirmed by the lipase with synthetic activity were strict inducible enzyme. This enzyme could be induced by oleic acid, and the synthetic activity could increase sharply only a little triolein (1 g/L) was added to the medium. The synthetic activity could be maintained at 650 U/g when more than 20 g/L triolein was added to the medium. Two requirements must be met at same time for high quantity of enzyme production: One is that the medium must contained oleic acid or it corresponding ester; another one is that the lipids must adhere to the fungi cell. Biosynthesis of this enzyme would be stopped once the adherence was eliminated. The regulation of enzyme activity was reflected by the change of hydrolytic activity for whole cell lipase. The whole cell with high synthetic activity exhibited low hydrolytic activity, whereas, hydrolytic activity was 55 U/mg for the membrane bound lipase which with high synthetic activity. Based on the analysis of protein for the intracellular enzymes obtained by different fermentation times, the allosteric regulation for the membrane lipase was suggested to explain the different between synthetic activity and hydrolytic activity for the whole cell lipase.(6) Fed batch cultivation was developed base on the study of effect of triolein initial concentration on the lipase production. The synthetic activity could reach 650 U/g, and the activity yield was about 13000 U/g, when triolein was fed at certain times in the shake flask. The final oil comsuption is 15 g/L, which is low than 20 g/L. Cell growth and enzyme production were different between the shake flask and fermentor, lipase production in fermentor by the triolein feeding could not get the same level as in the shake flask.(7) The changes in the macromorphology and micromorphology for mycelium were found to have remarkable effect on the membrane bound lipase production both in shake flask and fermentor process via observation and analyzing the mycelia macromorphology and micromorphology. During the shake flask process, the diameter of fully entangled filaments (clump, pellet or immobilized) was larger, the growth units were bigger, and no differentiations were found in the mycelia compare with the dispered filaments. When the mycelia morphology was fully entangled, the lipase production was 3 times higher than the dispered filaments.This result indicated that the mycelia aggregation was favor of membrane bound lipase production. Except for the effect on lipase biosynthesis and secretion directly, the changes in mycelium morphology also affected the mass transfer and hot transfer in the fermentation system. During the fermentor process, pellet morphology benefited the lipase production, and dispersed morphology was not. Result of broth rheology study in fermentor reveal that pelleted morphology could decreased the viscosity of the culture fluid, and improved mixing and mass transfer properties. Oil can dispersed completely under this condition. Cell immobilization on biomass support particles could improve the lipase production effectively, 400 U/g activity could be abtained in fermentor using immobilization.(8) In order to validate the catalytic ability of whole cell lipase produced by R. chinensis Y92-M in nonaqueous system. Comparison of ester synthesis ability between the whole cell lipase and two commercial lipases Novo435 and Amano PS-C were performed. The result indicated that whole cell lipase produced by R. chinensis Y92-M had excellence ester synthesis ability in organic solvent. Compare with the two commercial lipases, even though the activity and thermostabilization is little lower for this whole-cell lipase, but the lipase is easy to produce, more cheaper and suitable for industrialization.(9) The catalytic ability for fatty acid ester synthesis in heptane was investigated, when the concentration of fatty acid is 0.6 mol/L, and carbon number is more than 6, more than 90% of conversion was obtained. The whole cell lipase also able to catalyze esterification for ethylcaprylate and ethyloleate formation in solvent free system. Biodiesel productions in solvent free system by the enzyme were also studied. The enzyme was proved to be efficient in catalyzing many oils, such as waste oil, jatropha curcas oil and soybean oil to producing biodiesel, and 85% conversion could be realized. Immobilized mycelia also exhibited good ability in biodiesel production, 75% conversion could be abtained in shake flask, and could be improved to 80% in packed-bed reactor system. Even in the inner of immobilized biomass support particles in packed-bed reactor system, the 88% conversion could be achieved. This result indicated that the immobilized mycelia have potential application for industrial process.