Synthetic Lipases Produced Under Solid-state Fermentation By Rhizopus Chinensis Isolated From Chinese Daqu

The mycelium-bound lipases from filamentous fungi Rhizopus chinensis CCTCC M201021 possess excellent catalysis ability for esterification in non-aqueous phase.This thesis mainly aims at the study of the synthetic lipases by R.chinensis from solid-state fermentation(SSF).Firstly,in order to provide large amounts of whole-cell synthetic lipases for the following investigations,process factors affecting the production of mycelium-bound synthetic lipases from SSF were studied.Then,the lipases from SSF was purified and compared with the synthetic lipases from submerged fermentation(SmF),and a solid-state specifc lipase(named as SSL1) was proved to be uniquely expressed in SSF.Finally,western blot and indirect Elisa were used to monitor the fermentation process,with the aim of identifying which solid-state environmental factors are involved in the induction of SSL1(1) Investigation of process factors influencing the production of mycelium-bound synthetic lipases of R.chinensis during SSFMajor process parameters affecting the production of whole-cell synthetic lipase(WCSL) by R.chinensis in SSF were optimized based on synthetic activity.A combined substrate of wheat flour with wheat bran(3/2,w/w) supported both good biomass growth and enzyme production.The maximum synthetic activity of 24447 U/kg substrate was reached by selecting a moisture content of 70%,initial pH of 6.5,supplementation of lactose(2%,w/w) as additional carbon source,supplementation of peptone(2%,w/w) as additional nitrogen source and olive oil(2%,v/w) as inducer,after 72 h of incubation.This production represented a 4.1-fold increase of lipase synthetic activity compared to the original medium.(2) Purification of the synthetic lipases from SSFR.chinensis was able to produce two synthetic lipases isoenzymes under SSF condition. These lipases were extracted from cell membrane using Triton X-100,and purified to homogeneity through ammonium sulfate precipitation,hydrophobic interaction chromatography and gel filtration chromatography.SDS-PAGE estimated the molecular weights of these two lipases,with the result of 64 and 33 kDa.SSL1 was obtained with the yield and purification fold of 2.6%and 55.2,respectively,while purification of SSL2 resulted in 0.7%in activity recovery and 138.3 in purification fold.In addition,two synthetic lipases from SmF were also purified to homogeneity by the same procedure,separately named as SML1 and SML2.Judging from N-terminal sequences and peptide mapping between these enzymes,it was found that SSL2 was shared by both fermentation techniques,however,SSL1 might be a special protein that can only expressed in SSF.(3) Enzymatic characteristics and catalytic properties of the purified SSL1In term of hydrolytic activity,SSL1 exhibited maximum values at pH 8.0 and 40℃,and retained most of the original activity in pH range of 6.5～8.0 and temperature range of 30～50℃.K⁺ obviously increased the activity of SSL1,on the contrary,Fe²⁺ and Hg²⁺ strongly inhibited the activity.Moreover it was suggested that SSL1 was not a metalloenzyme,and disulfide bond had not a significant effect on the lipase active conformation.Purified SSL1 showed clear preference for long-chained p-nitrophenyl esters,yielding maximum activity towards p-nitrophenyl palmitate.In term of synthetic activity,lyophilized SSL1 gave the highest values at 30℃and pH memory of 7.5,respectively.This lipase can catalyze the esterification reaction using various substrates,and showed optimal substrate specificity on fatty acids with moderate or long carbon chain(C＞8) using ethanol as acyl-receptor.Most of ethyl esters synthesized by SSL1 achieved good yields(＞90%),and tetradecanoic acid served as the best acyl donors.In addition,the biochemical properties of SML1 were investigated and compared with SSL1.And results demonstrated that SSL1 was more pH tolerant and thermostable than SML1,but the other characteristics between these two synthetic lipases are distinctly different, including effect of ions,substrate specificity,pH memory and best acyl donors in non-aqueous esterifications.(4) Preparation and purification of polyclonal antibody against SSL1Large amount of SSL1 from solid-state fermentation was purified and used as antigen to immunize rabbits.After four times of immunization,the ELISA antibody titer of rabbit antiserum reached 1:10000.Then,the obtained antiserum was purified by Protein A Sepharose FF chromatography.To confirm the specificity of SSL1,the polyclonal antibody was emplyed to react with the extracellular,intracellular and membrane-bound extracts from submerged fermentation,and results of western blotting revealed that no clear band was generated,thus proving that SSL1 only can be produced in the solid-state fermentation..In addition,the production of the polyclonal antibody against SSL1 provides a useful tool for investigating the relationship between SSL1 and typical solid-state environmental conditions.(5) Enzymatic characteristics and catalytic properties of the purified SSL2In term of hydrolytic activity,maximum hydrolytic activity of SSL2 was obtained at pH 8.0-8.5 and 40℃using pNPP as substrate.Slight activation of the enzyme was observed when Mn²⁺ is present,Fe²⁺,Fe³⁺,especially Hg²⁺ strongly inhibited the activity.SSL2 was not a metalloenzyme,and disulfide bond had not a significant effect on the lipase active conformation.Moreover,this lipase was stable in some solvents and was most active on p-nitrophenyl laurate(C12).In term of synthetic activity,lyophilized SSL2 exhibited maximum synthetic activity at pH memory of 6.0 and 30℃.Most of ethyl esters synthesized by this enzyme achieved good yields(＞90%),and caprylic acid served as the best acyl donor. In addition,this lipase presented a particular affinity for ethanol,n-propanol and n-hexanol, with conversion of 92,93 and 92%,respectively,after 20 h-incubation.In addition,the biochemical property of SSL2 was compared with SML2 which was purfied from SmF.And results showed that these two enzymes had the same performance on most characteristics under investigation,whereas SSL2 was more active at high temperature.(6) Regulation of environmental factors on the expression of SSL1 by western blot and indirect ElisaSSL1 from R.chinensis was found to be uniquely expressed in SSF.To identify which solid-state environmental factors are involved in the induction of this enzyme,we modified certain culture parameters and monitor the fermentation process using western blot and Elisa. Results demonstrated that neither presence of solids nor high culture temperature had effects on induction of SSL1.Further studies proved that the a low water activity played a significant role in the induction of SSL1,as evidenced by the increased expressions of target lipase (20-46μg/g dry cell) along with the decrease with water activity(0.927-0.967).Physical barrier against hyphal extension was found to be another required factor for the maximal expression of SSL1,since the expression of SSL1 was enhanced evidently by 3-fold using a membrane with smaller pore size(0.45 and 0.22μm).This investigation shed a light on the understanding of the regulation of special enzymes and allowed us to gain more information about enzyme production in SSF.