The Lipase With Synthetic Activity In Non-aqueous Phase From Rhizopus Chinensis And Its Characteristics

Lipases from Rhizopus sp.play an important role in the lipase enzymology and its applications of catalysis in non-aqueous phase.Because of its multiformity and complexity, there are many issues to be considered in the basic theory research and applications of lipases. More studies need to be done to make further clarification and understanding on them,so that to enrich and develop lipases subject and explore its industrial application.The mycelium-bound lipase from filamentous fungi Rhizopus chinensis CCTCC M201021 possesses the excellent catalysis ability for esterificarion and transesterification reactions in non-aqueous phase,and has a good potential in many industry applications.This thesis mainly aims at the issues of the R.chinensis lipase with synthetic activity in non-aqueous phase(sRCL).According to its characteristics and application purposes,sRCL fermentation was investigated to improve its production firstly,using an analysis for lipase synthetic activity in non-aqueous phase.Then the lipase activity was enhanced by some organic solvents treatment.After isolation and purification of the target protein,the lipase characteristics and catalytic properties were investigated in protein level.(1) Investigation of important factors influencing the mycelium-bound sRCL productionIn order to facilitate the control of the lipase fermentation and its downstream process, the solid-state soybean meal used as nitrogen source in industrial fermentation was substituted by soluble peptone,and the production of the mycelium-bound sRCL was improved several times.It was suggested that the solid-state fermentable composition contained in fermentation media would induce this fungus to grow in the way like solid-state fermentation and lead to its morphological differentiation,which could decrease the lipase production.Oleic acid and its related substrates with proper concentration can be used directly as effective carbon source and inducer for the lipase.However,peptone and olive oil were optimal nitrogen source and carbon source for the lipase production,respectively.During the submerged culture of R. chinensis,the effect of macro-morphological patterns on the mycelium-bound sRCL was investigated.It was found that the smooth but compact clump of mycelium facilitated the lipase production,although the growth was slowly.Hence,the morphology of the filamentous fungus can be used as an indicator for the lipase production in submerged cultivation.(2) Optimization of the composition of fermentation media and fermentation conditions to improve the mycelium-bound sRCL productionThe composition of fermentation media and fermentation conditions were investigated and optimized in order to improve the synthetic activity of the lipase.Using orthogonal test and response surface methodology,the composition of fermentation media was further optimized.The optimized media for lipase synthetic activity and activity yield was composed of peptone 57.94 and 55.58 g/L,olive oil 21.94 and 22.99 g/L,maltose 12.91 and 14.34 g/L respectively,with K₂HPO₄ 3 g/L,MgSO₄·7H₂O 5 g/L and initial pH 6.0.Under the optimal conditions,the lipase activity and the activity yield were improved 61.5%and 93.4% comparing the results before optimization,respectively.The adequate models obtained had predicted the lipase production successfully.The optimal fermentation conditions in shaking flask were 30℃and 200 r/min shaking speed,and fermentation time was shortened 12 hours. However,the lipase production in fermentor was lower because of limited mycelium morphology by usually used fermentors,especially stirred tank reactors.(3) Localization of the mycelium-bound sRCL and its pretreatments with organic solventsThe mycelium-bound sRCL with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase^TM and detergent extraction firstly.In order to improve its synthetic activity in non-aqueous phase further,the pretreatments of this mycelium-bound enzyme with various organic solvents were investigated.The pretreatment with isooctane improved evidently the lipase synthetic activity,and acetone pretreatment can also improved its synthetic activity effectively but with lower recovery,and be considered a method of lipase "concentration" or "purification".The pretreatment mechanism of organic solvents was discussed.Similar with the Yatalase treatments,the morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound sRCL by the breakage of cell wall and the exhibition of its synthetic activity.The pretreatment conditions with isooctane and acetone were further investigated,and the optimum effect was obtained by the isooctane pretreatment at 4℃for 1 h,resulting in 156%in relative synthetic activity and 126%in activity recovery.When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane,compared with the untreated lyophilized one.It was suggested that the pretreatment of the membrane-bound lipase with isooctane or acetone could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.(4) Purification of the membrane-bound sRCL and its molecular characterizationThe further study in protein level needs to isolate this membrane-bound lipase.The membrane-bound sRCL can be isolated effective by extraction with 1.5%Triton X-100 solution for 4 h,but its further purification was quite difficult.The lipase extract was sensitive to temperature and pH with low stability.It also exhibited high hydrophobicity,and Triton X-100 was required to dissolve it.When Triton X-100 was removed by absorption with Bio-Beads SM-2,the lipase extract would be aggregated.Based on the results of solvents pretreatment,acetone pretreatment can "purify" the membrane-bound sRCL,and break the mycelium.After that,washing with buffer could remove an amount of impurity,and the lipase purification could be improved.According to this strategy,purified target lipase was obtained indeed,and purification fold was close 17. Followed by ultrafiltration and DEAE-sepharose ion-exchange chromatography,the membrane-bound sRCL was purified.SDS-PAGE of the purified membrane-bound lipase showed that its molecular mass was～32 kDa.The N-terminal sequence of the lipase protein was identified as SDSCEVVQ,and no lipase with high homology had been reported.The pI of the lipase was～4.5,it was an acidic protein.Its apparent molecular mass and electric charge were influenced by Triton X-100.(5) The enzymatic characteristics and catalytic properties of the purified membrane-bound sRCLThe enzymatic characteristics of the purified membrane-bound sRCL were investigated based on the lipase synthetic and hydrolytic activities,respectively.It was found that different effects of condition factors such temperature,pH and chemicals on the lipase synthetic and hydrolytic activities.Among them,Cl^- inhibited its synthetic activity strongly,but PO₄^3- increased the activity;while Zn²⁺ increased its hydrolytic activity obviously,Fe²⁺,Fe³⁺, especially Hg²⁺ inhibited this activity strongly.Moreover it was suggested that this lipase was not a metalloenzyme,and disulfide bond had not a significant effect on the lipase active conformation.There might be a Ser in its catalytic center.The sRCL can catalyze the esterification reaction using various substrates.It showed optimal substrate specificity on octanoic acid and also other fatty acids with moderate or long carbon chain(C＞8) using ethanol as acyl-receptor.Using octanoic acid as acyl-donator,n-butanol had the optimal substrate alcohol specificity for it.Except methanol,various fatty alcohols(C＜10) can be used as its substrates.The purified lipase exhibited high hydrolytic activities against various fatty acid esters with moderate or long carbon chain and showed high specificity on laurate (C₁₂).However,unlike other Rhizopus lipases reported,the sRCL did not show the position selectivity on triolein.Based on these results,the membrane-bound sRCL can be regarded as a "true" lipase,but exhibiting different properties with other Rhizopus lipases.It should be a new lipase.The catalytic properties of the purified sRCL in organic phase were investigated further. It was stable in acetone and apolar solvents(3.5＜Log P＜5.1).The treatment with organic solvents cannot improve its synthetic activity.This result confirmed that the synthetic activity enhancement of the mycelium-bound lipase was mainly caused by morphological changes of mycelia surface with organic solvents pretreatment.Similarly,the lipase showed high synthetic activity in apolar solvents(Log P＞3.5) as reaction media,and heptane was the optimal one.It was also found water activity(a_w) had not significant effect on the lipase catalysis in organic phase.It can be concluded that this lipase was applicable in non-aqueous phase catalysis.The effects of detergent Triton X-100 and some substrates of lipase on the lipase activity regulation were discussed based the recent reports relative with it.It was suggested that the mechanism of lipase activity regulation by some factors might be very complex and need to be improved.