【作者】 王栋；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【摘要】 根霉脂肪酶在微生物脂肪酶酶学研究和非水相生物催化中占有重要地位。由于脂肪酶存在广泛的多样性和复杂性,在脂肪酶的基础理论研究和实际应用中仍存在着许多问题,对这些问题的认识和理解仍需要大量的研究积累,以此不断丰富和发展脂肪酶学,并指导和拓展其在工业上的应用。丝状真菌华根霉（Rhizopus chinensis CCTCCM201021）菌体结合脂肪酶具有良好的催化非水相酯化反应的能力和工业应用潜力。本论文主要针对该华根霉非水相合成活性脂肪酶（sRCL）在生产和应用实践中出现的问题,根据其特点和应用目的,采用针对性的表征脂肪酶非水相催化酯合成能力的分析方法,通过对影响该脂肪酶发酵的关键因素研究,在发酵制备一定数量目的脂肪酶的基础上,利用有机溶剂处理等方法修饰提高其非水相合成活性,分离纯化目的蛋白并对其酶学性质和催化特性进行考察,在细胞水平和蛋白质水平进行了较为系统的研究,得到了一些新的发现。（1）影响菌体结合sRCL生产的关键因素研究首先为便于发酵过程控制,降低下游处理难度,以可溶性氮源蛋白胨替代工业应用的固态氮源豆饼粉,结果使菌体结合sRCL发酵水平成倍提高。研究表明豆饼粉等固态可发酵性组分的添加可能使华根霉以类似固态发酵条件下的代谢方式进行生长和代谢及形态分化,从而影响了菌体结合sRCL的产生。各种油脂及其相关物质对该脂肪酶发酵也有较大影响,适当浓度的油酸及其相关物质可直接作为有效的碳源和诱导物。在培养基主要组成中,蛋白胨和天然产物橄榄油是该脂肪酶发酵生产的最佳氮源和碳源底物。研究也发现华根霉液态培养中菌体表观形态对菌体结合sRCL的发酵有重要影响,表面光滑紧密的大菌团虽然菌体生长较慢,却有利于菌体结合sRCL的生产,可作为根据菌体表观形态判断sRCL生产水平的一个表观指征。（2）培养基组成含量及培养条件优化提高菌体结合sRCL的生产为进一步提高sRCL的生产水平,获得一定数量的目的脂肪酶用于后续研究,对发酵培养基各主要成分含量和部分发酵条件进行了考察和优化。在单因素研究的基础上,先后利用正交试验和响应面分析方法对该脂肪酶发酵培养基组成进行了优化。分别以脂肪酶合成活性和脂肪酶产率为指标,得到了优化的培养基组成为:蛋白胨分别为57.94和55.58g/L,橄榄油21.94和22.99g/L,麦芽糖12.91和14.34g/L,K₂HPO₄ 2g/L,MgSO₄·7H₂O 0.5g/L,起始pH 6.0。在优化条件下,脂肪酶合成活性和脂肪酶产率分别提高了61.5%和93.4%。同时得到了较为准确的脂肪酶生产的相关数学模型。该脂肪酶在摇床水平的最适发酵温度为30℃,200 r/min的摇床转速不仅可以得到较高的脂肪酶合成活性,并且使脂肪酶最佳发酵时间缩短约12小时。由于通常使用的发酵罐、特别是机械搅拌发酵罐培养方式限制了特定表观菌体形态的形成,该脂肪酶的发酵罐生产水平较低。（3）sRCL在华根霉细胞上的定位及其有机溶剂处理的影响为了对菌体结合sRCL进行深入研究,首先对其在菌体细胞上的位置进行了确定。利用真菌破壁酶Yatalase处理和去污剂提取分析,该脂肪酶被定位为膜结合蛋白。Yatalase处理可破坏菌体细胞壁结构,增加底物与酶的接触,从而提高该膜结合sRCL的活性。发酵得到的sRCL利用一些有机溶剂处理合成活性也得到了改善,其中异辛烷处理使脂肪酶合成活性增强最为显著。丙酮处理也可显著提高单位质量脂肪酶活性,但回收率降低,可用于该脂肪酶的“浓缩”或“纯化”。通过电镜观察,异辛烷等适当的有机溶剂处理与Yatalase处理作用相似,可使菌体表面结构发生改变,使膜结合sRCL更多地暴露,从而提高该膜结合sRCL的合成活性。对有机溶剂处理条件的研究表明,异辛烷4℃下处理1小时增强膜结合sRCL合成活性的效果最好,脂肪酶合成活性可提高56%,而活性回收率也提高26%。将其应用于催化庚烷中己酸乙酯的合成,催化效果确实得到了提高。利用这种处理方法,异辛烷和丙酮都可以制备用于非水相催化的“干”的脂肪酶制剂,而异辛烷处理更有利于提高该膜结合sRCL非水相中的合成活性。（4）膜结合sRCL的分离纯化及其分子特性研究进一步对该膜结合sRCL进行分离纯化,以便深入至蛋白质水平进行研究。采用常用的去污剂洗脱对膜结合蛋白进行分离提取,以含1.5%去污剂Triton X-100的缓冲液洗脱4小时,可以得到较好的提取效果,但进一步的纯化却相当困难。考察该脂肪酶粗酶提取液的性质发现,该膜结合sRCL对温度和pH都较为敏感,稳定性较差。而且该脂肪酶具有很强的疏水性,Triton X-100对维持其溶解性有重要作用,利用Bio-Beads SM-2树脂吸附去除Triton X-100易引起脂肪酶的聚集沉淀。普通的纯化方法对该膜结合脂肪酶的纯化有较大困难。根据有机溶剂处理的研究结果,丙酮处理可以“纯化”该膜结合sRCL,并使菌体结构破坏。利用缓冲液对其进行充分洗涤,能够去除大量杂蛋白,再经Triton X-100溶液提取,有可能提高该膜结合蛋白的纯化效率。采用上述纯化策略,确实得到了相当纯的目的蛋白,纯化倍数提高接近17倍。进一步通过超滤浓缩和阴离子交换层析,使目的蛋白sRCL得到了纯化。利用此纯化方法,可以简便而快速地纯化得到膜结合sRCL。纯化的膜结合sRCL分子量约为32 kDa,其蛋白N-末端氨基酸序列为SDSCEVVQ,Blast分析未发现与其同源性较高的已报道脂肪酶。该蛋白等电点约在4.5左右,为一个酸性蛋白。Triton X-100的存在对该脂肪酶分子大小、电荷性等表观分子特性有较大影响。（5）纯化的sRCL酶学性质和催化特性研究分别以脂肪酶合成活性和水解活性为表征,对纯化的膜结合sRCL酶学性质进行考察。结果表明:温度、pH、各种化合物等环境因素对该脂肪酶合成活性和水解活性的影响差异较大。sRCL合成活性主要受阴离子的影响,而金属离子则明显影响其水解活性。其中,Cl^-强烈抑制该脂肪酶合成活性,而磷酸根离子则对该活性有一定的提升。Zn²⁺显著提高了该脂肪酶的水解活性,但Fe²⁺和Fe³⁺,特别是Hg²⁺,却强烈抑制这一活性。研究表明该脂肪酶不是金属酶,二硫键在维持其活性构象中的作用也不大,该脂肪酶合成活性中心可能含有Ser。该脂肪酶催化有机相酯化反应,以乙醇为酰基受体时辛酸是最适的底物,对碳链长度大于8的中长链脂肪酸也有较高的催化酯合成的能力。以辛酸为酰基供体,脂肪醇底物特异性最好的是正丁醇;除了甲醇,该脂肪酶对碳链长度小于10的脂肪醇都有较高的合成活性。该脂肪酶的水解活性对月桂酸酯(C₁₂)的脂肪酸链长特异性最高,对中长链的脂肪酸酯也具有较高的水解活性。该脂肪酶不表现出对三甘酯的位置选择性。这些性质表明,膜结合sRCL是一种通常意义上的真正脂肪酶,但与一般的根霉脂肪酶有较大差异,结合其分子特性可以认为是一种新型脂肪酶。对纯化的sRCL有机相催化特性进行研究,结果表明,该脂肪酶对丙酮和非极性烷烃类有机溶剂（3.5＜Log P＜5.1）有较好的耐受性。与菌体结合脂肪酶相比有机溶剂处理基本没有增强纯化的sRCL合成活性,这一结果进一步证实,有机溶剂处理对菌体结合脂肪酶活性的影响主要是由于菌体结构的改变。同样,sRCL在非极性溶剂（Log P＞3.5）介质中的合成活性也相对较高,其中庚烷是该脂肪酶的最适反应介质。此外,有机相反应体系中初始水活度对该脂肪酶催化活性影响不大。以上结果表明,该膜结合sRCL比较适合非水相催化酯合成反应的应用。对近来发现的去污剂Triton X-100和三甘酯及其水解产物等对脂肪酶活性的调节进行了探讨。结果表明,上述活性调节因子对该脂肪酶活性的调节机制可能相当复杂,现有的脂肪酶催化机制尚需完善。更多还原

【Abstract】 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.更多还原