【作者】 滕云；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【摘要】 非水相中脂肪酶催化的酯合成反应已广泛用于各种酯的合成,并且也成为近二十年来脂肪酶催化的研究热点。由于并不是所有的脂肪酶都能催化非水相中的酯化反应,造成了现有脂肪酶中能有效催化酯合成反应的酶并不多,必须开发新的具有高酯合成活性的脂肪酶。本文以获得高酯活性脂肪酶为目标,首先建立了一种能快速筛选具有酯合成能力脂肪酶的筛选和酶活测定方法,然后以一株根霉Rhizopus chinensis Y92为出发菌,采用离子束和亚硝基胍进行诱变,以建立的方法进行初筛,最终筛选得到一株脂肪酶高产菌R. chinensis Y92-M,并对其培养条件进行了优化。深入研究了影响具有酯合成活性脂肪酶合成的主要因素,并对该菌所产胞内脂肪酶进行了详细分析,揭示了全细胞脂肪酶水解与合成活力之间的关系,找到了控制酯合成活性脂肪酶大量合成的手段。针对丝状真菌形态变化对生产的影响,详细研究了摇瓶和发酵罐中形态变化对酶合成的影响。并将全细胞脂肪酶用于酯的合成和生物柴油的生产,具体内容和结果:(1)为了能筛选得到酯合成活性高的脂肪酶生产菌,本文建立了一种非水相中用于具有合成活性脂肪酶筛选和活力测定的新方法。该方法的原理是利用脂肪酶在正庚烷中催化对硝基苯酚棕榈酸酯(pNPP)和乙醇之间的转酯化反应,然后用NaOH溶液快速萃取生成的对硝基苯酚并在410nm检测吸光度。该法与其它已经报道的类似方法相比,所需仪器简单,底物廉价,操作简单,因此筛选效率得到了有效提高。(2)以R. chinensis Y92为出发菌,采用两轮离子束注入诱变,并结合亚硝基胍诱变,以所建立的方法作为初筛方法,以气相测定酯合成能力作为复筛方法,筛选得到一株酯合成活性显著提高的菌株R. chinensisY92-M,该菌单位菌体的合成活性由200 U/g,提高到400 U/g。(3)为了进一步提高该菌的产酶能力,对培养条件进行了优化。首先采用田口设计对培养条件进行了初步优化,对四个培养基组成因素(麦芽糖、橄榄油、蛋白胨、和K2HPO4)和四个环境因素(摇床转速、接种量、pH和装液量)进行了考察,结果分析表明环境因素对产酶影响更显著。通过响应面对培养条件进一步优化得到最优培养条件:接种量4.25×108 spores/L,橄榄油2.37% (w/v),装液量21 mL/250mL,蛋白胨4.06% (w/v),麦芽糖0.5% (w/v),K2HPO4 0.3% (w/v),摇床转速200 r/min,pH 5.5。预测全细胞脂肪酶的最大酶活产率为14349.2 U/L,验证性实验在最优条件下,R. chinensisY92-M产具有酯合成活性全细胞脂肪酶可达13875 U/L。(4)通过培养基中添加不同类型、不同量的脂肪酸或脂肪酸酯,考察了发酵过程中全细胞脂肪酶水解活性与合成活性的关系。结果表明,全细胞的水解活性受各种脂肪酸和脂肪酸酯的诱导作用并不明显,而碳链大于18的脂肪酸或者脂肪酸酯对全细胞合成活性有显著的诱导作用,最高酶活力是未加油脂的培养基条件的17倍。(5)对细胞各部分的蛋白组分的分析结果表明,胞内脂肪酶具有水解活性而不具备合成活性。膜结合蛋白具有很好的酯合成活性,同时也具有水解活性。该微生物对具有酯合成活性脂肪酶的调节既表现在酶量的调节,也表现在酶活性的调节。酶量的调节体现在具有酯合成活性的脂肪酶为典型的诱导型酶:当诱导物三甘酯添加量大于20 g/L时,能维持酶活在650 U/g,当诱导物量不足时酶活迅速下降;具有合成活性脂肪酶的高产的条件除必须有油脂添加外,细胞还必须与油脂充分接触,解除这种接触状态,酶的合成即停止。酶活性的调节则体现在全细胞水解活力的变化上:具有高合成活性的全细胞的水解活性并不高,而实际上具有合成活性的膜结合脂肪酶水解活力可达56 U/mg。对不同培养时间的细胞各部分的蛋白组分的分析,推测造成全细胞表观水解活力和合成活力不对应的原因是膜结合脂肪酶存在变构调节。(6)研究了不同三油酸甘油酯添加量对产酶的影响,提出了油脂流加策略。摇瓶中采用油脂流加方式也可以使得酶活力保持在650 U/g,酶活产率13000 U/L左右,而油脂总添加量只需15 g/L。发酵罐中的菌体生长与产酶过程和摇瓶相比存在较大差距,油脂补料发酵并不能达到摇瓶的产酶效果。(7)通过对摇瓶过程和发酵罐过程中菌丝微观和宏观形态的观测和分析,发现菌丝微观和宏观形态变化对膜结合脂肪酶的合成影响非常显著。摇瓶过程中,聚集状态(块状,球状,固定化)下的菌丝直径稍宽,菌丝生长单位多,菌丝未出现分化现象;而松散状态下的菌丝直径稍窄,菌丝体到生长后期出现分化现象,并有膈出现。聚集状态细胞的酶活力是松散状态细胞酶活的3倍,说明菌丝聚集有利于膜结合脂肪酶的合成。通过对发酵罐中菌体形态变化以及发酵液流变特性的研究,发现小球状菌丝细胞有利于产酶,而松散状菌丝细胞不利于产酶。对发酵液流变特性的测定结果表明,球状菌体下的发酵液流动性要明显好于松散状菌体,有利于油脂的分散。载体固定化同时满足了菌体形态呈聚集状和细胞必须和油脂充分接触两个酶高产的必要条件,因此固定化细胞经发酵罐培养后酶活能达到400 U/g。(8)为了验证R. chinensisY92-M所产全细胞脂肪酶在非水相中的催化能力,将该酶和两种具有高酯合成活性的商品化脂肪酶Novo435和Amano PS-C的酯合成能力进行比较,结果显示Y92-M所产全细胞脂肪酶具有很好的酯合成能力。(9)考察了该酶在正庚烷体系中催化脂肪酸乙酯的能力,当直链脂肪酸浓度为0.6 mol/L时,该酶对碳链大于6的脂肪酸有较好的选择性,转化率可达90%以上。实验结果还表明,该脂肪酶与出发菌所产脂肪酶相比,在无溶剂体系中能有效催化辛酸乙酯和油酸乙酯的合成。还考察了该酶在无溶剂体系中合成生物柴油的能力,结果表明该全细胞脂肪酶能有效催化地沟油,豆油,麻枫籽油等各种油脂生产生物柴油,转化率均在85%以上。固定化细胞也表现出很好的催化能力,摇瓶中转化率可达75%,填充柱中的转化率可达80%,而载体内部的转化率可达到88%。若能对填充柱体系进行更为系统的研究,有望实现工业化生产。更多还原

【Abstract】 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.更多还原