【作者】 王凤超；

【导师】 刘巍峰； 张伟欣；

【作者基本信息】 山东大学 ， 生物与医药（专业学位）， 2025， 博士

【摘要】 近年来,全球能源价格整体上处于上升态势,同时全球范围内推动实施的减碳方案使生物燃料再一次成为全球热点。生物柴油是生物燃料的重要组成部分,可替代传统的石化柴油,是国际公认的可再生清洁燃料。国外生物柴油的原料多来自于食用植物油,而我国由于人口众多,为避免与人争粮,生物柴油行业必须寻找丰富廉价的可替代性原料。酸化油是一种废弃油,主要来源于食用植物油精炼过程,产量较大,国内仅大豆酸化油年产量大约在60万吨。以废弃酸化油生产生物柴油具有更高的减碳属性,有利于推动经济社会发展绿色化、低碳化。脂肪酶因能催化完成生物柴油生产过程中的酯化和酯交换反应,是合成生物柴油的有效催化剂。然而,由于废弃酸化油的酸价高,还不适于使用目前市场上已有的中性或碱性脂肪酶产品来直接催化转化,因此亟需开发出耐酸性高的特色脂肪酶产品,促进和引领国内生物柴油生物行业的可持续发展。本论文针对利用废弃酸化油转化生产生物柴油过程中,脂肪酶催化活性低、转化效率不高等主要问题,在新型耐酸脂肪酶产品的研发和脂肪酶规模化生产工艺优化等方面进行研究,取得主要结果如下:1.成功构建脂肪酶TLL在丝状真菌里氏木霉中的表达分泌体系首次将市场上商品化脂肪酶产品的主要酶活性组分—源于疏棉状嗜热丝孢菌的TLL(Thermomyceslanuginosus lipase,TLL)在丝状真菌里氏木霉中成功进行了分泌表达,重组表达菌株的摇瓶发酵酶活达3268 U/mL,20 L生物反应器中发酵后酶活达到9665 U/mL。酶性质表征分析发现TLL的最适反应pH为9.5,且耐酸性较差,在pH5.0的缓冲液中孵育1h后丧失了 90%的酶活。进一步分析发现,TLL虽然对食用大豆油的转化效果较好,生成的脂肪酸甲酯含量达90 wt%,但是作用于酸化油时,转化得到的脂肪酸甲酯含量低于80 wt%,并且随着反应时间延长而下降,推测是由TLL具有较差的耐酸性导致的。2.通过构建TLL随机突变体文库,结合毕赤酵母表达体系和高通量筛选工作站,筛选得到耐酸性显著提升的脂肪酶突变体TLL-T3,并构建获得TLL-T3的里氏木霉高效分泌表达菌株KDN201为了获得耐酸性能强的脂肪酶以应用于废弃酸化油的高效转化,采用定向进化联合高通量突变体筛选的策略,通过易错PCR结合毕赤氏酵母表达体系获得TLL基因的突变体表达文库,对发酵液在pH5.0和pH7.0条件下脂肪酶酶活比值上升的转化子进行高通量筛选,从7000余个转化子中筛选得到了 TLL耐酸性显著增强的突变体TLL-T3,其最适pH由9.5降低至8.0且在pH 4.0-6.0下孵育1小时后的残留活性较野生型提高了 2-6倍。序列分析表明TLL-T3中发生了三个氨基酸的改变,分别为A69S、V150P、N222G。使用Propka3计算分析,TLL-T3中催化位点D223的pKa值较野生型蛋白显著降低,这可能是其耐酸性增强的原因。进一步将TLL-T3在里氏木霉底盘中成功进行了分泌表达,获得高产菌株KDN201。菌株的摇瓶发酵液酶活为2683 U/mL,20 L发酵罐中发酵液酶活达12031 U/mL。对TLL-T3转化酸化油生产生物柴油的分析结果表明,其转化能力较野生型酶显著提高,脂肪酸甲酯产量达到90 wt%以上。3.KDN201菌株发酵生产工艺的优化及TLL-T3液体产品的开发、稳定性能和应用效果测试首先通过对菌株KDN201发酵表达TLL-T3的诱导时间、补糖速率、补料培养基组分等方面进行优化,确定了菌株在20 L发酵罐中最优的发酵工艺:以溶氧回升10%为诱导起点;补料速率由起始2.0 g/L/h逐步降低至后期的1.0 g/L/h;诱导物为淀粉水解液组合物(还原糖300 g/L和15 g/L的玉米浆及无机盐)。诱导培养基的优化及补料速率的调整不但解决了发酵后期酶活不再增长的问题,而且提升了整个发酵周期酶活的增长速率,最终工艺优化后的发酵酶活上升至16123 U/mL。其次,相继通过发酵液的固液分离、超滤膜选型、保护剂配方等后处理环节的优化,最终确定了发酵中试及生产试验的提取工艺为:通过板框过滤进行固液分离,进一步根据脂肪酶收率及处理周期,选型截留分子量为20 kDa的超滤膜进行浓缩最终获得液体产品。研究发现,在产品保护剂中通过添加0.5%的海藻糖,35%的山梨醇及其它成分,可以使液体产品室温放置1年的酶活保留率达到91%,最终产品的稳定性能具备了市场销售的条件。最后通过小试转化试验确认液体脂肪酶10,0000 U/ml产品的最佳添加量为0.19 wt%。4.KDN201菌株发酵生产工艺的放大及TLL-T3固体产品的开发、稳定性能和应用效果测试。固体酶产品在稳定性能及储运成本等方面优于相应液体产品,因此也需在中试放大中开发固体剂型的产品,以满足热带地区市场需求。在KDN201菌株小试发酵工艺参数的基础上继续进行放大发酵实验。根据尾气分析仪测定的摄氧率(OUR)及二氧化碳释放速率(CER)数据,对相关生产工艺参数进行微调,包括在前期提高补料速度(由2.0 g/L/h提升至2.6 g/L/h)以提升前期营养供给,增加初始菌体湿重,而在中期适当降低补料速度(1.8 g/L/h)以降低菌体代谢负担,并将菌体湿重维持在较为稳定的水平,最终在30 T生产罐上进行发酵的酶活达到17054 U/mL。按照中试提取工艺,发酵液至超滤阶段的提取收率为86.74%,达到了试生产目标,制备的液体脂肪酶产品可直接供生物柴油厂家试用。同时在放大发酵基础上,通过选型麦芽糊精为辅料,优化喷雾工艺参数如进料压力为3 Mpa、进风温度160±2℃、出风温度80±2℃、喷片及旋片采用2.0 mm/3.0 mm,成功完成了可溶性固体产品的开发,产品酶活指标为30,0000 U/g,水分<8 wt%,且固体脂肪酶产品即使在37℃放置一年,酶活保留率仍在90%以上,可满足市场要求。本研究所开发的液体脂肪酶产品在应用实验的基础上,与下游用户厂家合作陆续完成了生物柴油生产的5 T和100 T放大及试生产实验,所得到的生物柴油产品的酸价和甲酯含量能够满足生物柴油生产行业的实际要求,产品已成功推向市场销售,年销售额约500万元。同等酶活的固体产品在多个生物柴油生产厂家的应用效果也与液体产品相当,并出口至东南亚地区,年销售额约300万元。更多还原

【Abstract】 In recent years,global energy prices have generally been on the rise,and the global push for carbon reduction initiatives has brought biofuels back into the spotlight.Biodiesel,an important component of biofuels,can replace traditional petrochemical diesel and is internationally recognized as a renewable and clean fuel.In foreign countries,biodiesel is primarily produced from edible vegetable oils,while in China,due to the large population,it is crucial to avoid competing with food sources,necessitating the search for alternative raw materials for the biodiesel industry.Acidified oil is a type of waste oil,mainly derived from the refining process of edible vegetable oils.It is produced in large quantities,with the annual production of acidified soybean oil alone being approximately 600,000 tons.The domestic production of waste acid oils is substantial,and using waste acid oils to produce biodiesel has a higher carbon reduction potential,making it an ideal raw material for biodiesel production.Lipase can catalyze the esterification and transesterification reactions in the biodiesel production process and serves as an effective catalyst for biodiesel synthesis.However,waste acid oils have a high acid value,which makes them unsuitable for catalysis by currently available neutral or alkaline lipase products on the market.Therefore,there is an urgent need to develop lipase products with high acid resistance to lead the industry forward.This paper addresses the major issues of low enzyme activity and low conversion efficiency of lipase during the processing of waste acid oils in biodiesel production.It focuses on the research and development of novel acid-resistant lipase products and the optimization of lipase production processes,with the following key results:1.Efficient expression and secretory production of the lipase TLL in Triochoderma reesei.For the first time,the main component protein of the commercial lipase product,TLL(derived from Thermomyces lanuginosus),was successfully expressed and secreted in the filamentous fungus Trichoderma reesei.The enzyme activity of the recombinant strain in shake flask fermentation reached 3268 U/mL,and after fermentation in a 20 L bioreactor,the enzyme activity reached 9665 U/mL.Analysis revealed that the optimal reaction pH for TLL was 9.5,but its acid resistance was poor,as it lost 90%of its enzyme activity after being incubated for 1 hour in a pH 5.0 buffer.Therefore,while TLL showed good conversion efficiency with edible soybean oil,its ability to convert acid oils into biodiesel only reduced the acid value to 13.0 mg KOH/g,with a methyl ester content below 80 wt%,and the yield declined with increasing reaction time.This is consistent with TLL’s poor acid resistance.2.Construction of a random mutant library of the tll gene,identification of the lipase mutant TLL-T3 with significantly improved acid tolerance through the Pichia pastoris expression system and high-throughput screening workstation,and development of the efficient TLL-T3-expressing T.reesei strain KDN201.In order to obtain a lipase with strong acid resistance for the efficient conversion of waste acidified oil,a strategy combining directed evolution and high-throughput mutant screening was employed.Using error-prone PCR coupled with a Pichia pastoris expression system,a mutant library of the TLL gene was generated.High-throughput screening was performed on the transformed colonies based on the increased lipase activity ratio at pH 5.0 and pH 7.0.From over 7,000 transformants,a mutant with significantly enhanced acid resistance,TLL-T3,was selected.Its optimal pH decreased from 9.5 to 8.0,and after incubation at pH 4.0-6.0 for 1 hour,its residual activity increased 2-6 times compared to the wild type.Sequence analysis revealed three amino acid changes in TLL-T3:A69S,V150P,and N222G.Propka3 analysis indicated that the pKa value of the catalytic site D223 in TLLT3 was significantly lower than that of the wild-type protein,which may contribute to its improved acid resistance.Further secretion expression of TLL-T3 in the Trichoderma reesei platform was successfully carried out,yielding a high-producing strain,KDN201.The enzyme activity in the shake flask fermentation reached 2683 U/mL,and in a 20 L fermentation tank,it reached 12,031 U/mL.Analysis of TLL-T3 in the transesterification of acidified oil to produce biodiesel demonstrated a significant improvement in performance compared to the wild-type enzyme,with the fatty acid methyl ester yield exceeding 90 wt%.3.Optimization of the fermentation production process of the KDN201 strain and development of the liquid product of TLL-T3 with excellent performance instability and application that meet market requirements.Firstly,the fermentation conditions for expressing TLL-T3 in the strain KDN201 were optimized,including induction time,sugar feeding rate,and the composition of the feeding medium.The optimal fermentation process in a 20 L fermenter was determined as follows:induction began when dissolved oxygen increased by 10%;the feeding rate was gradually reduced from an initial 2.0 g/L/h to 1.0 g/L/h in the later stages;the inducer was a starch hydrolysate composition containing 300 g/L of reducing sugar,15 g/L of corn syrup,and inorganic salts.Optimization of the induction medium and adjustment of the feeding rate not only resolved the issue of no further increase in enzyme activity during the late fermentation phase but also enhanced the overall fermentation rate of enzyme activity,ultimately achieving a maximum enzyme activity of 16,123 U/mL after process optimization.Subsequently,the downstream processing steps,including solid-liquid separation,ultrafiltration membrane selection,and the formulation of protectants,were optimized.The final extraction process for pilot-scale and production-scale trials was established as follows:solid-liquid separation was carried out by plate-and-frame filtration;based on lipase yield and processing time,an ultrafiltration membrane with a molecular weight cut-off of 20 kDa was selected for concentration;to maintain product stability,a protectant containing 0.5%trehalose,35%sorbitol,and other components was added.This formulation ensured that the enzyme activity of the liquid product remained at 91%after one year of storage at room temperature.The stability of the final product met the requirements for market commercialization.Finally,small-scale trials confirmed that the optimal concentration of liquid lipase at 100,000 U/mL was 0.19 wt%.4.Scale-up of the fermentation production process for the KDN201 strain and the development of the TLL-T3 solid product with excellent stability and application performance meeting market demands.Solid products outperform liquid products in terms of stability and storage/transportation costs.To meet the market demand in tropical regions,the development of solid dosage forms was essential during the pilot scale-up process.Based on the fermentation parameters of the KDN201 strain from the laboratory scale,further fermentation experiments were conducted at a larger scale.The oxygen uptake rate(OUR)and carbon dioxide evolution rate(CER),measured using an exhaust gas analyzer,were used to fine-tune relevant production process parameters.This included increasing the feeding rate in the early stage(from 2.0 g/L/h to 2.6 g/L/h)to enhance nutrient supply and boost initial wet weight.In the mid-stage,the feeding rate was appropriately reduced(to 1.8 g/L/h)to alleviate the metabolic burden on the microorganism,maintaining a relatively stable wet weight.Ultimately,fermentation in a 30 T production fermenter achieved an enzyme activity of 17,054 U/mL.Using the same extraction process as at the laboratory scale,the yield at the ultrafiltration stage reached 86.74%,meeting the pilot production target.During the trial production phase,liquid lipase products were prepared for testing by biodiesel manufacturers.Additionally,by selecting maltodextrin as an excipient and optimizing spray drying parameters-such as feed pressure at 3 MPa,inlet air temperature at 160±2℃,outlet air temperature at 80±2℃,and using nozzle and rotary discs of 2.0 mm/3.0 mm-a soluble solid product was successfully developed.The enzyme activity of the product reached 300,000 U/g,with a moisture content of<8 wt%Even when stored at 37℃ for one year,enzyme activity retention remained above 90%,meeting market requirements.Building on the liquid lipase product application experiments,collaborations with downstream application manufacturers led to the successful completion of both 5 T and 100 T scale-up and trial production experiments for biodiesel production.The resulting biodiesel products had acid values and methyl ester contents that met the practical requirements of the biodiesel industry.The product has been successfully launched to the market and is currently in use by biodiesel manufacturers in Hebei,Shanghai,and other regions,generating an annual sales revenue of approximately 5 million RMB.Solid products with the same enzyme activity have demonstrated comparable performance to liquid products in several biodiesel production plants and are exported to Southeast Asia,with an annual sales revenue of around 3 million RMB.更多还原