【作者】 林志坚；

【导师】 郑裕国；

【作者基本信息】 浙江工业大学 ， 生物化工， 2012， 博士

【摘要】 微生物腈水合酶作为腈转化酶家族的重要一员,能够催化腈类化合物的水合反应生成高附加值的酰胺,被广泛应用于制备医药、农药中间体和精细化工产品等。2-氨基-2,3-二甲基丁酰胺是广谱、高效的咪唑啉酮除草剂合成的关键中间体。腈水合酶催化转化2-氨基-2,3-二甲基丁腈制备2-氨基-2,3-二甲基丁酰胺具有产率高、反应条件温和、环境友好等特点,符合“绿色化学”的要求。本论文以该工艺路线为核心,从生物催化剂的发现、表征、制备与应用,产物的分离、提纯等方面展开研究。本文首先创建了一个以α-氨基腈或α-氨基酰胺为底物的腈水合酶和酰胺酶高通量筛选模型。研究并提出了其显色反应的机理,是基于亚铁离子和铁离子与α-氨基腈溶液中的氰离子或α-氨基酰胺溶液中的氢氧根离子的反应。通过该模型,从418株菌种中筛选到了3株能转化2-氨基-2,3-二甲基丁腈生产2-氨基-2,3-二甲基丁酰胺的腈水合酶产生菌。其中两株筛选自土壤样品,编号ZA0707和P4;另外一株菌Rhodococcus boritolerans CCTCC M 208108来自本实验室的微生物菌库。该模型是第一个直接、快速的腈水合酶高通量筛选模型,同时也是第一个集腈水合酶和酰胺酶的筛选于一体的筛选模型。通过形态学、生理生化试验、API自动鉴定系统、16S rDNA序列及系统发育分析,菌株ZA0707被鉴定为庆笙红球菌（Rhodococcus qingshengii）。这是该种内首次报道的产腈水合酶的菌株。进而研究并对比了菌株ZA0707和R. boritolerans CCTCC M 208108胞内腈水合酶的氰离子耐受性、产物耐受性、热稳定性和对2-氨基2,3-二甲基丁腈活力等方面的特性,优选R. boritolerans CCTCC M 208108作为酶法转化2-氨基2,3-二甲基丁腈制备2-氨基2,3-二甲基丁酰胺的生物催化剂并用于后续实验。通过单因素和响应面优化法对R. boritolerans CCTCC M 208108产腈水合酶的培养基成分进行了优化。确定了较佳的产酶培养基组分为（g/l）:蔗糖7.00,柠檬酸钠3.04,牛肉膏5.13,酵母粉5.00,己内酰胺1.50,KH2PO4 1.0,K2HPO4 1.0,NaCl 1.0,FeCl3 0.005,CoCl2 0.005,MnSO4 0.005。随后研究并选定了较适宜的培养条件为:初始pH6.5,30°C,装液量40 ml/250 ml,接种量1.5 ml。在上述培养条件下培养60 h后,生物量达到6.21 g/l,腈水合酶的活力达到5393 U/g CDW。论文还研究了酶催化反应的环境和介质对该腈水合酶活力的影响。结果表明,最适宜的缓冲液体系是Tris-HCl溶液,pH8.9;最高酶活出现在30?35°C范围内;向反应体系中添加30%（v/v）正己烷和1 mM Ni2+,酶活力可分别提高30%和23%。对酶促反应动力学参数的研究发现,反应的Vmax和Km分别为:15.73μmol·min?1·mg?1和44.30 mM。发现了降低温度可以使2-氨基-2,3-二甲基丁腈溶液更加稳定,特别是在10°C,由底物自发解离产生的氰离子浓度仅为2.01 mM,不足30°C下的四分之一。在深入了解影响反应的主要因素以后,放大反应体系至800 ml,建立了水相和以正己烷为共溶剂的两相反应体系;对关键的反应参数优化后,有效地减轻了氰离子和底物对腈水合酶的抑制,使得产物浓度、产率和催化剂生产力分别达到50 g/l、91%和6.3 g product/g catalyst。在优化条件下,菌体至少可以重复利用两次,使得催化剂生产力进一步提高到12.3 g product/g catalyst。论文随后选用膨胀型珍珠岩作为固定化的载体,采用吸附培养法固定R. boritolerans CCTCC M 208108细胞,以提高细胞的氰离子耐受性和重复使用性能。从提高酶活的角度,优化珍珠岩吸附固定化细胞的制备条件,确定较佳条件为:初始培养pH值7.0,固定化培养温度30°C,载体用量40 ml（培养基用量40 ml,250 ml三角瓶）,最佳培养时间54 h。在此条件下,固定化细胞和游离细胞相比氰离子耐受性和重复使用次数分别提高了16%和3批次。这是有关珍珠岩用于固定化腈水合酶或其产生菌的首次报道。研究了固定化细胞转化2-氨基-2,3-二甲基丁腈的动力学参数:Vmax-固= 13.59μmol·min^-1·mg^-1,Km-固= 47.98 mM;并求得固定化细胞催化反应的达姆科勒数,Da = 4.9×104。固定化细胞的Km-固值大于游离细胞的Km值,而且Da远大于1,说明外部传质阻力（底物从主体溶液到固定化载体上的细胞表面所受的阻力）影响了固定化细胞转化2-氨基-2,3-二甲基丁腈,并且可能成为反应的限速因素。论文最后开发了一个从反应体系中分离、纯化产物2-氨基-2,3-二甲基丁酰胺的新方法。利用离心、活性炭吸附除杂、减压蒸馏、乙酸乙酯/正己烷重结晶等分离手段对酶催化反应体系中的重结晶产物进行了有效分离、提纯（纯度达98.5%）。并通过FT-IR、1H NMR和13C NMR等分析方法对产物结构进行了表征,进一步确定该结晶产物为2-氨基-2,3-二甲基丁酰胺。更多还原

【Abstract】 Nitrile hydratase （NHase） is one of the important enzymes of nitrile metabolism in numerous microbes that catalyses the hydration of nitriles to higher-value amides, and has been successfully adopted in the chemical industry for production of pharmaceuticals, agrochemicals and fine chemicals. 2-Amino-2,3-dimethylbutyramide （ADBA） is a key intermediate of highly potent and broad spectrum imidazolinone herbicides. Enzymatic hydration of 2-amino-2,3-dimethylbutyronitrile （ADBN） by NHase offers alternative to conventional ADBA production due to the high yield, mild conditions and environment-friendly nature. This paper focused on the bioconversion of ADBN to ADBA and investigated in respect of screening, identification, cultivation and application of the biocatalysts, as well as separation and purification of product.A combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase withα-amino nitriles andα-amino amides as substrates, respectively. Mechanisms of color changes were further proposed. It was due to the reactions involved ferrous ions, ferric ions, cyanide ions dissociated spontaneously fromα-amino nitriles solution and hydroxyl ions in theα-amino amides solution. Using this method, 3 strains with nitrile hydratase activity towards ADBN were screened from 418 isolates. 2 of them named ZA0707 and P4 were isolated from soil samples and the other one, R. boritolerans CCTCC M 208108 was selected from culture collections in our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both NHase and amidase.Strain ZA0707 screened from soil samples was identified as Rhodococcus qingshengii on the basis of its morphological and physiological features along with API identification system and 16S rDNA sequencing method. This is the first report on strains in this species with NHase activity. In addition, the cyanide-resistance, product tolerance, thermostability and NHase activity towards ADBN of strain ZA0707 and R. boritolerans CCTCC M 208108 were investigated. Consequently, R. boritolerans CCTCC M 208108 with the better results in all these aspects were selected as the optimum catalyst for the following investigation.The medium composition for NHase formation in R. boritolerans CCTCC M 208108 was optimized by single factors and response surface methodology. The optimum medium composition was as follows （g/l）: sucrose 7.00, sodium citrate 3.04, beef extract 5.13, yeast extract 5.00, caprolactam 1.50, KH2PO4 1.0, K2HPO4 1.0, NaCl 1.0, FeCl3 0.005, CoCl2 0.005, MnSO4 0.005. Furthermore, the satisfactory culture conditions for cell growth and NHase production were obtain as bellow: initial pH6.5, temperature 30°C, medium volumetric ratio 16% （v/v）, inoculum size 3.75% （v/v）. Under these conditions, 6.21 g/l biomass and 5393 U/g CDW specific NHase activity were achieved after 60 h cultivation.The influences of reaction conditions and media on NHase activity were also investigated. It was indicated that the NHase exhibited maximal activity in Tris-HCl buffer （pH 8.9） at 30°C. The addition of 30 % （v/v） n-hexane and 1 mM Ni2+ in the reaction mixture resulted in approximately 30 and 23 %, respectively, enhancement in NHase activity. Kinetic studies on the NHase catalyzed reactions were performed and the kinetic constants were as follows: Vmax = 15.73μmol·min?1·mg?1, Km = 44.30 mM. Moreover, ADBN was found to be more stable in water at lower temperature. Especially, cyanide accumulated very slowly in the initial 10 h at 10 oC, giving concentrations of 2.01 mM, which was less than a quarter of that at 30 oC. Based on these findings, a preparative scale （800 ml） process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control （at 10 oC）. The product concentration, yield and catalyst productivity were further improved to 50 g·l^-1, 91% and 6.3 g product/g catalyst using a 30/100 （v/v） n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst.Next, in order to improve the cyanide-resistance and reusability of the biocatalyst, expanded perlite was employed to immobilize R. boritolerans CCTCC M 208108. After optimization in terms of NHase activity, the optimum preparation conditions were set as follows: initial pH7.0, temperature 30 oC, perlite loading 40 ml （medium volume 40 ml in 250 ml flask）, 54 h of immobilization time. As a result, the cyanide-resistance and reusability were greatly improved, which were 16% and 3 batches higher than those of free cells. It was the first report on immobilization of NHase-producing strain using perlite. The kinetic constants of immobilized cells including maximum reaction velocity （Vmax-固） and Michaelis constant （Km-固） were calculated to be 13.59μmol·min^-1·mg^-1 and 47.98 mM, respectively. Free cells showed lower Km value of 44.30 mM compared to 47.98 mM for immobilized cells. It indicated that the substrate ADBN was more accessible to free cells than immobilized cells. This also suggested that external mass transfer （ADBN from bulk to cell in immobilized matrix） resistance affected hydration of ADBN by immobilized cells and could be the limiting step in comparison to the reaction rate. It was further confirmed by calculating the values of Damk-hler number at different ADBN concentrations, which were very much higher than 1.Finally, a new method for separation and purification of ADBA was developed. Purified ADBA （98.5%） was obtained by centrifugation, impurity removal using activated carbon, reduced pressure distillation and recrystallization from ethyl acetate/n-hexane. The structure of ADBA was further confirmed by FT-IR, 1H NMR and 13C NMR spectra.更多还原