摘要
D-对羟基苯甘氨酸(D-pHPG)是合成p-内酰胺类抗生素如阿莫西林、头孢羟氨苄等的重要中间体,其主要的制备方法包括化学法和生物酶法。其中最安全环保、步骤简单的方法是一菌双酶法。本文所研究的恶臭假单胞菌在其生长代谢过程中能同时产生D-海因酶(DHase)和N-氨甲酰-D-氨基酸酰胺水解酶(DCase),此双酶能将DL-对羟基苯海因一步催化生成D-对羟基苯甘氨酸。
本文主要的研究内,容包括：产双酶菌种的验证及中间体N-氨甲酰-D-对羟基苯甘氨酸(NC-D-pHPG)的制备；紫外(UV)结合硫酸二乙酯(DES)复合诱变选育高产菌株；低能民+注入诱变选育高产菌株；高产变异菌株发酵培养条件的优化；双酶的提取纯化和DHase的固定化；DHase固定化酶与DCase纯化酶两酶一步法制备D-对羟基苯甘氨酸。
通过对出发菌株spMF0507的初步验证,确定其发酵20h-50h为对数生长期,最适生长温度为28℃-32℃,产双酶最适温度为28℃-30℃；其最适生长pH为6.5-7.5,产双酶最适pH为7.0-7.5。DHase和DCase在不同的温度和pH下表现出不同的酶活力,其中DHase稳定性较高,而DCase对热不稳定,在高温高pH条件下易失活。通过升温55℃-60℃处理菌悬液30min可使DCase失活而DHase基本保持原活力,把底物DL-pHPH配成6%浓度,与DCase失活的菌悬液等体积混合,控制温度33℃,pH9.0-9.5进行转化反应制备NC-D-pHPG,转化后结晶率达97.9%,制备的NC-D-pHPG纯度为98.5%。
采用UV结合DES复合诱变出发菌株spMF0507,选择紫外灯照射30s并结合1.5mg/mL的DES处理20min后再通过20μg/mL5-氟尿嘧啶(5-FU)选择性筛选和发酵摇瓶初筛复筛并通过继代遗传稳定性实验考察,诱变菌spUD0612具有较高的产酶稳定性,其DHase酶活力达1.784U/mL,DCase酶活力达0.865U/mL,分别比出发菌株提高了87.39%和58.420%。
用低能N+离子注入对spUD0612菌株进行诱变选育,在注入能量30 KeV,注入剂量为8×1015ions/cm2,真空10-3Pa,电流强度10mA,脉冲8s条件下进行矿注入,通过在含有25μg/mL5-FU的选择培养基上筛选和摇瓶初筛复筛后再通过继代遗传稳定性实验考察,诱变菌spUDN0706具有较高的产酶稳定性,其DHase活力达2.482U/mL,DCase酶活力、达1.253U/mL,分别比出发菌株spUD0612提高了46.69%和53.93%。
通过对发酵培养基和发酵控制条件进行优化,确定了在接种量为8%-10%,培养温度为30℃,初始pH为7.5,摇瓶装量为100mL时,高产变异株spUDN0706发酵有较高的生物量和双酶活力。对发酵配方进行正交实验优化后通过综合比较双酶活力,得出最佳发酵培养基配方为：玉米浆2.0%；甲硫乙基海因(10%)3.5%；葡萄糖2.5%; NaCl0.3%; MgSO40.05%; CoCl20.01%; (NH4)2SO40.1%。在新配方和优化的控制条件下进行高产变异株spUDN0706摇瓶发酵, DHase和DCase酶活力分别为2.591U/mL和1.622U/mL,分别比原发酵条件下DHase和DCase酶活力提高了14.04%和28.63%。将高产变异株spUDN0706进行5L自动发酵罐发酵,DHase活力为3.0.78U/mL, DCase活力为1.953U/mL,分别比摇瓶活力提高了18.80%和20.41%。
收集高产诱变株spUDN0706发酵液的菌体,采用高压均质机进行破碎,确定了最适匀浆压力为60MPa,最适破碎时间为15min。采用先20%(NH4)2SO4纯化DCase再34%(NH4)2SO4纯化DHase可实现双酶的分离纯化。通过粗酶液絮凝、两级(NH4)2SO4纯化以及经截留量为一万分子量的超滤膜处理,DHase纯化总收率为69.2%,纯化倍数达6.4；DCase纯化总收率为76.4%,纯化倍数达5.45。通过对DHase固定化条件的研究,确定了TJS载体投量为1g：133U,固定化温度、pH、固定化时间分别为25℃、7.5和15h,在此条件下进行DHase的固定化,固定化活力为48.7U/g,酶回收率为34.8%。
通过测定不同pH和不同温度下DHase固定化酶及DCase纯化酶的酶活力,DHase固定化酶最适pH为9.5,最适温度为60℃；DCase纯化酶最适pH为7.5,最适温度为40℃。DHase固定化酶对热和酸碱的稳定性均明显优于DCase纯化酶。Mn2+、Ca2+、Mg2+、Ba2+对DHase固定化酶和DCase纯化酶均有激活作用；Co2+对DHase固定化酶有激活作用而对DCase纯化酶有抑制作用；Fe2+、Zn2+、Hg2+对DHase固定化酶及DCase纯化酶均有抑制作用,其中重金属Hg2+对酶有强烈的抑制作用。DHase固定化酶的半衰期为210d,其表观米氏常数Km为20.14mmol/L; DCase纯化酶半衰期为6d,其表观米氏常数Km为8.40mmol/L。在底物浓度3%,温度38℃,DHase固定化酶投入量5kU/L, DCase纯化酶投入量3kU/L条件下进行多批次转化,固定化酶半衰期在256批以后,每批的转化率和收率均在97%左右。用DHase固定化酶结合DCase纯化酶两酶一步法转化DL-pHPH制备D-pHPG后,经截留量为一万分子量的超滤膜过滤浓缩精制,D-pHPG的含量为99.2%,比旋度为-159.8°,达到了较高的质量水平。
D-hydroxyphenylglycine (D-pHPG) is the most important intermediate used for the synthesis of Semisynthetic antibiotic including amoxicillin and Cefadroxil. The two main synthesis methods for D-pHPG are chemical processes and enzymatic processes, among which the process using one strain with twin enzyme is the best for the environmental and economic concerns. Pseudomonas putida studied in this paper can produce both D-Hydantoinase (DHase) and D-Carbamoylase (DCase) in its growing period. Twin enzyme (DHase and DCase) can catalyse DL-p-hydroxyphenylhydantoin (DL-pHPH) to D-pHPG directly.
The main contents of this paper were:to identify the strain that can produce twin enzyme and produce the intermediate N^carbamoyl-D-p-hydroxyphenylglycine (NC-D-pHPG); to mutate and screen the strain with ultraviolet radiation(UV) and diethyl sulfate(DES); to mutate and screen the strain with N+ion implantation; to optimize the culture condition of mutated strain; to isolate and purify twin enzyme and immobilize DHase; to produce D-pHPG with immobilized DHase and purified DCase.
With the identification of the strain spMF0507, it was determined that its logarithm growing period was about 20-50hours; the best growing temperature is 28-32℃, the best enzyme producing temperature is 28-30℃; The best growing pH was 6.5-7.5. The best enzyme producing pH was 7.0-7.5. DHase and DCase showed different activity under different temperature and pH. DHase was more stable than DCase. DCase will be inactivated by being heated at 55-60℃for about 30 min. DL-pHPH can translate to NC-pHPG by reacting with the strain of inactivated DCase under following conditions:DL-pHPH concentration 6%, temperature 33℃and pH9.0-9.5. The crystallized rate of NC-D-pHPG was 97.9% and the purity of NC-D-pHPG was 98.5% by HPLC.
A high twin enzyme producing, strain spUD0612 with genetic stability was obtained from Pseudomonas putida spMF0507 by mutagenesis with UV 30s and diethyl sulfate for 20min,followed by resistance selection with substrate analogue 20μg/mL 5-FU. After the screening of fermentation with a shake flask, its activity of DHase and DCase reached 1.784 U/mL and 0.865 U/mL, respectively. Compared with starting strain, the enzyme activity enhanced 87.39% and 58.42%, respectively. A high twin enzyme producing strain spUDN0706 with genetic stability was obtained from Pseudomonas putida spUD0612 by mutagenesis with N+ ion implantation (30KeV,8×1015ions/cm2,10-3Pa,10mA,8s)followed by resistance selection with substrate analogue 25μg/mL 5-FU. After the screening of fermentation using a shake flask, its. activity of DHase and DCase reached 2.482 U/mL and 1.253 U/mL, respectively.Compared with spUD0612, the enzyme activity enhanced 46.69% and 53.93%, respectively.
The culture medium and the fermentation conditions were optimized based on the former culture medium and formal fermetation conditions. The results of the experiments showed the best fermentation conditions were as following:inoculum amount 8-10%, loading amount 100mL,culture temperature 30℃, initial pH7.5.And the best composition were as following:corn steep liquor 2.0%,glucose 2.5%, revulsent 3.5%,NaCl 0.3%,MgSO40.05%,CoCl20.01%,(NH4)2SO40.1%. The spUDN0706 grew well on the optimized medium and fermentation condition.Its activity of DHase and DCase reached-2.591U/mL and 1.622U/mL, respectively. Compared with the formal medium and fermentation condition, the enzyme activity enhanced 14.04% and 28.63%, respectively. By validating on 5L auto-fermentor, its activity of DHase and DCase reached 3.078U/mL and 1.953U/mL, respectively. Compared with using shake flask, the enzyme activity enhanced 18.80% and 20.41%, respectively.
Collecting the strain of spUDN0706 fermentation broth and breaking up the strain making use of homogenizer, the best pressure was 70MPa and the best breaking up time was 15min. Purifying twin enzyme with 20%(NH4)2SO4 and 34%(NH4)2SO4 can isolate and purify both DHase and DCase. After flocculation, purification and ultrafiltration, the total purified yield and purification folds of DHase were 69.2% and 6.4, respectively; and the total purified yield and purification folds of DCase were 76.4% and 5.45, respectively. The effects of some parameters on DHase immobilization were investigated, and the optimized conditions were obtained as follows:carrier TJS input 133U/lg, immobilization time 15h, temperature 28℃, pH7.5 and the activity of immobilized enzyme was 48.7U/g, the immobilization yield was 34.8% under the optimized conditions.
By checking the activity of immobilized DHase and purified Dcase, it was determined that the best pH and temperature for immobilized DHase were 9.5 and 60℃respectively; and the best pH and temperature for DCase were 7.5 and 40℃, respectively. Immobilized DHase was more stable than purified DCase against temperature and pH. Ions including Mn2+, Ca+, Mg2+, Ba2+could activate both immobilized DHase and purified DCase.Co2+activated immobilized DHase but inhibited purified Dcase. Fe2+, Zn2+, and especially Hg2+inhibited both immobilized DHase and purified DCase. The half life of immobilized DHase was 210 days and its apparent Km was 20.14mmol/L; the half life of purified DCase was 6 days and its apparent Km was 8.40mmol/L. Immobilized DHase and purified DCase can catalyse the conversion of DL-pHPH to D-pHPG in one step under following conditions: substrate conc.3%,temperature 38℃,immobilized DHase input 5kU/L,purified DCase input 3kU/L. Immobilized DHase could be used over 256 cycles and conversion rate and yield were all over 97% averagely. After purification by ultrafiltration and concentration, the content of the translated D-pHPG was 99.2% and its polarity was-159.8°, achieving a satisfactory reliability.
引文
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