基因工程酶法生产N-氨基甲酰-D-对羟基苯甘氨酸反应条件的优化(英文)

为了实现利用生物酶转化法生产D 对羟基苯甘氨酸 ,以工程菌E .coliBL2 1 pMD T7 dht细胞作酶源 ,对底物对羟基苯海因到中间体N 氨基甲酰 D 对羟基苯甘氨酸的酶转化条件进行了优化 .酶转化的最适温度为 37℃ ,最适pH为 9 0 .在Tris HCl、磷酸盐、碳酸盐和硼酸盐 4种缓冲体系中 ,底物对羟基苯海因的转化率相近 .菌体细胞经适当冻融后 ,底物对羟基苯海因的转化率被提高 .水溶性有机溶剂DMSF、DMF和Tween 80使对羟基苯海因的转化率降低 .转化时底物和菌体的合适比例为 30g L对羟基苯海因和 10g L湿菌体 .经工程菌E .coliBL2 1 pMD T7 dht细胞催化 ,底物的转化率在 13h内可达到 96 % .所制备的产物熔点、旋光性和红外光谱等与标准品一致     

假单胞菌海因酶基因在大肠杆菌中的高效表达(英文)

为实现利用生物酶转化法进行D 对羟基苯甘氨酸的工业化生产 ,构建了 3株海因酶基因工程菌 .利用PCR技术从恶臭假单胞菌 (Pseudomonasputida)CPU 980 1染色体DNA中扩增得到长约1.8kb的含编码区和自身启动子的海因酶全基因 .通过将海因酶全基因插入pMD18 T质粒、海因酶基因的编码区与pET 17 b质粒重组、海因酶基因编码区和T7强启动子一起插入pMD18 T质粒分别得到重组质粒pMD dht、pET dht和pMD T7 dht.将上述重组质粒分别转化大肠杆菌 (Escherichiacoli) ,通过地高辛标记菌落原位杂交和海因酶活力测定两种方法 ,筛选出具有海因酶活力的阳性转化子 .结果表明 ,大肠杆菌的RNA聚合酶能够识别和结合来自恶臭假单胞菌海因酶基因的自身启动子 ,该启动子在大肠杆菌中能够工作 .基因工程菌E .coliBL2 1 pMD dht、E .coliBL2 1 pET dht和E .coliBL2 1 pMD T7 dht的海因酶活力分别为 170 0U L、190 0U L和 2 5 0 0U L ,比野生菌P .putidaCPU 980 1的海因酶活力分别提高了 8倍、9倍和 12倍 .薄层扫描结果显示 ,这些工程菌的海因酶表达量分别约占菌体总可溶性蛋白质的 2 0 %、31%和 5 7%.SDS PAGE显示 ,海因酶的单体分子量约为 5 0kD .经工程菌E .coliBL2 1 pMD T7 dht催化 ,底物对羟基苯海因的转化率在 13h内可达到 9     

构建重组枯草芽孢杆菌催化制备D-对羟基苯甘氨酸

目的: 在Bacillus subtilis中表达异源D-海因酶基因(hyd)和D-氨甲酰水解酶基因(adc),构建重组细胞作为催化剂,用于生产D-对羟基苯甘氨酸(D-HPG)。方法: 构建hyd表达质粒,考察培养基中二价金属离子对D-海因酶活性的影响。过表达acoR基因,考察AcoR蛋白胞内水平与P_acoA-hyd基因拷贝数的关系。筛选表达adc基因的启动子,构建hyd和adc基因共表达质粒,考察双酶活性菌株的催化特性。结果: 成功构建了海因酶表达质粒pHPS和pUBS,培养基中添加0.8mmol/L的MnCl₂·4H₂O,使168N/pUBS菌株的D-海因酶活性达到956U/gDCW。整合表达P_cdd-acoR基因,使LSL02/pUBS菌株的D-海因酶活性达到1 470U/gDCW。单拷贝P_AE-adc基因的表达水平相对最高。双酶共表达质粒pUBSC被成功构建,菌株LSL02/pUBSC的最适催化温度为40℃45℃,催化活性能够持续12h,当底物起始浓度为20g/L时,反应12h生成的D-HPG达到14.32g/L,转化率达到95%,收率超过80%。结论: 构建具有D-海因酶和D-氨甲酰水解酶双酶活性的重组Bacillus subtilis作为全细胞催化剂,用于海因酶法生产D-HPG,具有技术上的可行性和优势。     

海因酶法制备D-对羟基苯甘氨酸的研究进展

D-对羟基苯甘氨酸(D-HPG)主要用于合成β-内酰胺类半合成抗生素,是国内最紧缺的医药中间体之一。微生物酶法是目前获得光学纯D-HPG的重要途径,微生物中起催化作用的主要是D-海因酶和N-氨甲酰水解酶。文章综述了产酶微生物的来源,酶的理化性质,以及培养条件的优化、基因工程、酶的固定化技术生产D-HPG的研究进展。     

聚苯乙烯树脂固定化D-海因酶的初步研究

D-海因酶广泛用于D-氨基酸的制备研究和生产中,目前已有许多固定化D-海因酶及含D-海因酶细胞的研究报道。尝试了不同功能基团的聚苯乙烯树脂进行D-海因酶的固定化,结果表明功能基为伯氨基和仲氨基效果较好,并选取聚苯乙烯树脂D92进行了固定化D-海因酶的研究。采用该树脂制备固定化酶的最优条件是：酶质量浓度6mg／mL、温度25℃、固化时间12h。所得固定化酶的最适作用温度45℃,最适作用pH为8.5,且作用温度及适宜pH较广,Km为游离酶的1,8倍,且储存稳定性、操作稳定性较好。45℃下半衰期为11d。     

Eupergit C250L固定化D-海因酶及其催化性质

D-海因酶是海因酶法制备D-氨基酸的关键酶。利用Burkholderic cepecia1003菌发酵产酶,所得海因酶纯化后,以Eupergit C250L为载体进行共价固定化。分别考察了酶液蛋白浓度、固定化时间对蛋白固定量和酶活回收率的影响以及固定化前后海因酶催化性质的变化。结果表明:较高的酶液蛋白浓度和较长的固定化时间均有助于改善海因酶的固定化效果;固定化可显著提高海因酶的最适作用温度,但对其最适作用pH影响不大;固定化后海因酶对D,L-BH和MH的米氏常数均有较大幅度的降低。固定化酶反应器的实验表明:40℃下,底物(D,L-BH)1.0 g.L-1,体积流速1.0 mL.min-1,经21 h转化,产物N-Phe质量浓度可达0.47 g.L-1,转化率达43.21%。     

来源于Bacillus sp. AR9 D-海因酶半理性设计的研究

D-对羟基苯甘氨酸是一种重要的精细化工品，在制药行业具有广泛的应用前景。酶法是生产D-对羟基苯甘氨酸的主要手段，但由于缺乏高催化效率的酶而限制了D-对羟基苯甘氨酸的生产。为了提高来自Bacillus sp. AR9的D-海因酶（HYD）的催化效率，进而提高D-对羟基苯甘氨酸的产量，对HYD的底物结合通道进行分析，选取底物通道瓶颈处的氨基酸进行饱和突变和筛选，以提高HYD的催化效率。结果显示，突变体F159S、F159A和F65V的活性相较于野生型HYD分别提高了51%、40%和17%，通过对突变体F65V、F159S和双位点突变F65V/F159S的酶动力学研究发现，突变体的Km值基本与野生型HYD相似，而kcat是野生型HYD的1.3、1.9和2.0倍，最终双位点突变F65V/F159S的催化效率kcat/Km是野生型HYD的2.4倍。高催化效率突变体的获得，以及对突变体动力学的分析，对酶法制备D-对羟基苯甘氨酸具有重要的研究意义和应用价值。     

异源D-海因酶和N-氨甲酰水解酶共表达重组枯草芽孢杆菌的构建

【目的】构建异源D-海因酶和N-氨甲酰水解酶共表达的重组枯草芽孢杆菌,探讨其作为全细胞催化剂合成D-对羟基苯甘氨酸的可行性。【方法】采用P_(aco)表达盒表达D-海因酶基因hyd或sd1,采用P_(AE)表达盒表达N-氨甲酰水解酶基因adc。分别以质粒pHP13和pUB110为载体,构建D-海因酶和N-氨甲酰水解酶共表达质粒pHCS、pHCY和pUCS。在受体菌中整合表达了acoR和sigL基因,敲除了skf和sdp基因。将共表达质粒分别转化不同的受体菌,通过测定全细胞催化活性,表征D-海因酶和N-氨甲酰水解酶共表达的效果。【结果】带有质粒pHCY和pHCS的重组菌,全细胞催化活性分别为0.21 U/mL和0.31 U/mL。整合表达acoR和sigL基因以及高拷贝质粒pUCS,使全细胞催化活性达到1.0 U/mL。【结论】异源D-海因酶和N-氨甲酰水解酶在枯草芽孢杆菌中能够正确表达。基因拷贝数、acoR和sigL基因表达水平,及skf和sdp基因缺失对重组菌的催化活性具有显著影响。     

D-(-)-苯甘氨酸酶法制造工艺的研究

D-(-)-苯甘氨酸是半合成抗菌素氨苄青霉素及先锋四号的重要侧链,制造工艺有化学法和酶法。酶法制造工艺是八十年代新工艺,具有工艺简便、收率较高、生产周期短、原材料成本低等特点。为了探讨D-(-)-苯甘氨酸酶法制造工艺的可行性,通过筛选获得了D-乙内酰脲水解酶产生菌B—BM—17,并进行了发酵、转化、水解等一系列的制造工艺的研究,取得了较好结果。以D。L-苯海因为原料,B—BM—17菌体转化成氨基甲酰-D-苯甘氨酸,再经亚硝酸水解制备D-(-)苯甘氨酸,30克投料规模,连批平均收率为69.1％(以苯甲醛计其收率为62.23％)。周期为24小时(发酵15小时转化5小时水解1小时)。以上述收率计算制备每公斤D-(-)苯甘氨酸的原材料成本不超过30元。     

利用基因工程菌HC01固定化细胞转化生产D-对羟基苯甘氨酸

对一菌两酶工程菌HC01转化底物DL-对羟基苯海因(DL-HPH)的最适条件及其细胞固定化进行了研究,HC01游离细胞转化DL-HPH的最适条件为40°C、pH7.5。通过对固定化细胞酶活力测定,确定细胞固定化的最优条件为海藻酸钠浓度2.5%、细胞浓度0.029g/mL、钙离子浓度3%。固定化HC01的热稳定性比游离细胞高5°C,二价金属离子Mn2+、Mg2+、Cu2+、Co2+和Ni2+在浓度为0.1mmol/L时对固定化细胞中D-海因酶(HYD)和N-氨甲酰-D-氨基酸酰胺水解酶(CAB)两酶的活力无显著影响,Mn2+和Mg2+可分别使游离细胞中CAB活力提高至原来的2.1和2.7倍。在氮气保护下,当初始pH为9.0、转化温度为40°C、转速为80r/min,利用固定化HC01转化30g/L的DL-HPH时,36h后转化率可达97%左右,产物D-HPG经纯化后光学纯度达到99.7%,得率可达85%。     

Optimization of a heterogeneous reaction system for the production of optically active D-amino acids using thermostable D-hydantoinase

A thermostable D-hydantoinase from Bacillus stearothermophilus SD-1 was previously mass-produced by batch cultivation of the recombinant E. coli harboring the gene encoding the enzyme (Lee et al., 1997). In this work, we attempted to optimize the process for the production of N-carbamoyl-D-p-hydroxyphenylglycine, which is readily hydrolyzed to D-p-hydroxyphenylglycine under acidic conditions, from 5-(4-hydroxyphenyl)hydantoin using the mass-produced D-hydantoinase. In an effort to overcome the low solubility of the substrate, enzyme reaction was carried out in a heterogeneous system consisting of a high substrate concentration up to 300 g/L. In this reaction system, most of substrate is present in suspended particles. Optimal temperature and pH were determined to be 45 degrees C and 8.5, respectively, by taking into account the reaction rate and conversion yield. When the free enzyme was employed as a biocatalyst, enzyme loading higher than 300 unit/g-substrate was required to achieve maximum conversion. Use of whole cell enzyme resulted in maximum conversion even at lower enzyme loadings than the free enzyme, showing 96% conversion yield at 300 g/L substrate. The heterogeneous reaction system used in this work might be applied to the enzymatic production of other valuable compounds from a rarely water-soluble substrate.     

Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase

We developed a fully enzymatic process employing D-hydantoinase and N-carbamoylase for the production of D-amino acid from 5'-monosubstituted hydantoin. For the comparison of the reaction systems using two sequential enzymes, D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoyl-D-amino acid amidohydrolase (N-carbamoylase) of Agrobacterium tumefaciens NRRL B11291 were separately expressed in each host cell and coexpressed in the same host cell. A high level and constitutive expression of both enzymes in Escherichia coli in a soluble form was achieved using a promoter derived from B. stearothermophilus SD1. The expression levels of both enzymes ranged from 17% to 23% of the total soluble protein, depending on the expression system. In the case of employing separately expressed enzymes, the product yield of D-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin and productivity were 71% and 2.57 mM/g-cell/h in 15 h, respectively. The accumulation of N-carbamoyl-D-hydroxyphenylglycine was significant over the reaction time. On the other hand, use of coexpressed enzymes resulted in 98% product yield of D-hydroxyphenylglycine in 15 h, minimizing the level of intermediates in the reaction mixture. The productivity of coexpressed whole cell reaction was estimated to be 6.47 mM/g-cell/h in 15 h. The coexpressed system was tested for an elevated concentration of D,L-p-hydroxyphenylhydantoin, and efficient production can be achieved.     

β-脱卤酶的基因克隆表达及其酶学性质

根据GenBank中的序列设计引物,克隆芽孢杆菌中的β-脱卤酶基因(命名为bhd)。以pET30a(+)为载体、Escherichia coli BL21(DE3)-CondonPlus为宿主菌,实现了bhd的高效表达。使用HisTrapTMFF亲和层析柱纯化重组β-脱卤酶,分子量约为23.1 kD。酶学性质研究表明,纯化的重组β-脱卤酶水解3-氯丙酸制备3-羟基丙酸的最适反应体系为30°C,100 mmol/L,pH 7.0的磷酸钠缓冲液。在最适反应条件下,重组β-脱卤酶的比活为16.2 U/mg,Km和Vmax分别为3.26μmol/L和17.86 mmol/(min.g protein)。在最适反应条件下,以10 mmol/L 3-氯丙酸为底物,反应36 h的转化率在93%以上。     

Adsorptive removal of inhibitory by-product in the enzymatic production of optically active D-p-hydroxyphenylglycine from 5-substituted hydantoin

Summary One-step enzymatic production of D-p-hydroxyphenylglycine (D-HPG) from 5-substituted hydantoin was carried out using a bacterium Agrobacterium sp I-671 which possesses D-hydantoinase and N-carbamoylase. From the inhibition study, it was found that N-carbamoylase was severely inhibited by ammonium ions which is co-produced with D-HPG. In order to increase the conversion yield of D-HPG, simultanious removal of inhibitory by-product from reaction mixture was carried out using the specific adsorbents for ammonium ions. The conversion yield of D-HPG reached about 98% in the presence of adsorbents in 27 hours, while 50% conversion was observed in the absence of adsorbents.     

重组大肠杆菌全细胞催化合成莱鲍迪苷D

莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。     

Microbial production of d‐lactic acid from dried distiller's grains with solubles

d ‐Lactic acid production is gaining increasing attention due to the thermostable properties of its polymer, poly‐d ‐lactic acid . In this study, Lactobacillus coryniformis subsp. torquens, was evaluated for its ability to produce d ‐lactic acid using Dried Distiller's Grains with Solubles (DDGS) hydrolysate as the substrate. DDGS was first subjected to alkaline pretreatment with sodium hydroxide to remove the hemicellulose component and the generated carbohydrate‐rich solids were then subjected to enzymatic hydrolysis using cellulase mixture Accellerase® 1500. When comparing separate hydrolysis and fermentation and simultaneous saccharification and fermentation (SSF) of L. coryniformis on DDGS hydrolysate, the latter method demonstrated higher d ‐lactic acid production (27.9 g/L, 99.9% optical purity of d ‐lactic acid), with a higher glucose to d ‐lactic acid conversion yield (84.5%) compared to the former one (24.1 g/L, 99.9% optical purity of d ‐lactic acid). In addition, the effect of increasing the DDGS concentration in the fermentation system was investigated via a fed‐batch SSF approach, where it was shown that the d ‐lactic acid production increased to 38.1 g/L and the conversion yield decreased to 70%. In conclusion, the SSF approach proved to be an efficient strategy for the production of d ‐lactic acid from DDGS as it reduced the overall processing time and yielded high d ‐lactic acid concentrations.     

Enzymatic Production of Pure D-Mannitol at High Productivity

An enzymatic, NAD(H)-dependent process for the efficient production of D-mannitol from D-fructose as one single product is described and optimized with respect to productivity at high substrate conversion. Stereospecific reduction of D-fructose is catalyzed by recombinant mannitol dehydrogenase from Pseudomonas fluorescens DSM 50106, overexpressed in Escherichia coli. Regeneration of NADH is accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂, thus avoiding byproduct formation and yielding total turnover numbers for the coenzyme of approximately 1000 for a single round of D-fructose conversion. In optimized batchwise reduction of D-fructose, a D-mannitol productivity of 2.25 g/(L h) was obtained for a final product concentration of 72g/L and a D-fructose conversion of 80%. D-Mannitol was crystallized from the ultrafiltered product solution in 97% purity and 85% recovery, thus also allowing reuse of enzymes for repeated batchwise production of D-mann!itol!.     

Expression and purification of soluble B lymphocyte stimulator from recombinant Escherichia coli

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.