三酶偶联全细胞生物合成L-酪氨酸

通过将醛缩酶(ALD)、D-丝氨酸脱水酶(SDH)和酪氨酸酚裂解酶(TPL)三酶偶联,催化甘氨酸、甲醛和苯酚合成L-酪氨酸(L-Tyr)。并以L-Tyr的质量浓度为指标对转化工艺进行了考察。结果表明:三酶偶联最佳反应条件为pH=8.5、温度35℃、乙酸铵质量分数6 g/L、磷酸吡哆醛(PLP)质量浓度0.1 g/L、3种酶细胞配比m(ALD)∶m(SDH)∶m(TPL)=6∶3∶5。当甘氨酸加量为50 g/L,利用上述工艺进行10000 L放大,反应11 h,L-Tyr质量浓度可达117 g/L,苯酚转化率为97%,转化体系放大后苯酚转化率提高4%,收率为85%。     

重组酪氨酸酚裂解酶全细胞催化合成L-酪氨酸

利用基因工程手段重组表达了弗氏柠檬酸杆菌来源的酪氨酸酚裂解酶(TPL),以丙酮酸和L-丝氨酸为底物全细胞催化合成L-酪氨酸,考察了pH、温度、表面活性剂、金属离子、铵盐种类和氯化铵浓度等因素对L-酪氨酸合成的影响,并比较了两种底物合成L-酪氨酸的转化率。结果表明,TPL的最适反应条件是45℃,pH=8.0,4mmol/L PLP,氯化铵浓度350 mmol/L。1 mmol/L triton-x 100对TPL酶活有促进作用,金属离子对TPL酶活都有明显的抑制作用。0.1 mol/L丙酮酸的转化率约是L-丝氨酸的1.8倍。     

重组酪氨酸酚裂解酶全细胞酶法合成L-酪氨酸

利用基因工程手段重组表达了弗氏柠檬酸杆菌来源的酪氨酸酚裂解酶（TPL）,以丙酮酸和L-丝氨酸为底物酶法合成L-酪氨酸,考察了pH、温度、表面活性剂、金属离子、铵盐种类和氯化铵浓度等因素对L-酪氨酸合成的影响,并比较了两种底物合成L-酪氨酸的转化率。结果表明,TPL的最适反应条件是45℃,pH 8.0,4 mmol/l PLP,氯化铵浓度350 mmol/l。1 mmol/l triton-x 100对TPL酶活有促进作用,金属离子无明显影响。质量浓度1%丙酮酸的转化率约是L-丝氨酸的1.8倍。     

双酶连续催化生物合成D-丙氨酸

建立了天冬氨酸消旋酶和D-氨基酸转氨酶双酶连续催化制备D-丙氨酸的方法。利用天冬氨酸消旋酶全细胞催化L-天冬氨酸消旋得到DL-天冬氨酸,离心去除天冬氨酸消旋酶全细胞后升温灭活残留的游离天冬氨酸消旋酶,再加入经镍柱亲和纯化的D-氨基酸转氨酶酶液,催化D-天冬氨酸(D-Asp)和丙酮酸(PA)经转氨反应生成D-丙氨酸。经单因素实验得到天冬氨酸消旋酶最佳催化条件为:反应温度40℃,0.2 mol/L磷酸钾缓冲溶液(pH=7.0),底物L-天冬氨酸质量浓度为100 g/L。D-氨基酸转氨酶最佳催化条件为:反应温度42℃,0.2 mol/L磷酸钾缓冲液(pH=7.0),4 mmol/L磷酸吡哆醛,DL-天冬氨酸质量浓度为50 g/L,底物n(PA)∶n(D-Asp)=1∶10。转化产物经等电点结晶和阳离子树脂分离得到D-丙氨酸。在该条件下,D-天冬氨酸转化率达94%,D-丙氨酸收率为84%,对映体过量值(ee值)=98%。     

L-苯丙氨酸酶法制备中的动力学研究

为研究酶法生产L-苯丙氨酸工艺中影响收率的主要因素,提高L-苯丙氨酸的收率,对该反应过程的动力学进行了研究。研究中采用实验检测和动力学模拟的方法获取了转化反应中的主要动力学参数。研究这些参数和L-苯丙氨酸收率间的关系发现,达到L-苯丙氨酸最大收率的前提条件是草酰乙酸的浓度趋近于0。在此前提条件下,将苯丙酮酸的浓度选在0.10~0.15 mol/L,提高体系中酶活性,降低苯丙酮酸的降解常数,可以提高体系中L-苯丙氨酸的收率。以此规律为指导,构建了合适的基因工程菌,其转氨酶活性提高4.6倍,苯丙酮酸的降解常数降低到0.0158 h-1,选择c(苯丙酮酸)=0.137 mol/L充N2的条件下进行反应,其L-苯丙氨酸收率提高到90.2%。     

草生欧文氏菌中酪氨酸酚裂解酶的体外定向进化及结构模拟

以来源于草生欧文氏菌(Erwinia herbicola)的酪氨酸酚裂解酶(Eh-TPL)为目的基因,通过易错PCR定向进化技术构建突变体文库,利用高通量筛选方法获得了比酶活提高的突变体S20C和E202R,再通过定点突变得到突变体S20C/E202R/N161S。经摇瓶发酵培养发现,突变体S20C/E202R/N161S比野生型Eh-TPL的比酶活提高了30.61%;酶学性质研究结果表明,突变体S20C/E202R/N161S的最适温度仍为37℃,最适pH值从8.2升至8.5,热稳定性、pH稳定性均得到明显提高;结构模拟结果表明,突变体S20C/E202R/N161S的三维结构无明显变化,突变位点附近氢键和表面电势有所变化。体外定向进化技术能有效提高Eh-TPL的催化效率,对酶法生产L-酪氨酸具有重要应用价值。     

双酶级联生物合成D-赖氨酸

建立了氨基酸消旋酶和赖氨酸脱羧酶双酶级联高效生产D-赖氨酸的方法。利用氨基酸消旋酶全细胞催化消旋L-赖氨酸得到DL-赖氨酸,去除氨基酸消旋酶后再加入赖氨酸脱羧酶,将消旋产物中的L-构型脱羧生成1,5-戊二胺和CO2,剩下D-赖氨酸;最终,通过阳离子交换树脂吸附洗脱得到D-赖氨酸。经考察双酶级联催化的最佳条件为:反应温度40℃,0.2 mol/L磷酸钾缓冲溶液(p H=5.8),底物L-赖氨酸质量浓度为50 g/L,氨基酸消旋酶全细胞和赖氨酸脱羧酶全细胞质量浓度均为10 g/L,4 mmol/L磷酸吡哆醛,0.1 g/L Triton X-100,反应时间12 h,其中消旋反应2 h和脱羧反应10 h,最终D-赖氨酸收率达42%,对映体过量值(e.e.值)=98%。     

重组酪氨酸脱羧酶酶学性质

酪氨酸脱羧酶能以L-酪氨酸为底物脱羧生成酪胺。该文利用pET28a为载体在宿主细胞E.coli BL21(DE3)中重组表达了短乳杆菌来源的酪氨酸脱羧酶,并研究了其酶学性质,考察了起始pH、温度、辅酶、底物浓度等因素对酶活的影响。结果表明,酪氨酸脱羧酶重组表达成功,酶促反应工艺为:在1 mL转化液中含有0.18 g L-酪氨酸,0.02 g湿菌体,0.2 mol/L的醋酸缓冲溶液和0.2 mmol/L的5'-磷酸吡哆醛,40℃,pH=5.5,反应7 h,L-酪氨酸的摩尔转化率达到99%。酪氨酸脱羧酶酶活为29.2 U/g,Km值和Vmax为0.71 mmol/L和9.31mol/(L·min·g)。     

天冬氨酸酶基因工程菌固定化细胞制备L-天冬氨酸合成条件优化

以反丁烯二酸和氨水为原料,采用天冬氨酸酶基因工程菌固定化细胞生物催化法合成L-天冬氨酸。通过响应面法考察反丁烯二酸浓度、温度、p H对合成L-天冬氨酸的影响。结果表明,固定化天冬氨酸酶基因工程菌合成L-天冬氨酸最佳条件为:底物反丁烯二酸的质量浓度为300 g/L,反应温度为37℃,底物p H为7.5,L-天冬氨酸的产率为96.7%。固定化细胞可连续使用10批次。通过电镜观察发现天冬氨酸酶基因工程菌均匀分布于载体,天冬氨酸酶基因工程菌固定化细胞具有良好的稳定性。     

L-丙氨酰-L-酪氨酸二肽化学合成研究

以L-酪氨酸和L-丙氨酸为原料,苄氧羰基保护丙氨酸,甲酯保护酪氨酸,EDCI-HOBt法合成丙酪二肽,总摩尔收率（以酪氨酸计）为42%,结构经IR、APCI-MS、NMR表征。     

Influence of Preoperative Serum Aspartate Aminotransferase (AST) Level on the Prognosis of Patients with Non-Small Cell Lung Cancer

The purpose of this work is to analyze preoperative serum aspartate aminotransferase (AST) levels and their effect on the prognosis of patients with non-small cell lung cancer (NSCLC) after surgical operation. These analyses were performed retrospectively in patients with NSCLC followed by surgery; participants were recruited between January 2004 and January 2008. All clinical information and laboratory results were collected from medical records. We explored the association between preoperative serum AST and recurrence-free survival (RFS), and the overall survival (OS) of NSCLC patients. Kaplan–Meier analysis and Cox multivariate analysis, stratified by the AST median value, were used to evaluate the prognostic effect. A chi-squared test was performed to compare clinical characteristics in different subgroups. A p-value of ≤0.05 was considered to be statistically significant. A total of 231 patients were enrolled. The median RFS and OS were 22 and 59 months, respectively. The AST levels were divided into two groups, using a cut-off value of 19 U/L: High AST (>19 U/L), n = 113 vs. low AST (≤19 U/L), n = 118. Multivariate analysis indicated that preoperative serum AST > 19 U/L (hazard ratio (HR) = 0.685, 95% confidence interval (CI): 0.493–0.994, p = 0.046 for RFS, HR = 0.646, 95% CI: 0.438–0.954, p = 0.028 for OS) was an independent prognostic factor for both RFS and OS. High preoperative serum AST levels may serve as a valuable marker to predict the prognosis of NSCLC after operation.     

重组大肠杆菌全细胞催化合成S-苷甲硫氨酸

从大肠杆菌(E.ColiK12)基因组DNA中克隆出甲硫氨酸腺苷转移酶基因,构建了能高效表达甲硫氨酸腺苷转移酶的重组大肠杆菌E.ColiJM109(pBR322-MAT)。将重组大肠杆菌细胞用于催化合成S-腺苷甲硫氨酸(SAM)的研究中。实验发现,用φ(甲苯)=2%的水溶液对重组细胞进行通透化处理后,能大幅提高SAM的产率。探讨了底物浓度、温度、pH、反应时间以及菌体密度对反应转化率的影响。最佳反应条件为:底物浓度(ATP)30 mmol/L,反应温度35℃,pH=7.0,反应时间8 h,细胞密度80 g湿细胞/L反应液。在此条件下,底物ATP的转化率超过95%。     

Effects of Tyrosine and Tryptophan in Rats with Diet-Induced Obesity

Amino acids tyrosine (Tyr) and tryptophan (Trp) play a significant role in the regulation of energy metabolism, locomotor activity, and eating behavior. We studied the possibility of modulating these processes in obesity by increasing the pool of Tyr and Trp in the experimental diet. As a model of obesity, we used Wistar rats fed a diet with an excess specific energy value (HFCD) for 64 days. Trp led to a normalization of the rats’ body weight almost to the control level, but increased anxiety-like behavior and decreased long-term memory. The consumption of amino acids resulted in increased grip strength and impairment of short-term memory. The locomotor activity of animals decreased with age as a result of Tyr consumption, while Trp, on the contrary, prevented this. The Tyr supplementation led to the normalization of triglycerides and LDL. In the spleen cell lysates, amino acids suppressed the production of proinflammatory cytokines. The liver tissue morphology showed that the consumption of Tyr noticeably weakened the signs of fatty degeneration. The addition of Trp, on the contrary, led to an unfavorable effect, consisting of the appearance of a high number of large rounded fatty vacuoles. The data obtained indicate a more pronounced anti-inflammatory effect of Tyr as compared to Trp.     

Biochemical,Enzymatic, and Computational Characterization of Recurrent Somatic Mutations of the Human Protein Tyrosine Phosphatase PTP1B in Primary Mediastinal B Cell Lymphoma

Human protein tyrosine phosphatase 1B (PTP1B) is a ubiquitous non-receptor tyrosine phosphatase that serves as a major negative regulator of tyrosine phosphorylation cascades of metabolic and oncogenic importance such as the insulin, epidermal growth factor receptor (EGFR), and JAK/STAT pathways. Increasing evidence point to a key role of PTP1B-dependent signaling in cancer. Interestingly, genetic defects in PTP1B have been found in different human malignancies. Notably, recurrent somatic mutations and splice variants of PTP1B were identified in human B cell and Hodgkin lymphomas. In this work, we analyzed the molecular and functional levels of three PTP1B mutations identified in primary mediastinal B cell lymphoma (PMBCL) patients and located in the WPD-loop (V184D), P-loop (R221G), and Q-loop (G259V). Using biochemical, enzymatic, and molecular dynamics approaches, we show that these mutations lead to PTP1B mutants with extremely low intrinsic tyrosine phosphatase activity that display alterations in overall protein stability and in the flexibility of the active site loops of the enzyme. This is in agreement with the key role of the active site loop regions, which are preorganized to interact with the substrate and to enable catalysis. Our study provides molecular and enzymatic evidence for the loss of protein tyrosine phosphatase activity of PTP1B active-site loop mutants identified in human lymphoma.