Engineering of Corynebacterium glutamicum to Enhance L-ornithine Production by Gene Knockout and Comparative Proteomic Analysis

Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.

The effect of L-ornithine hydrochloride ingestion on human growth hormone secretion after strength training

This study aimed to examine the effect of L-ornithine hydrochloride ingestion on serum growth hormone secretion response after strength training in young men who did not regularly engage in high intensity exercise. Ten healthy young males without workout habits (age: 22.2 +/- 1.0 yr). Subjects performed biceps curl strength training after L-ornithine hydro- chloride and placebo ingestions. They participated in both of the above conditions randomly with a week interval in between. Serum growth hormone and ornithine levels were measured before L-ornithine hydrochloride or placebo ingestions and at 30 minutes after strength training. Serum growth hormone and ornithine level were measured. A change magnitude of serum growth hormone was significantly larger in the L-ornithine hydrochloride condition than in the placebo condition, and the effect size was also large (t = 1.91, p = .044, ES = .75). A significant interaction (F = 280.98, p = 0.000, ηp2 = 0.96) was found in serum ornithine and a multiple comparison test showed that it was greater in the L-ornithine hydrochloride condition. Serum growth hormone level after strength training increases by L-ornithine hydrochloride ingestion in untrained young males.

L-ornithine hydrochloride ingestion increased carbohydrate oxidation but not lipid oxidation during submaximal endurance exercise following resistance exercise

This study aimed to examine the effect of L-ornithine hydrochloride ingestion on lipid oxidation during submaximal endurance exercise following resistance exercise. Ten healthy young male adults with no history of regular resistance exercise (age: 26.0 +/- 4.4) conducted resistance exercise after L-ornithine hydrochloride or placebo ingestion (0.1 g/kg). Subjects exercised for 60 min on an ergometer at 50% VO2peak 120 min after resistance exercise. Plasma ornithine concentrations measured immediately, 120min and 180min after resistance exercise in the L-ornithine hydrochloride ingestion condition were significantly greater than those in the placebo condition. No significant difference was found in serum growth hor mone concentrations between both conditions (F = 4.4, p = 0.065). Serum free fatty acid concentrations were significantly greater immediately after submaximal ergometer bicycle exercise in both conditions than those before ingestion, immediately after resistance exercise and 120min after resistance exercise (F = 43.4, p .001, 300% - 508%), but no significant difference was found between both conditions (F = 3.6, p = 0.090). A similar trend was observed in serum ketone bodies as well. Although total energy production during submaximal ergometer exercise did not significantly differ (t = 0.74, p = 0.238), a significant difference was found in energy production via carbohydrate and lipid oxidation;the former was greater in the Lornithine hydrochloride ingestion condition (t = 1.89, p = 0.046, d = 0.44, 106%), and the latter was greater in the placebo condition (t = 1.89, p = 0.046, d = 0.78%, 145%). From the above, L-ornithine hydrochloride ingestion may not affect lipid metabolism during submaximal endurance exercise following resistance exercise. It may be involved in energy production via carbohydrate oxidation with glucogenic amino acid.

L-盐酸赖氨酸对局部脑缺血大鼠的治疗作用

目的：研究Ｌ盐酸赖氨酸对大鼠局限性脑缺血的治疗作用。方法：采用电凝阻断大鼠一侧大脑中动脉造成局限性脑缺血模型。结果：一侧大脑中动脉阻断后１５ｍｉｎｉｐＬ盐酸赖氨酸６２１５及３１０８ｍｇ·ｋｇ－１，能显著降低局限性脑缺血大鼠的脑梗塞面积，减少相关脑区变性、坏死神经元的数量，而且Ｌ盐酸赖氨酸６２１５ｍｇ·ｋｇ－１还能明显改善大鼠的神经功能缺失，降低行为评分。结论：Ｌ盐酸赖氨酸对脑缺血有治疗作用。

L-瓜氨酸和L-高瓜氨酸的制备

以L-鸟氨酸盐酸盐为原料与碱式碳酸铜反应生成二鸟氨酸合铜保护α-NH2,用尿素对δ-NH2进行甲酰化,得到二瓜氨酸合铜,后用H2S除铜离子得到L-瓜氨酸。用同样方法以L-赖氨酸盐酸盐为原料制备了L-高瓜氨酸,通过IR,元素分析和高效液相色谱证明了其结构和含量。

康脑灵对大鼠缺氧状态下脑保护作用的定量脑电图

目的：通过皮层脑电（ＥＣｏＧ）的定量分析评价康脑灵（ＫａｎｇＮａｏＬｉｎ，ＫＮＬ）对缺氧状态下大鼠的脑保护作用。方法：用定量方法分析在体脑电在正常供气与停止呼吸情况下的变化，判断药物的脑保护作用。结果：ｉｇ康脑灵可以显著延长停止供气后脑电平均振幅到达最低点的时间，促进重新供气后脑电活动的完全恢复。这种作用在０６３ｇ·ｋｇ－１和１２６ｇ·ｋｇ－１间呈剂量依赖性，与对照组有极显著差异，与氟桂嗪组（２５ｍｇ·ｋｇ－１）无显著性差异。其中康脑灵１２６ｇ·ｋｇ－１组在缺氧２ｍｉｎ时脑电不完全消失。ｉｇ康脑灵可以不同程度地降低皮层脑电的平均振幅。结论：ｉｇＫＮＬ在一定剂量范围内有明显的抗脑缺氧的作用。

盐酸环丙沙星三元配合物体系的光谱研究及应用

研究了盐酸环丙沙星、茜素和稀土镧离子所形成的三元配合物体系。结果表明，以水-乙醇作为溶剂，在pH为5．8的KH2PO4-NaOH缓冲液中，该三元配合物最大吸收波长为280nm，表观摩尔吸收系数为4．23×10^4L·mol^-1·cm^-1，盐酸环丙沙星在0．3～34．5mg／L范围内符合郎伯-比耳定律，方法的检出限为0.01mg／L。已用于市售盐酸环丙沙星片和胶囊的测定。

一种新型食品防腐剂——L-月桂酰胺精氨酸盐酸盐乙醇酯

阐述了L-月桂酰胺精氨酸盐酸盐乙醇酯的物化性质、抑菌活性、抑菌机理、安全性以及在食品和化妆品等领域的应用,为我国研究和开发新型、安全、高效的食品防腐剂提供参考。

L-瓜氨酸与L-高瓜氨酸的合成

以L-鸟氨酸盐酸盐为原料与五水硫酸铜反应生成二鸟氨酸合铜保护α-NH2,用尿素对δ-NH2甲酰化得到二瓜氨酸合铜,后用二水合草酸脱α-NH2保护得到目标产物L-瓜氨酸,总收率49.5%。用同样的方法以L-赖氨酸盐酸盐为原料制备了L-高瓜氨酸,总收率53.5%。产物经IR、熔点、旋光确证。该方法具有操作方便、安全、经济和环保等特点。

配种前母体营养影响子代雄性比率的研究

以初产昆明小鼠为材料,配种前在其母鼠日粮中加入不同组合的3种物质。在3次实验的635只母鼠中得到395窝仔鼠。结果表明:日粮中添喂25%葡萄糖能显著降低子代雄性比率,且产仔数不受影响;添喂3%氯化钠使性比和产产仔数均降低;0.7%精氨酸盐的添加中未显示出对性比的影响;上述3种物质以不同浓度的组合都显示出性比降低、产仔数未受影响的效应。80只母鼠配种前血清睾酮含量分析,发现添喂葡萄糖或葡萄糖、精氨酸盐酸盐、氯化钠混合物使母鼠血清睾酮含量降低。

1,3-二氨基胍盐酸盐的高效液相色谱分析

提出用高效液相色谱法测定1，3-二氨基胍盐酸盐的含量，选用甲醇与乙酸铵缓冲盐体积比75：25为流动相，c8色谱柱，检测波长314nm。试验结果表明：测试结果的相对标准偏差小于2．03％；回收率在97．8％～100．2％；1，3-二氨基胍盐酸盐和三氨基胍盐酸盐得到较好的分离。该法操作简单、实用且定量分析结果准确稳定。

微波辅助合成L-瓜氨酸

以L-鸟氨酸盐酸盐为原料,经配位反应保护α-NH2,后经δ-NH2甲酰化,在微波辐射下去除铜离子,制得目标产物L-瓜氨酸,总收率90.3%。探讨了配合反应中配合试剂的选择、脱保护反应中除铜试剂的选择以及微波辐射功率、微波辐射时间对反应的影响,确定最佳合成条件。产物经红外光谱、熔点和旋光确证。该工艺更安全、环保,且操作方便。

N-保护鸟氨酸的合成

以L-鸟氨酸盐酸盐为原料,经碱式碳酸铜配合α-氨基,在δ-氨基上引入苄氧羰基,用EDTA-2Na脱去α-氨基上的铜离子,得到Nδ-苄氧羰基-L-鸟氨酸,再在α-氨基上引入叔丁氧羰基,得到Nδ-苄氧羰基-Nα-叔丁氧羰基-L-鸟氨酸,用Pd/C与甲酸铵选择性脱去苄氧羰基,得到Nα-叔丁氧羰基-L-鸟氨酸,共得到3个N-保护鸟氨酸产物,收率分别为73.1%、61.2%、52.8%(均以L-鸟氨酸盐酸盐计)。探讨了EDTA-2Na作为脱铜试剂以及催化转移加氢选择性脱苄氧羰基反应的最佳合成条件,产物结构经1H NMR及元素分析确证。该合成工艺操作简便,环境污染较少,产品收率与纯度均较好。

LMH对脑损伤后大鼠胰岛细胞及血糖含量的影响

目的 观察L 盐酸赖氨酸(LMH)对脑损伤后大鼠胰高血糖素细胞(A)、胰岛素细胞(B)和生长 抑素细胞(D)及血糖含量的影响。方法 采用落体法致大鼠脑损伤模型;伤后立刻一次腹腔注射LMH,24h 后断头处死全部大鼠,用免疫组织化学方法观察LMH对脑损伤后大鼠胰岛A、B和D细胞的影响;用生化分 析仪测定血糖含量。结果 与对照组比,脑损伤组大鼠胰岛A、D细胞免疫反应明显增强(P<0.05),胰岛B 细胞免疫反应明显减弱(P<0.05),血糖含量明显增高(P<0.05)。LMH组大鼠胰岛A细胞免疫反应明显 弱于脑损伤组(P<0.05),但与对照组相比无明显差异(P>0.05);胰岛B、D细胞免疫反应与脑损伤组无 明显差异(P>0.05);LMH组血糖含量低于脑损伤组(P<0.01)。结论 LMH在大鼠脑损伤过程中可能 参与了胰岛A细胞功能的调节,并产生显著的降血糖作用。

咪苯嗪酮对花生四烯酸环氧酶途径代谢产物的选择性影响

兔iv咪苯嗪酮(CI—914)20 mg/kg,用RIA法测定其血浆中TXB_2和6—kcto—PGF_(1α)含量,给药后30 min和60 min.6—keto—PGF_(1α)/TXB_2比值显著升高(p<0.05).在用放射性TLC法进行的洗涤大鼠血小板^(14)C—AA代谢实验中,CI—914在2～500 μmol/L范围内以剂量依赖方式抑制大鼠血小板TXB_2生成,IC_(50)为51.5μmol/L,对HHT生成的抑制明显弱于对TXB_2生成的抑制作用;在相同剂量范围内,CI—914同时以剂量依赖方式使PGE_2,PGF_(2α)和PGD_2生成增加.在相同实验条件下,50μmol/L的已知的TXA_2合成酶抑制剂DAZ,与大剂量CI—914的实验结果类似.上述结果提示,CI—914对血小板TXA_2合成酶可能具有选择性抑制作用.

(L)-2-氧代四氢噻唑-4-羧酸的合成研究及工艺改进

以L-半胱氨酸盐酸盐和氯甲酸苯酯为主要原料在碱性环境中进行反应,经分液、萃取、重结晶,得到反应产物。对反应产物进行了结构表征,通过优化,确定了最优的反应工艺条件:n(氯甲酸苯酯)∶n(L-半胱氨酸盐酸盐)=1.6,反应温度40℃,反应时间2h,pH=14,解析了反应机理,完成了(L)-2-氧代四氢噻唑-4-羧酸的实验室合成。

L-高精氨酸盐酸盐的合成

以L-赖氨酸盐酸盐为原料与碱式碳酸铜反应生成二赖氨酸铜配合物保护α-NH2,从而对ω-NH2进行胍基化得到二高精氨酸合铜配合物,再经过脱铜、离子交换树脂除杂和酸化得到最终产物高精氨酸盐酸盐。产物经元素分析I、R表征其结构,HLPC测其纯度达99%以上。

硼酸对赖氨酸盐酸盐在半胱氨酸自组装膜上晶体生长的影响

研究了利用半胱氨酸自组装膜对诱导L-赖氨酸盐酸盐晶体生长的影响。实验发现,赖氨酸盐酸盐优先在水溶液内而不是在自组装膜表面成核生长为晶体,这是首次发现自组装单层对赖氨酸盐酸盐的成核有抑制作用。对自组装单层上的成核机理进行了探讨,2种氨基酸正电荷间的排斥作用可能是自组装膜抑制赖氨酸盐酸盐成核的原因。在赖氨酸盐酸盐溶液中加入了等摩尔硼酸,XRD结果表明,硼酸晶体优先生长在自组装单层上。而析出后的硼酸晶体又加速促进了赖氨酸盐酸盐的结晶析出。扫描电子显微镜表征发现,加入硼酸后赖氨酸盐酸盐晶体的形貌发生了很大变化,由原来的块状变为针状。实验结果表明,加入硼酸,可以加速赖氨酸盐酸盐的晶体生长并可以调控晶体的形貌和取向。

柱前衍生化RP-HPLC法测定L-鸟氨酸盐酸盐的有关物质

目的:采用9-芴甲氧羰酰氯(FMOC-Cl)为柱前衍生化试剂,建立FMOC-Cl柱前衍生化反相高效液相色谱法测定L-鸟氨酸盐酸盐的有关物质。方法:采用Agilent Extend C18(250 mm×4.6 mm,5μm)色谱柱,醋酸盐缓冲溶液(0.1 mol·L^(-1)醋酸铵,冰醋酸调节p H至5.0)-乙腈为流动相,梯度程序洗脱,流速:1 m L·min^(-1),检测波长:265 nm,柱温:35℃,测定L-鸟氨酸盐酸盐中的有关物质。衍生化方法与条件:需衍生化样品与0.05 mol·L-1硼砂缓冲液和8 mg·m L^(-1)FMOC-Cl乙腈溶液(v/v,1∶3∶3)室温反应20 min后正己烷萃取2次,4 mol·L^(-1)盐酸溶液调节p H至3~4。结果:该品及其潜在的12种氨基酸类有关物质衍生物色谱分离度良好;测得各氨基酸浓度在1.0~20μg·m L^(-1)范围内,线性关系良好,r均大于0.9995,加样回收率和精密度结果均良好。结论:该方法专属性好、准确度高,适用于该品有关物质的检测。

3-丁氧基-4-氨基苯甲酸-β-二乙氨基乙酯单盐酸盐的合成

利用自行改进的方法，合成了Ｎｏｖｅｓｉｎｅ，实验结果表明，综合技术指标转化率、选择性和收率比原合成方法有显著的提高。