解除硝酸盐呼吸和氮通量限制对钝齿棒杆菌产精氨酸的影响

精氨酸广泛应用于食品、医药和工业领域中，其生产方式主要是微生物发酵法。钝齿棒状杆菌(Corynebacterium crenatum)是精氨酸生产的重要工程菌株。该文针对课题组前期构建的钝齿棒杆菌(CCM01)进行代谢改造，探究了arnR和cgl2482的敲除对钝齿棒杆菌产精氨酸的影响。采用无痕敲除方法构建工程菌株并对其进行摇瓶发酵，测定菌体生长量、L-精氨酸产量、葡萄糖消耗量考察菌株产L-精氨酸的影响。硝酸盐能提高钝齿棒杆菌在氧气不足时的生长率，并且arnR的敲除有助于L-精氨酸的生产，arnR敲除菌株CCM02较对照菌株产量提高了8.58%,且当硝酸盐浓度在1.5 mmol/L时对积累L-精氨酸最为有利，产量达到17.09 g/L;cgl2482的敲除有助于氮源流入精氨酸合成途径，其敲除菌株CCM03精氨酸产量达到了17.88 g,较对照提高了4.9%;最终在CCM03发酵中添加1 g/L的尿素、50 g/L的谷氨酸钠以及1.5 mmol/L的硝酸盐时，L-精氨酸产量达到22.25 g/L,较CCM01提高了46.8%。     

L-瓜氨酸生产菌株的筛选及鉴定

通过选择性培养基初筛(利用精氨酸双水解酶筛选培养基)和复筛(高效液相色谱法测定L-瓜氨酸质量浓度),从杭州市勾庄地区西瓜大棚的土壤中筛选获得45株产L-瓜氨酸菌株.其中一株细菌(菌株编号11)转化L-精氨酸生产L-瓜氨酸的能力最强,产生的L-瓜氨酸浓度最高,0.344 g湿菌,在60 mL转化体系中转化一天,可将20 g/L的底物L-精氨酸转化为9.31g/L的L-瓜氨酸.对11号菌株分别进行形态学观察,生理生化试验及分子生物学鉴定,初步鉴定该菌株为粪肠球菌(Enterococcus faecalis).     

高产L-精氨酸谷氨酸棒状杆菌的构建

以产L-精氨酸诱变菌株谷氨酸棒状杆菌（Corynebacterium glutamicum）AJC为出发菌株,采用基因组编辑技术对其进行改造。 首先,敲除阻遏蛋白ArgR和FarR,解除反馈阻遏作用;然后,敲除乳酸脱氢酶编码基因ldh和整合鸟氨酸乙酰转移酶编码基因argJ,阻 断乳酸合成途径和增加前体物;最后,敲除谷氨酸分泌蛋白编码基因NCgl1221和整合乙酰谷氨酸激酶基因argB,减弱L-谷氨酸的胞 外分泌,筛选一株L-精氨酸高产菌株。 结果表明,获得一株高产L-精氨酸菌株AJC-4（C. glutamicum AJCΔargRΔfarRΔldh::PtufargJ ΔNCgl1221::PsodargB）,该菌株在5 L发酵罐中发酵64 h后,L-精氨酸产量和糖酸转化率分别为78.0 g/L和0.38 g/g,较出发菌株AJC分 别提高21.9%、18.8%;副产物乳酸和L-谷氨酸积累量分别为0.11g/L、0.16 g/L,较出发菌株AJC分别降低96.8%、96.1%。     

L-精氨酸高产菌株的亚硝基胍诱变选育和种子培养基的优化研究

为了筛选L-精氨酸的高产菌株,采用亚硝基胍对出发菌株A TCC 14067 （Brevibacterium flavum）进行诱变,结合抗精氨酸结构类似物D-精氨酸,S-甲基半胱氨酸平板抗性筛选高产菌株,并采用正交试验优化了种子培养基.结果显示,经过1 mg/mL的亚硝基胍诱变处理4min后,采用14 mg/mL的D-精氨酸抗性平板和8mg/mL的S-甲基半胱氨酸抗性平板筛选获得精氨酸高产菌株,由于解除精氨酸自身的反馈调节,L-精氨酸积累增大,产酸量达37.2 g/L,比野生菌株提高了128.2％,得到的高产菌株遗传性状稳定.通过对种子培养基进行正交试验优化,确定最终培养基配方为葡萄糖3.0％,玉米浆2.0％,硫酸铵2.0％,KH2PO4 0.10％,MgSO4·7H2O 0.05％,尿素0.15％,在此发酵条件下,L-精氨酸产量达37.8 g/L.     

精氨酸对Escherichia coli发酵生产L-色氨酸的影响

色氨酸是人和动物体内必须氨基酸,在食品、饲料及医药工业中具有广泛应用价值.以色氨酸生产菌株Escherichia coli TRTH为出发菌株,在培养基中添加精氨酸,以降低代谢副产物的积累量,提高L-色氨酸的产量.在30 L发酵罐上考察了精氨酸对L-色氨酸发酵的影响,结果表明:添加0.2 g/L的精氨酸时,菌体生物量、L-色氨酸产量和糖酸转化率分别为41.5 g/L,34.5 g/L和18.0％,较未添加时分别提高了5.46％,5.83％和2.86％,且乙酸积累量较未添加时降低了10.99％.     

3-磷酸甘油醛脱氢酶促进谷氨酸棒杆菌发酵生产L-精氨酸和L-鸟氨酸

在谷氨酸棒状杆菌(Corynebacterium glutamicum) SNK118中表达NADP~+依赖型的3-磷酸甘油醛脱氢酶编码基因,提高胞内NADPH水平,以提高L-精氨酸(L-Arginine)和L-鸟氨酸(L-Ornithine)发酵产量。通过NCBI数据库检索,选取了3个不同来源的3-磷酸甘油醛脱氢酶编码基因。经测定酶活力,最终选择糖丁基梭菌(Clostridium saccharobutylicum) DSM 13864来源的NADP~+依赖型的3-磷酸甘油醛脱氢酶基因(Csgap C)。构建了产L-精氨酸的重组菌SNK118/p XMJ19-Csgap C,当摇瓶发酵70 h时产L-精氨酸11. 55 g/L,糖酸转化率0. 13 g/g,与对照菌SNK118/p XMJ19相比,精氨酸产量和糖酸转化率分别提高了26%和10. 2%。在L-鸟氨酸生产菌株SNK118Δarg FΔargR中重组表达Csgap C,重组菌SNK118Δarg FΔargR/p XMJ19-Csgap C摇瓶发酵70 h产L-鸟氨酸27. 76 g/L,糖酸转化率0. 274 g/g,与对照菌SNK118Δarg FΔargR/p XMJ19相比,L-鸟氨酸产量和糖酸转化率分别提高了20. 1%和15. 6%。结果表明,异源表达Csgap C有助于提高谷氨酸棒杆菌发酵生产L-精氨酸和L-鸟氨酸的水平。     

基于钝齿棒杆菌argGH启动子改造的L-瓜氨酸高效合成

L-瓜氨酸是尿素循环的重要中间体,在argGH编码酶的催化下易分解为L-精氨酸,在微生物体内难以大量积累。作者通过启动子替换手段,以弱启动子P-dapAB6替换调控argGH表达的启动子P-argG,构建了弱化L-瓜氨酸分解代谢途径的重组菌株Corynebacterium crenatum H-7-PdapAB6:argGH。重组菌株的转录水平和酶活力结果显示,重组菌株分解途径中argG和argH的基因表达水平下调,精胺琥珀酸合成酶ASS(argG编码)和精胺琥珀酸裂解酶ASL(argH编码)酶活分别降低91.80%和55.35%。摇瓶发酵结果表明,L-瓜氨酸的产量、糖酸转化率和生产强度分别为33.85 g/L、0.25 g/g和0.34 g/(L·h),较原始菌株分别提高了4.91、5.00和4.86倍。通过启动子替换策略,构建了L-瓜氨酸分解代谢途径弱化的工程菌株,初步实现了L-瓜氨酸的高效合成。     

黄色短杆菌B．003产L-精氨酸发酵条件的研究

目的通过对摇瓶发酵条件的研究,提高黄色短杆菌B-003 L-精氨酸的产量。方法在单因素实验的基础上,利用响应面法,以L-精氨酸的产量为响应值,研究L-精氨酸摇瓶发酵条件。结果 Plackett-Burman设计筛选出3个对L-精氨酸产量有显著影响的因素：葡萄糖、硫酸铵和磷酸二氢钾的浓度,通过Box-Behnken试验设计得出,葡萄糖的浓度为99.57 g/L,硫酸铵浓度为60.80 g/L,磷酸二氢钾浓度为2.18 g/L时L-精氨酸产量最高。结论在最佳培养条件下,L-精氨酸产量达24.10 g/L,与最初培养条件相比产量提高了58.55%。     

一株联产L-精氨酸和聚羟基丁酸酯的重组钝齿棒杆菌的构建

Corynebacterium crenatum SYPA 5-5是一株用于L-精氨酸(Arg)生产的工业菌株。聚羟基丁酸酯(PHB)是在细菌内部形成的聚合物,将PHB代谢途径引入细胞内可影响胞内的全局代谢网络,实现联产PHB和相关化工产品如琥珀酸、L-谷氨酸和L-色氨酸等。采用基因工程技术,将来源于Ralstonia eutropha H16的PHB合成基因簇phb CAB导入到L-精氨酸生产菌株C.crenatum SYPA 5-5中,进行组成型表达,旨在获胞内产PHB、胞外产L-精氨酸的效果,对出发菌和重组菌分别进行5 L容积发酵罐实验,并对发酵相关参数进行测定分析。结果表明,由于PHB合成基因簇的导入,重组菌中PHB质量为细胞干质量的12.8%,实现了胞外产L-精氨酸、胞内产PHB的效果,并发现胞内PHB的积累对菌体生产L-精氨酸有一定的正向作用,发酵96 h,L-精氨酸的产量达到43.21 g/L,相比于出发菌提高了20%。     

黄色短杆菌B-003产L-精氨酸发酵条件的研究

目的通过对摇瓶发酵条件的研究，提高黄色短杆菌B-003 L-精氨酸的产量。方法在单因素实验的基础上，利用响应面法，以L-精氨酸的产量为响应值，研究L-精氨酸摇瓶发酵条件。结果 Plackett-Burman设计筛选出3个对L-精氨酸产量有显著影响的因素：葡萄糖、硫酸铵和磷酸二氢钾的浓度，通过Box-Behnken试验设计得出，葡萄糖的浓度为99.57 g/L，硫酸铵浓度为60.80 g/L，磷酸二氢钾浓度为2.18 g/L时L-精氨酸产量最高。结论在最佳培养条件下，L-精氨酸产量达24.10 g/L，与最初培养条件相比产量提高了58.55%。     

基于代谢流分析的L-组氨酸产生菌定向选育

基于谷氨酸棒杆菌(Corynebacterium glutamicum)代谢网络和L-组氨酸合成代谢流分析,对L-组氨酸产生菌谷氨酸棒杆菌TQ2223(Phe-,Tyr-,8-AGr,SGr,CINr)进行多次硫酸二乙酯(DES)定向诱变,依次赋予其6-MPr、5-MTr、2-TAr和L-Hisase-(L-组氨酸酶缺陷)遗传标记后得到突变株TL1106(5-MTr,SGr,5-FTr,8-AGr,6-MPr,2-TAr,Hisase-)。经验证突变株TL1106可在10%葡萄糖的发酵培养基上积累L-组氨酸6.0g/L,比出发菌株提高了2.5倍。结果表明:通过赋予目的遗传标记而改变代谢流分布,能够促进L-组氨酸的合成与积累。     

肌苷高产菌的转化育种及均匀设计在发酵条件试验中的应用

应用感受态细胞转化法，以ＴＸ２为供体，以ＴＳＡ１９为受体，成功地选育出了具有目的遗传标记Ａｄｅ－＋Ｈｉｓ－＋Ｘａｎ－＋８－Ａｇｒ＋６－ＭＰｒ＋ＳＧｒ，产肌苷高的转化子ＴＳＸＢ２９，在发酵条件试验中，引进均匀设计理论，利用微机对试验数据进行统计处理，找出了最佳条件。在该条件下，转化子ＴＳＸＢ２９摇瓶发酵平均产肌苷２７．８５ｍｇ／ｍｌ，最高达２９．６７ｍｇ／ｍｌ，经斜面传代１５次及连续摇瓶传代１０次，发现该转化子是稳定的。经连续１０批稳产试验，发现其１０批平均产肌苷２７．３８ｍｇ／ｍｌ，完全可用于工业生产中。     

L-精氨酸生产菌的诱变育种研究

实验采用亚硝基胍对谷氨酸棒杆菌(Coynebacterumglutamicum)进行诱变处理,以添加了L-精氨酸结构类似物磺胺胍的筛选平板,筛选黄胺胍抗性突变菌株,将突变菌株进行摇瓶发酵实验,选育出1株L-精氨酸产量较高和产酸性能比较稳定的突变菌株,该菌株L-精氨酸产量比出发菌株提高了56.7%。     

生物素对L-缬氨酸发酵的影响

为研究生物素添加量对谷氨酸棒状杆菌（Corynebacterium glutamate）发酵生产L-缬氨酸的影响,以谷氨酸棒状杆菌XV0505（Leu－＋Ile－＋2-TAr＋α-ABr＋SGr）为供试菌株,考察不同生物素添加量条件下菌体量、耗糖、产酸以及副产物L-丙氨酸的情况,确定了生物素最适添加量为50 μg/L;利用膜偶联透析发酵方式有效解除了发酵生产过程中产生的反馈抑制现象,降低了副产物的产量,提高了L-缬氨酸的转化率及产量。与原单批次发酵的工艺相比,新工艺的最终L-缬氨酸总量达到106.1 g/L,产量提高了47.4%,糖酸转化率提高到34.5%。     

由L-精氨酸发酵液制备结晶型精酮合剂

精酮合剂(L-精氨酸-α-酮戊二酸盐,AAKG)是目前国际市场上销量日益增长的一种氨基酸盐类功能制品,通常的产品形式是混合型而非结晶型。实验直接以L-精氨酸(L-Arg)发酵液为原料,通过加入α-酮戊二酸(α-KG)进行络合,制备出结晶型精酮合剂,其主要工艺步骤和控制参数如下:首先对发酵液进行絮凝和过滤,并减压浓缩至L-Arg浓度为1.0 mol/L,再等摩尔加入α-KG进行络合,进一步减压浓缩使AAKG浓度达到1.5mol/L;浓缩液以2℃/h的速率缓慢降温至10℃,恒温结晶24 h,获得粗晶体,并再在同样的条件下进行重结晶。其精酮合剂纯度为99.2%,分子中L-Arg∶α-KG的摩尔比为1∶1.02,产品总收率为62.9%。     

Arginine infusion stimulates prolactin, growth hormone, insulin, and subsequent lactation in pregnant dairy cows

L-Arginine or saline was administered intravenously by rapid infusion into 16 late-pregnant Holstein cows to study changes of prolactin, growth hormone, insulin, total protein, urea nitrogen, and subsequent lactation. Arginine was infused daily at .1 g/kg body weight starting about 7 days prior to predicted calving until calving. Blood was sampled via a jugular cannula at 0700, 0715, 0730 (infusion immediately followed 0730 h sample), 0745, 0815, 0845, 1100, 1300, 1500, 1700, and 1900 h. Arginine infusion evoked dramatic but transient increase of concentrations of blood serum prolactin, growth hormone, and insulin. Urea nitrogen also was elevated in blood serum but not total protein. The secretory response of prolactin, growth hormone, and insulin to daily arginine infusion during the entire prepartum period was not diminished. Milk production for the first 22 wk of lactation tended to be higher (by about 10%) for cows infused with arginine as compared to cows infused with saline. Therefore, repeated arginine infusion in late-pregnant cows dramatically increased prolactin, growth hormone, and insulin and tended to increase subsequent milk yield.     

Manipulating interfacial behaviour and emulsifying properties of myofibrillar proteins by L-Arginine at low and high salt concentration

The present work examined the impact of L-Arginine (Arg) on the emulsifying properties, interfacial behaviour and conformational characteristics of myofibrillar proteins (MPs) at high (0.6 m ) and low (0.15 m ) salt concentration to maintain good emulsifying properties of MPs at low salt concentration. The data indicated that Arg increased the emulsifying activity index/emulsion stability index (EAI/ESI) and decreased the CI and droplet size of emulsions regardless of salt concentration. Raman spectra revealed that the α-helix content decreased from 60.30% to 51.26% at high salt concentration, and from 60.20% to 54.82% at low salt concentration in the presence of Arg. In addition, MPs treated with Arg exhibited a higher interfacial pressure and more rapidly diffusion to the oil surface. Meanwhile, Arg increased the interfacial protein loading. The results demonstrated that Arg caused the unfolding of MPs, promoting the adsorption of proteins and decreasing the interfacial tension, ultimately improving the stability of emulsions at low salt concentration.     

Effect of timing of prepartum vaccination relative to pen change with an acidogenic diet on lying time and metabolic profile in Holstein dairy cows

The objective was to assess the effect of prepartum vaccination timing relative to pen change with an acidogenic diet at 28 or 21 d before expected parturition (dpp) on lying time (LT), prepartum serum energy status (glucose, IGF-1, and nonesterified fatty acids), urine pH, and serum Ca at calving in pregnant Holstein dairy cows. Pregnant multiparous Holstein cows (n = 308) from 1 large dairy herd were randomly allocated into 1 of 3 treatment groups at 35 ± 3 dpp as follows: (1) vaccination at 28 dpp and pen change at 21 dpp (V28PC21; n = 108), (2) vaccination and pen change at 28 dpp (V28PC28; n = 99), and (3) vaccination and pen change at 21 dpp (V21PC21; n = 101). When cows changed pens, an acidogenic diet was introduced. Every other week, a group of 43 to 53 animals were enrolled and electronic data loggers (IceQube, IceRobotics) were fitted to the hind leg of individual cows to assess their LT. Blood samples were collected at 28, 26, 21, 19, 14 dpp and at calving. Parity, body condition score, days dry, and gestation length were not different among groups. Overall, V28PC28 cows had 7 additional days in prepartum pens consuming an acidogenic diet compared with V28PC21 or V21PC21 cows. Regardless of treatment group, cows in the far-off pen had 43 min/d less LT (709 vs. 753 min/d) and increased day-to-day coefficient of variation of LT (0.21 vs. 0.10) compared with cows within the prepartum pen. On average, for the 7 d following vaccination alone (28 to 22 dpp period), V28PC21 cows had ~22 min/d less LT compared with V21PC21 cows. Serum concentrations of glucose, nonesterified fatty acids, and IGF-1 were altered following vaccination alone, pen change alone, or vaccination plus pen change with an acidogenic diet before calving. At calving, V28PC21 cows had greater glucose concentrations (6.45 mmol/L) compared with V21PC21 cows (5.76 mmol/L), with V28PC28 cows intermediate (6.11 mmol/L). The assessment of Ca status at calving revealed that V28PC21 cows had greater Ca concentration (2.34 mmol/L) with lower subclinical hypocalcemia (<2.0 mmol/L; 17.3%) compared with V21PC21 cows (2.17 mmol/L and 31.9%), with V28PC28 cows intermediate (2.28 mmol/L and 25.2%). Serum concentrations of IGF-1 at calving were also greater for V28PC21 (3.43 nmol/L) cows compared with V21PC21 (2.69 nmol/L), with V28PC28 cows intermediate (3.07 nmol/L). Overall, V28PC21 cows had greater serum glucose, IGF-1, and ~46% reduction in subclinical hypocalcemia (from 31.9 to 17.3%) compared with V21PC21 cows but did not differ from V28PC28 cows (25.2%). These findings provided evidence that vaccinating cows at 28 dpp, followed 7 d later by pen change with an acidogenic diet at 21 dpp, would be beneficial.     

Effects of prepartum vaccination timing relative to pen change with an acidogenic diet on serum and colostrum immunoglobulins in Holstein dairy cows

The objective of the present study was to assess the effects of prepartum vaccination timing relative to pen change with an acidogenic diet at 28 or 21 d before expected parturition (dpp) on colostral and serum IgG concentrations at calving in pregnant Holstein dairy cows. Pregnant multiparous Holstein cows (n = 308) from one large dairy herd were randomly allocated into 1 of 3 treatment groups at 35 ± 3 dpp: (1) vaccination at 28 dpp and pen change at 21 dpp (V28PC21; n = 108), (2) vaccination and pen change at 28 dpp (V28PC28; n = 99), and (3) vaccination and pen change at 21 dpp (V21PC21; n = 101). An acidogenic diet was fed when cows changed pens at 28 or 21 dpp. Blood and colostral samples were collected within 1 h following parturition. The total number of clinical mastitis (CM) cases within the first 150 d in milk (DIM) were recorded. The V28PC21 cows had greater colostral IgG concentrations at calving (160.4 ± 7.0 g/L) compared with V21PC21 cows (134.4 ± 7.0 g/L), and V28PC28 cows were intermediate (148.3 ± 7.2 g/L). At calving, V28PC21 cows had lower serum IgG concentrations (29.1 ± 1.2 g/L) compared with V21PC21 cows (32.2 ± 1.2 g/L) or V28PC28 cows (32.6 ± 1.3 g/L). Overall, 41% of V21PC21 cows received the booster vaccinations with at least 21 d before actual calving compared with V28PC21 or V28PC28 cows (88 and 86% respectively). The shorter the interval from prepartum booster vaccination to calving, the lower the colostral IgG at calving, regardless of treatment groups. Vaccinating at 28 dpp and pen change with an acidogenic diet at 21 dpp tended to reduce the rate of CM within the first 150 DIM compared with V21PC21. These findings provide evidence that vaccinating cows at 28 dpp, followed by pen change with an acidogenic diet at 21 dpp, improved the concentrations of colostral IgG at calving and tended to reduce the rate of CM. The interaction of prepartum vaccination timing relative to feeding an acidogenic diet should be considered when implementing an effective vaccination program to enhance overall herd health.     

离子交换法从发酵液中提取L-精氨酸

介绍了从发酵液中提取L精氨酸的工艺方法。确定了絮凝法预处理发酵液的最佳条件:聚丙烯酰胺的用量为400mg/L,发酵液pH值为10.0,温度为30℃。固定床离子交换的最佳工艺条件:上柱发酵液pH值为2.0,上柱流速为2mL/min,氨水洗脱浓度为0.1mol/L,流速为2mL/min。脱色工艺的最佳条件:活性炭用量为10g/L,脱色温度为80℃,pH值为5.0。整个提取过程中,L精氨酸的总收率为91.2%。