国产717阴离子树脂对酶法合成 L-谷氨酰胺的分离与纯化

目的:从酶法反应液中分离和纯化谷氨酰胺,并为工业化生产谷氨酰胺提供一定的依据;方法:根据谷氨酸和谷氨酰胺电离常数的差异,采用国产717强碱性阴离子Cl-型拄,经预处理.离交分离,浓缩结晶,75%乙醇洗涤并干燥;结果:反应液中谷氨酰胺总提取收率为60.3%,纯度为97%;结论:此方法能有效地降低纯化的成本,并维持较高得率和纯率.     

谷氨酸棒杆菌中谷氨酰胺合成酶的克隆表达及固定化

对谷氨酸棒杆菌(C.glutamicum)来源的谷氨酰胺合成酶进行腺苷酰化位点定点突变,并在大肠杆菌中进行异源表达,得到的重组酶活为6.215 U/mg。分离纯化重组突变酶后,对其固定化条件及固定化酶的性质进行研究。结果得到固定化条件为:以LX-1000EP树脂作为固定载体,载体量0.176 g/U、p H值8.0、温度30℃、吸附时间16 h。固定化酶活力达到3.658 U/g,酶活回收率达到67.17%。重组酶固定化后,反应最适温度没有变化,最适p H略向碱性偏移,储藏稳定性提高,转化谷氨酸生产谷氨酰胺的水平与游离酶相当,对50 mmol/L谷氨酸的转化率为92.83%,为酶法生产谷氨酰胺后续研究提供了参考。     

毛细管电泳法检测转化液中谷氨酰胺和谷氨酸

建立了适用于发酵液、微生物/酶法氨基酸转化液、食品及血液样品中谷氨酰胺和谷氨酸分离分析的芴甲基氯甲酸酯(Fmoe-C1)衍生、毛细管电泳方法.采用通过优化得到的最佳衍生条件,以扁桃酸为内标,0.1mol/L硼酸,硼砂缓冲液(pH8.0)为电泳缓冲液,在分离电压15kV下,于16min实现了内标、两种氨基酸的良好分离;谷氨酰胺和谷氨酸保留时间RSD分别为0.81%和1.14%,峰面积RSD分别为0.71%和0.79%;检测线分别为86μtmol/L和169μmol/L.应用该法测定微生物转化液中谷氨酰胺和谷氨酸的加样回收率分别为96.7%～99.3%和95.9%～97.9%.     

L-谷氨酰胺的发酵生产方法

本文对L-谷氨酰胺(L-Gln)用发酵生产的三种方法作了综述.即采用野生型谷氨酸产生菌通过改变发酵条件来生产谷氨酰胺,采用各种谷氨酸生产菌的变异菌株和酶法生产.     

小麦麸皮阿魏酸的制备

本文以小麦麸皮为原料,采用碱法和酶法制备阿魏酸,通过正交试验确定出制备阿魏酸的最佳工艺参数,碱液浸提法阿魏酸最高释放量为3.08mg/g麦麸,最优组合是碱液浓度为1%,温度为80℃,提取时间为4h,麸皮粒度为80目;酶法提取的最高释放量为3.35mg/g麦麸,最优组合是料液比1∶20,混合酶浓度1%,混合酶作用时间3h。另外,碱处理后的反应液颜色为深棕色,褐变较为严重;混合酶处理后反应液颜色较浅。酶法提取效果较佳。     

解淀粉芽孢杆菌SWJS22固体发酵产谷氨酰胺酶的优化研究

以南海海泥中筛选出来的解淀粉芽孢杆菌SWJS22(Bacillus amyloliquefaciens SWJS22)为菌种,优化固体发酵产谷氨酰胺酶的工艺。选择发酵条件(接种量、发酵时间、发酵温度)和发酵培养基(淀粉原料、料水比、产酶诱导剂)进行单因素试验,通过正交试验对料水比、产酶诱导剂、接种量进行进一步优化,将谷氨酰胺酶活力从(83.10±4.64)U/mg干基提高到了(2 690.02±28.80)U/mg干基。优化后的发酵条件为:接种量2.0%(V/m),温度37℃,时间48h;优化后的发酵培养基为:豆粕20g,小麦粉5g,蔗糖脂肪酸酯(SE-1170)0.02g,料水比1.0∶0.6(m∶m)。对固体曲料中的谷氨酰胺酶进行提取工艺的优化,在料液比为1∶8(m∶V)、37℃下摇床提取1h最优。将粗酶液与同等酶活力的商业酶对谷氨酰胺进行酶解,通过测定谷氨酸和谷氨酰胺的含量变化,验证了粗酶液将谷氨酰胺转化成谷氨酸的能力较强。     

L-谷氨酰胺对提取有什么影响?

在生产实践中,发酵液含L-谷氨酰胺0.25%时,就会明显地影响谷酸酰的提取,L-谷氨酰胺含量越高,谷氨酸结晶β-型的出现就越严重,所以应积极控制L-谷氨酰胺的生成。它来源于L-谷氨酸发酵过程,当谷氨酸形成时,在NH4+1过量情况下,及pH值低时(尤其是pH在5.5～     

酶法提取蛋壳膜中的角蛋白

以鸡蛋蛋壳膜为原料,酶法提取蛋壳膜中角蛋白的研究,对酶的用量、提取时间、提取温度、提取的最佳pH值、反应的固液比进行了探讨,在最佳工艺条件下:酶用量为蛋膜量的3%(W/W),固液比1:15,pH9～10,提取温度55～65℃,提取时间60min,角蛋白产率可达到26%。     

正交试验优化谷氨酰胺酶改性米谷蛋白工艺

研究谷氨酰胺酶对米谷蛋白改性的工艺。以脱酰胺度、溶解度为考察指标进行了工艺条件的优化,探讨谷氨酰胺酶与米谷蛋白质量比、反应温度、反应pH值3个工艺参数对改性米谷蛋白溶解度及脱酰胺度的影响,确定了谷氨酰胺酶改性最佳工艺条件,用正交试验法对米谷蛋白酶法改性工艺条件进行了优化,得到最佳工艺条件为:谷氨酰胺酶与米谷蛋白的质量比1:7、酶解脱酰胺改性的反应温度37.0℃、时间24h、pH7.0。优化后的米谷蛋白脱酰胺度为52.76%,溶解度为93.78%。     

NCgl1221敲除和pyc过表达对谷氨酰胺发酵的影响

谷氨酰胺在生命活动中具有重要的作用,已被广泛应用于食品、医药等诸多领域。谷氨酸是谷氨酰胺生物合成的前体物质,也是谷氨酰胺生产过程中最常见的副产物。研究发现,NCgl1221基因的编码蛋白是谷氨酸分泌的重要载体,丙酮酸羧化酶是谷氨酸棒杆菌回补途径中的关键酶。为减少谷氨酰胺发酵过程中谷氨酸的积累量并提高谷氨酰胺产量,本研究利用同源重组技术敲除谷氨酰胺生产菌GM34的谷氨酸分泌载体编码基因NCgl1221,获得GM34ΔNCgl1221菌株;构建了丙酮酸羧化酶基因pyc过表达菌株GM34-pXMJ19pyc。5 L罐发酵实验表明,NCgl1221基因缺失可以使得谷氨酸积累量降低19.05%,过表达pyc基因使得谷氨酰胺产量和转化率分别提高5.54%和2.37%。可见,敲除NCgl1221、过表达pyc能够有效降低谷氨酸分泌并提高谷氨酰胺产量。     

谷氨酰胺的分离纯化研究

L-谷氨酰胺是人体内最重要的游离氨基酸，它既是一种重要的生化制剂，也是一种极有前途新药，目前已作为一种新型的饲料添加剂备受关注。发酵法生产谷氨酰胺也正成为国内外的研究热点。本文综述了从发酵液中提取纯化L-谷氨酰胺的多种工艺方法，为开展L-谷氨酰胺的工业化生产提供了一定的依据。     

Effect of glutamine supplementation on splanchnic metabolism in lactating dairy cows

The suggestion that glutamine (Gln) might become conditionally essential postpartum in dairy cows has been examined through increased postruminal supply of Gln. Net nutrient flux through the splanchnic tissues and mammary gland was measured in 7 multiparous Holstein cows receiving abomasal infusions of water or 300 g/d of Gln for 21 d in a crossover design. Milk yield increased significantly (by 3%) in response to Gln supplementation, but the 2.4% increase in milk protein yield was not statistically significant. Glutamine treatment had no effect on portal or hepatic venous blood flows. Net portal appearance of Gln and Glu was increased by Gln supplementation, accounting for 83% of the infused dose with, therefore, only limited amounts available to provide additional energy to fuel metabolism of the portal-drained viscera. The extra net portal appearance of Gln was offset, however, by a corresponding increase in hepatic removal such that net Gln splanchnic release was not different between treatments. Nonetheless, the Gln treatment resulted in a 43% increase in plasma Gln concentration. Infusions of Gln did not affect splanchnic flux of other nonessential amino acids or of essential amino acids. Glutamine supplementation increased plasma urea-N concentration and tended to increase net hepatic urea flux, with a numerical increase in liver hepatic O₂ consumption. There were no effects on glucose in terms of plasma concentration, net portal appearance, net liver release, or postliver supply, suggesting that Gln supplementation had no sparing effect on glucose metabolism. Furthermore, mammary uptake of glucose and amino acids, including Gln, was not affected by Gln supplementation. In conclusion, this study did not support the hypothesis that supplemental Gln would reduce glucose utilization across the gut or increase liver gluconeogenesis or mammary glutamine uptake to increase milk protein synthesis.     

赖氨酸与谷氨酰胺在谷氨酰胺转胺酶作用下的交联反应

本文研究了赖氨酸与谷氨酰胺在谷氨酰胺转胺酶催化下的交联反应。探讨了底物比、加酶量、pH值、反应温度和反应时间对聚合度的影响。利用Design-Expert6.0.3软件优化得出交联反应的最佳工艺条件为底物比2.15、加酶量14.39U/g、pH值8.0、温度42℃和反应时间94min。同时,分析了各因素之间的交互效应。     

环糊精葡萄糖基转移酶分离纯化及酶学性质研究

通过筛选和诱变获得一株高产环糊精葡萄糖基转移酶芽孢杆菌菌株,研究该菌所产环糊精葡萄糖基转移酶的分离纯化及酶学性质,结果显示：发酵液经离心处理后,采用硫酸铵溶液分步盐析、DEAE-cellulose 52 离子交换层析、Sephadex G-200 凝胶过滤层析方法得到电泳级环糊精葡萄糖基转移酶,SDS-PAGE 电泳显示该酶分子量为33kD,纯化倍数为10,得率为14.4%。该酶反应的最适温度为50℃,在40～60℃基本稳定,最适pH 值为8.0,在pH6.0～10.0 范围内基本稳定。Fe2+、Cu2+、Mg2+ 对该酶活力有明显的抑制作用。     

Stability of Glutamine and Pyroglutamic Acid under Model System Conditions: Influence of Physical and Technological Factors

The stability of glutamine and pyroglutamic acid as regards pH, temperature and oxygen was studied during storage and two thermal processings, the residual products and those which appeared being determined. Contrary to glutamic acid, the two derivatives are labile below pH 2 and above pH 13 and alteration occurs much faster. Temperature increase promotes this phenomenon. Glutamic and pyroglutamic acids convert reversibly into one another, more or less quickly and completely according to the conditions. Glutamine changes during heating in two steps, first into glutamic acid, then into pyroglutamic acid if the pH values are not extreme. The influence of pH conditions on the direction and the rate of the reaction are indicated.     

酸解-酶膜联用法检测谷氨酰胺

研究了谷氨酰胺在酸性条件下的水解特性后 ,建立了酸解 酶膜联用的谷氨酰胺定量分析方法。此方法的线性范围为 10～ 40mmol/L ,相关系数R =0 .999,分析误差为 0 .1mmol/L ,测量所需时间约为 30min。     

急性热应激条件下肉鸡骨骼肌谷氨酰胺代谢的变化

研究急性热应激对肉鸡骨骼肌（胸肌和腿肌）谷氨酰胺（Gln）代谢和血清相关指标的影响。选择常规饲养至35日龄（d）的公AA鸡50只,预饲至42 d时,选取30只随机分成5组（每组6只）,分别进行0（Ⅰ组,室温对照组）、4（Ⅱ组）、8（Ⅲ组）、12（Ⅳ组）、16（Ⅴ组）h的急性热应激处理,采血清和骨骼肌样分析。结果表明：与对照组相比,急性热应激显著（P<0.01或0.05）降低了血清的TP、ALB、GLOB、A/G、Gln、ALP,增加了血清Glu、GOT、GPT、CK,影响程度均随着时间的延长而增加。对照组中,腿肌的CP、Gln、Glu、GS高于胸肌（P<0.01或0.05）,Glnase低于胸肌（P<0.01）。急性热应激对骨骼肌CP、Gln、Glu和Glnase有降低作用,对GS有增加作用,在处理16 h时均有显著以上差异。试验说明,34℃急性热应激对肉鸡血清和骨骼肌中蛋白质和Gln代谢产生了显著的影响,且存在明显的组织特异性和时间依赖性。     

谷氨酰胺和甘氨酰-谷氨酰胺对断奶仔猪小肠黏膜的影响

选用21日龄断奶的杜大长仔猪108头,按试验要求分为3组,每组3个重复,每重复12头.分别饲喂含1%谷氨酰胺（Gln）和0.3%的甘氨酰-谷氨酰胺（Gly-Gln）的日粮,试验期14天,在断奶前、断奶后7天和14天分别屠宰取样.试验结果表明：（1）断奶后7天,日粮中添加Gln和Gly-Gln可显著提高小肠各段绒毛的高度,降低隐窝深度,断奶后14天添加Gly-Gln可显著提高十二指肠、回肠绒毛高度.（2）断奶后7天和14天,添加Gln和Gly-Gln均可显著提高空肠黏膜蔗糖酶和麦芽糖酶活性,对乳糖酶活性无显著影响.（3）断奶后7天,添加Gln和Gly-Gln可降低血浆二胺氧化酶（DAO）活性,提高空肠黏膜DAO活性,断奶后14天,添加Gly-Gln可显著提高空肠黏膜DAO活性.     

Distribution of stable free radicals among amino acids of isolated soy proteins

Application of deuterium sulfide to powdered isolated soy proteins (ISP) was used to quench stable free radicals and produce a single deuterium label on amino acids where free radicals reside. The deuterium labels rendered increases of isotope ratio for the specific ions of radical-bearing amino acids. Isotope ratio measurements were achieved by gas chromatography/mass spectrometry (GC/MS) analyses after the amino acids were released by acidic hydrolysis and converted to volatile derivatives with propyl chloroformate. The isotope enrichment data showed the stable free radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp but not on Val, Pro, Met, Phe, Lys, and His. Due to the low abundance of Ser, Thr, and Cys derivatives and the impossibility to accurately measure their isotope ratios, the radical bearing status for these amino acids remained undetermined even though their derivatives were positively identified from ISP hydrolysates. The relative isotope enrichment for radical-bearing amino acids Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp were 8.67%, 2.96%, 2.90%, 3.94%, 6.03%, 3.91%, and 21.48%, respectively. Isotope ratio increase for Tyr was also observed but further investigation revealed such increase was mainly from nonspecific deuterium-hydrogen exchange not free radical quenching. The results obtained from the present study provide important information for a better understanding of the mechanisms of free radical formation and stabilization in "dry" ISP.     

不同来源谷朊粉谷氨酰胺肽释放特性的比较分析

从国内谷朊粉生产厂家收集了12种商品谷朊粉,在对其进行基本组成分析基础上,研究了不同来源谷朊粉的酶解及谷氨酰胺肽释放特性。结果表明：所收集谷朊粉样品的蛋白质平均含量为77.59%,样品间差异较小（CV:2.68%）;淀粉含量差异较显著（CV:21.65%）;脂肪含量虽然较低（平均1.08%）,但样品间差异较大（CV:69.60%）;氨基酸组成中谷氨酸/谷氨酰胺平均含量较高,为412.58 mg/g蛋白,占总氨基酸组成的39.38%。综合考虑不同样品酶解产物中蛋白回收率、水解度及谷氨酰胺含量三个指标,以获得高谷氨酰胺含量的短肽为目的,最终确定1号（山东滨州）、9号（陕西宝鸡）、12号（河北邢台）样品为制备谷氨酰胺肽的理想原料,这三种样品的酶解产物中有效谷氨酰胺含量可达22%左右,水解度大于15%,蛋白回收率大于90%。