Partial Purification and Properties of Tea Leaf Ribonuclease

Four fractions with ribonuclease activity have been isolated from tea leaves by DEAE-cellulose column chromatography and designated as RNase Tf-1, RNase Tf-2, RNase Tf-3 and RNase Tf-4. The bigger fractions of both RNase Tf-3 and RNase Tf-4 have been partially purified by Sephadex G-100 column chromatography.

RNase Tf-3 and RNase Tf-4 were respectively found to have their optimum pH at 4.75 and 4.9 and molecular weights of approximately 13,000 and 16,000, as determined by gel filtration. Both enzymes were inhibited by Cu²⁺ and Hg²⁺, and inactivated by heating at over 50°C. By addition of yeast RNA to the two enzymes, however, their thermostabilities increased. The activities of the enzymes were stable in a pH range of 4.5 to 6.5. Like other plant RNases, RNase Tf-3 and RNase Tf-4 appeared to have no preference for base in RNA.     

大别山野生油茶中茶氨酸检测与茶氨酸合成酶基因克隆

茶氨酸是茶树叶片中最丰富的游离氨基酸,具有重要的生理药理功能,但迄今仅在蘑菇、蕈和一些山茶科(属)植物中检测到茶氨酸。茶氨酸因有一种独特的风味特色"umami鲜爽味"而被人类营养学广泛研究,并发现合成茶氨酸的植物不仅在植物分类上具有积极意义,而且对于植物资源的有效发掘有巨大经济价值;同时还可以间接去研究茶树中茶氨酸的代谢机理以及茶氨酸合成酶的分离纯化和TS基因的克隆表达。该文运用HPLC、LC-TOF/MS对大别山地区野生幼年与成年油茶根、叶中茶氨酸进行检测,并结合分子生物学手段对油茶中茶氨酸合成酶(theanine synthetase,TS)基因进行克隆与生物信息学分析。结果表明:在幼年的油茶根中检测到茶氨酸,含量为0.08mg·g-1(鲜重),而在幼年的油茶叶片和成年油茶根、叶中均未检测到茶氨酸;在幼年油茶根中克隆出一条长为1 071bp油茶TS基因开放阅读框,其基因序列与茶树谷氨酰胺合成酶(glutamine synthetase,GS,AB117934)基因和TS(DD410896)序列的同源性达到98%,氨基酸序列与茶树中GS(AB117934)和TS(DD410896)的相似性高达99%。经生物信息学分析,该序列编码的TS蛋白具有20个磷酸化位点,不存在信号肽序列与跨膜结构,含有卷曲螺旋结构的亲水性细胞质蛋白。该研究将为油茶新经济价值的发掘,为茶氨酸在油茶中合成代谢途径的研究提供一定的理论基础,同时也为进一步研究茶氨酸在茶树中代谢机理提供了新的研究思路。     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

Repeated Batch Production of Theanine by Coupled Fermentation with Energy Transfer Using Membrane-Enclosed γ-Glutamylmethylamide Synthetase and Dried Yeast Cells

γ-Glutamylmethylamide synthetase and dried baker’s yeast cells were enclosed together in a dialysis membrane tube to produce theanine repeatedly by coupled fermentation with energy transfer. The membrane-enclosed enzyme preparation (M-EEP) formed approximately 600 mM theanine from glutamic acid and ethylamine at a 100% conversion rate. M-EEP maintained its productivity of theanine during six consecutive reactions in a mixture containing NAD⁺.     

Pharmacokinetics of theanine enantiomers in rats

Theanine, first discovered in tea, is a chiral nonproteinic amino acid that has been reported to have cardiovascular, neurological, and oncological effects. It is being considered as a therapeutic/medicinal agent and additive in consumer products. The present study evaluated the pharmacokinetics of D-theanine, L-theanine, and D,L-theanine in plasma and urine using LC-ESI/MS in rats after oral (p.o.) and intraperitoneal (i.p.) administration. Oral administration data indicated that gut absorption of d-theanine was far less than that of L-theanine. However, after i.p. administration, plasma theanine concentrations of L- and D-theanine were similar. This indicated that D- and L-theanine may exhibit a competitive effect with respect to intestinal absorption. Regardless of the route of administration, p.o. or i.p., the presence of the other enantiomer always decreased theanine plasma concentrations, indicating D,L-theanine competition with respect to urinary reabsorption. Data on urinary concentrations of D-theanine suggested that the D-isomer may be eliminated with minimal metabolism. L-Theanine appeared to be preferentially reabsorbed and metabolized by the kidney while D-theanine was preferentially excreted. Clearly, the bioequivalencies of D,L-theanine and its enantiomers were found to be quite different from one another. Consequently, the efficacy of commercial theanine products containing D-theanine, L-theanine, or D,L-theanine may be quite different.     

茶树茶氨酸合成酶基因的酶活性验证与蛋白三维结构分析

针对NCBI上已登录的茶氨酸合成酶与谷氨酰胺合成酶基因序列进行克隆、原核表达与酶活性验证,利用多种生物信息学数据库和软件,对Cs TS与Cs GS基因进行结构、性质和功能预测,采用同源建模法对蛋白三维结构进行预测,比较并预测催化作用位点的差异;用系统进化树分析从裸子植物到高等被子植物的谷氨酰胺合成酶基因序列,推测其进化的演变过程;通过对原核表达的基因工程菌提取粗酶液进行酶活性测定。结果表明:尽管茶树TS与GS序列高度同源,但是原核表达后的融合蛋白仍然显示了不同的催化能力,蛋白一、二级结构分析显示Cs TS与Cs GS差异不大,但是通过同源建模形成的蛋白三级结构分析显示,Cs TS与Cs GS存在3个催化位点上的差异,这可能是导致其酶活性差异的关键。系统进化分析结果首次确定茶氨酸合成酶应为谷氨酰胺合成酶基因家族成员,按照其细胞定位预测应为胞质型GS,其亲缘关系与同为双子叶植物的葡萄、陆地棉、巴西橡胶树、拟南芥较接近。     

茶氨酸提取纯化工艺研究

系统研究了从茶多酚工业废液中提取纯化茶氨酸的工艺。采用絮凝、吸附、阳离子树脂交换、重结晶工艺来分离纯化茶氨酸。结果表明,絮凝能有效的去除茶多酚工业废液中的蛋白质等杂质,杂质的去除率为50％;吸附能进一步去除色素、多酚类物质及大分子有机物;阳离子交换树脂能较专-吸附氨基酸。茶多酚工业废液经絮凝→吸附→阳离子树脂交换工艺可得纯度50%的茶氨酸,得率为1．8％;通过重结晶可得到纯度90％的茶氨酸,得率为0．8%。     

茶氨酸制备工艺研究

研究了一种从提取茶多酚后的残留液中提纯茶氨酸的制备工艺;首先通过ZTC+1型天然澄清剂对茶多酚生产废液絮凝处理,随后应用一种特制的弱极性大孔树脂(JAD-1000)对处理液进行初步分离,制备质量分数在50%以上的茶氨酸粗品;结果表明絮凝处理,不仅对蛋白质去除率达80%以上,对果胶等杂质也有很好的去除效果(去除率达32.0%),杂质总去除率达36.2%,同时对茶氨酸的回收率达到98%以上;JAD-1000初步分离可得质量分数在53%以上的茶氨酸,回收率也达到78%以上,醇洗馏份中基本没有茶氨酸。     

Impact of nitrogen supply on carbon/nitrogen allocation: a case study on amino acids and catechins in green tea [Camellia sinensis (L.) O. Kuntze] plants*

The concentrations of free amino acids (AA) and polyphenols (PP) are important determinants of green tea quality. Levels of AA and PP are governed interactively by nitrogen (N) supply and carbon (C) status, so the impact of C/N allocation on green tea quality was investigated in saplings cultivated hydroponically with 0.3, 0.75, 1.5 or 4.5 mmol l^?1 N. Activities of glutamine synthetase (GS), phenylalanine ammonia lyase (PAL), and phosphoenolpyruvate carboxylase (PEPC) were determined, as were concentrations of AA, PP and soluble sugars. Concentrations of AA increased with increasing N supply, and the AA profile was shifted towards AA characterised by low C/N ratios (arginine, glutamine) and away from theanine, the unique non‐protein AA that is abundant in Camellia sinensis. High N supply significantly reduced the concentrations of PP in young shoots, and was accompanied by lower levels of carbohydrates (soluble sugars). Analysis of the C and N status and selected enzyme activities, combined with path coefficient analysis of variables associated with C and N metabolism, demonstrated increasing deviation of C flux to AA under abundant N supply. Accumulation of AA and PP depended strongly on N status, and the balance shifted toward increasing synthesis of AA associated with enhanced growth, while investment of C in secondary metabolites did not change proportionally under the condition of ample N supply.     

离子交换法提取茶氨酸的研究

本研究应用国产７３２阳离子交换树脂,从茶愈伤组织浸提液中提取茶氨酸。通过静态、动态试验,对洗脱液的ｐＨ、离子强度、样液浓度和流速等因素进行了考察,比较在不同条件下,树脂对茶氨酸的吸附情况。结果表明用ｐＨ９．１８,２／１５ｍｏｌ／ＬＮａ２ＨＰＯ４作洗脱液,控制流速在０．７ＢＶ／ｈ,可分离到茶氨酸,得率７０％。