天冬酰胺酶胞外表达的初步研究

[目的]为降低L-天冬酰胺酶Ⅱ的生产成本提供依据。[方法]通过PCR反应扩增大肠杆菌JM109成熟区的L-天冬酰胺酶II基因(ansB),将扩增片段与pMD18-T载体连接,构建重组质粒pMD18-T-asp。对pMD18-T-asp与质粒pET-22b进行双酶切后,将它们连接起来,转化感受态细胞,筛选重组表达载体pET-22b-asp。利用重组表达载体pET-22b-asp转化BL21(DE3),并对其发酵条件以及分离、纯化工艺进行了初步研究。[结果]成功克隆长度为960 bp的L-天冬酰胺酶II基因。重组表达载体pET-22b-asp也构建成功,并在BL21中得到表达。在重组菌接入TB培养基后2 h加入IPTG、培养基的初始pH值为7.2、装液量为12%,L-天冬酰胺酶Ⅱ酶活最高。重组天冬酰胺酶的纯化收率为82%,比活力达196 U/mg。[结论]重组天冬酰胺酶的胞外表达大大简化了其分离步骤。     

四种微生物产L-天冬酰胺酶及其发酵条件初探

L-天冬酰胺酶即L-天冬酰胺基水解酶,对白血病和淋巴瘤有很好的抑制作用.从发酵pH值、发酵温度以及添加葡萄糖等方面对L-天冬酰胺酶的发酵条件进行了探讨.结果发现,大肠杆菌、变形杆菌、金黄色葡萄球菌及枯草芽孢杆菌4种微生物中产酶最好的为大肠杆菌,大肠杆菌在pH值为7.0、温度为37℃条件下发酵产L-天冬酰胺酶效果最好,牛肉膏蛋白胨培养基以不加葡萄糖为宜.     

胞外表达L-天冬酰胺酶发酵条件的研究

[目的]优化胞外表达L-天冬酰胺酶工程菌的发酵条件。[方法]用正交试验研究培养基的组成,用单因素试验研究诱导条件以及通气量对酶产量的影响。[结果]发酵培养基采用8.0%玉米浆和1.0%甘油组成;诱导剂采用终浓度为0.5g/L的乳糖,扩大培养后10h加入,诱导时间为12h,通气量为0.83VVM。[结论]该研究为胞外表达L-天冬酰胺酶的工业化生产提供了参考。     

不同培肥措施对大豆不同生育时期复垦土壤氮代谢相关酶活性的影响

为了解有机肥、化肥、有机肥与化肥配施对复垦土壤氮代谢相关酶活性的影响并探索其作用机制,以大豆为供试作物进行大田试验,采用单因素完全随机设计,共设置4个处理(对照(CK)、化肥(CF)、有机肥(M)、有机肥+化肥(MCF)),研究不同培肥措施对土壤硝酸还原酶、亚硝酸还原酶、天冬酰胺酶、谷氨酰胺酶、蛋白酶、脲酶、几丁质酶、脱氢酶8种酶活性的影响。结果表明,在大豆整个生育期各施肥处理均可有效提高土壤中氮代谢相关酶活性,其中,MCF处理的硝酸还原酶、亚硝酸还原酶、天冬酰胺酶、谷氨酰胺酶、蛋白酶、脲酶、脱氢酶活性均最高,较CK提高了7.60%～322.03%;几丁质酶活性为M处理最高,比CK提高了37.70%～51.46%;土壤硝酸还原酶和亚硝酸还原酶活性生育期内变化趋势为成熟期苗期开花结荚期;土壤天冬酰胺酶、谷氨酰胺酶、蛋白酶、脲酶、几丁质酶和脱氢酶活性则随着大豆生育时期的推进而呈现递增趋势。     

植物氮代谢中天冬酰胺合成酶B的研究进展

天冬酰胺合成酶B(Asparagine synthetase B,AS-B,EC 6.3.5.4)是植物氮代谢中的关键酶,在植物氮素同化与分配上发挥着重要作用,其产物天冬酰胺(Asn)是植物体内有机氮运输和储存的主要形式之一。近年来,对AS-B的研究取得很大进展,本文就AS-B的结构、分类、反应机制、表达模式以及生物学功能进行全面的阐述,为利用分子育种方法提高作物或林木的氮素利用率奠定理论基础。     

培养条件对大肠杆菌产L-天冬酰胺酶的影响

研究了培养温度、培养时间、初始pH值和摇床转速等培养条件对所选大肠杆菌（E.coli No TN1.5885）产L-天冬酰胺酶的影响。实验结果表明,该菌株进行摇瓶培养的最适培养条件为37℃下培养16h,初始pH值在7.0-7.5间,摇床转速约为300r／min。其比酶活力约为406U／g,比优化前大约提高了20％。     

响应面法优化紫红红球菌的发酵培养基

采用响应面法优化腈水合酶产生紫红红球菌的发酵培养基。以天冬酰胺、磷酸氢二钾、磷酸二氢钾、酵母抽提物、脲素、氯化钴为影响因子,以菌体干质量和酶活为响应值,进行多元二次响应回归分析,结果显示,最优培养基为:天冬酰胺含量0.04%、磷酸氢二钾0.05%、磷酸二氢钾0.03%、酵母抽提物(FM818)5.48%、脲素0.55%、氯化钴0.01%;在摇瓶中进行验证试验,优化条件下菌体干质量为33.98mg·mL-1、酶活为167.57U·mL-1;对照条件下菌体干质量26.6mg·mL-1、酶活为125.63U·mL-1,此优化培养基使干质量、酶活分别提高了27.74%和33.38%。     

普鲁兰酶的反向合成特性

对普鲁兰酶催化反向合成反应的底物特异性进行了研究.研究结果表明,麦芽糖是普鲁兰酶催化反向合成反应的最小底物.以麦芽糖为底物分子,研究了普鲁兰酶的反向合成特性,结果表明,普鲁兰酶催化反向合成反应的最适pH值为4.5,反向合成活性在pH值4.0 ～6.0范围内稳定;普鲁兰酶催化反向合成反应的最适温度为65℃,反向合成活性在60～ 70℃范围内较高;Na+、K+、Li+和Co2+对普鲁兰酶反向合成活力的影响不大,Hg2+、Sn2+、Cu2+、Ag+和pb2+对普鲁兰酶反向活力有较强的抑制作用,Ca2+、Mn2+对反向活力有较为明显的激活作用,Ca2+可以提高热稳定性.     

蝴蝶兰ACC合成酶基因片段的克隆及其反义表达载体的构建

［目的］克隆蝴蝶兰ACC合成酶cDNA片段,构建其反义表达载体。［方法］根据已报道的蝴蝶兰ACC合成酶的基因序列,设计并合成了1对引物,通过RTPCR以蝴蝶兰总RNA中扩增出ACC合成酶的cDNA片段,将其连接到MD19T质粒载体上进行测序。用XbaⅠ和SacⅠ对重组质粒和pBI221酶切后连接,构建蝴蝶兰ACC合成酶的反义基因表达载体。［结果］获得了蝴蝶兰ACC合成酶的cDNA片段,测序结果显示,该片段与参考的已报道序列具有98.92%和76.36%的同源性。通过引入XbaⅠ和SacⅠ酶切位点,进行载体构建,构建了蝴蝶兰ACC合成酶的反义基因表达载体。［结论］成功构建了蝴蝶兰ACC合成酶的反义基因植物表达载体,为进一步研究其对蝴蝶兰花期和生长的影响打下了基础。     

赤豆种子萌发中酰脲累积动态及若干碳氮化合物对其形成的影响

赤豆种子萌发中,幼苗迅速合成酰脲,萌发第4d达到最大值.谷氨酰胺和甘氨酸促进尿囊素合成,谷氨酸和天冬酰胺对尿囊素合成影响较小.外源铵促进幼苗合成尿囊素.三羧酸循环的部分中间产物(特别是琥珀酸或延胡索酸)或高浓度的乙醛酸(6-10 mmol/L)不同程度地抑制幼苗的尿囊索合成.     

Genome-wide identification and expression analysis of asparagine synthetase family in apple

Asparagine is an efficient nitrogen transport and storage carrier. Asparagine synthesis occurs by the amination of aspartate which is catalyzed by asparagine synthetase(ASN) in plants. Complete genome-wide analysis and classifications of the ASN gene family have recently been reported in different plants. However, systematic analysis and expression profiles of these genes have not been performed in apple(Malus domestica). Here, a comprehensive bioinformatics approach was applied to identify MdASNs in apple. Then, plant phylogenetic tree, chromosome location, conserved protein motif, gene structure, and expression pattern of MdASNs were analyzed. Five members were identified and distributed on 4 chromosomes with conserved GATase-7 and ASN domains. Expression analysis indicated that all MdASNs mRNA accumulated at the highest level in reproductive organs, namely flowers or fruits, which may be associated with the redistribution of free amino acids in plant metabolic organs and reservoirs. Additionally, most of Md ASNs were dramatically up-regulated under various nitrogen supplies, especially in the aboveground part. Taken together, MdASNs may be assigned to be responsible for the nitrogen metabolism and asparagine synthesis in apple.     

Tertiary structure is a principal determinant to protein deamidation

The protein deamidation process involves the conversion of the amide side-chain moieties of asparagine and glutamine residues to carboxyl groups. This conversion is an unusual form of protein modification in that it requires catalysis by an intramolecular reaction where both the substrate (asparagine and glutamine side chains) and "catalytic site" (the peptide nitrogen of the succeeding residue) are constituents of several consecutive residues along the polypeptide chain. The stereochemical factors governing this process were studied with a data base derived from the neutron crystallographic structure of trypsin from which amide groups and oxygen can be unambiguously differentiated because of their different neutron scattering properties. The neutron structure allowed for the direct determination of those residues that were deamidated; 3 of 13 asparagine residues were found to be modified. These modified residues were clearly distinguished by a distinct local conformation and hydrogen-bonding structure in contrast to those observed for the other asparagine residues. No correlation was found between preference to deamidate and the chemical character of residues flanking the site, as had been proposed from previous peptide studies.     

Precision phenotyping of contrasting potato(Solanum tuberosum L.) varieties in a novel aeroponics system for improving nitrogen use efficiency: In search of key traits and genes

With increasing population, degrading soil health, limited arable land area, and high cost of nitrogen(N) fertilizers, improving nitrogen use efficiency(NUE) of potato is an inevitable approach to save the environment and achieve sufficient tuber yields with less N fertilizer supply. Recently, we have developed an aeroponics system to study NUE in potato using genomics, physiology, and breeding approaches. This study aims on precision phenotyping of plants of two distinct potato varieties(Kufri Gaurav, N efficient; Kufri Jyoti, N inefficient) in the novel aeroponics system. Plants were grown in aeroponics under controlled conditions with low N(0.75 mmol L~(-1) NO_3~-) and high N(7.5 mmol L~(–1) NO_3~-) levels. Plant biomass, root traits, total chlorophyll content, and plant N were increased with increasing N supply, whereas higher NUE parameters namely NUE, agronomic NUE(Ag NUE), N uptake efficiency(NUp E), harvest index(HI), and N harvest index(NHI) were observed at low N. An NUE efficient cv. Kufri Gaurav showed higher tuber dry weight, fresh tuber yield, tuber number per plant, early start of tuber harvesting, root traits, stolon traits, NUE parameters, and higher amino acid(aspartic acid and asparagine) content at low N supply. Higher expression of nitrate reductase(NR), nitrite reductase(NIR), and asparagine synthetase(AS) genes was observed in the leaf tissues of Kufri Gaurav at high N. Thus, aeroponics-based precision phenotyping enables identification of NUE efficient genotypes based on key traits and genes involved in improving NUE in potato. Further, this study suggests that the potential of aeroponics can be utilized to investigate N biology in potato under different N regimes.     

普通玉米和超甜玉米苗期蔗糖合成酶与磷酸蔗糖合成酶的活力比较

以普通玉米和超甜玉米为试材，比较两玉米中在苗期的蔗糖合成酶与磷本蔗糖合成酶的酶活力变化，结果表明，从胚芽期到五叶期，超甜玉米和普通玉米的蔗糖合成酶，磷酸蔗糖合成酶的活力逐渐升高，并且超甜玉米中这两种酶的酶活力都高于普通玉米。     

有机添加物对小麦成熟胚愈伤组织分化特性的影响

为了提高小麦成熟胚愈伤组织的分化率,以7个小麦品种(系)为试材探究不同质量浓度谷氨酰胺、天门冬酰胺及维生素B1对小麦成熟胚愈伤组织分化的影响。结果显示,谷氨酰胺和天门冬酰胺对小麦成熟胚愈伤组织的分化均有正向效应,低质量浓度的维生素B1可有效促进愈伤组织的分化,高质量浓度则抑制分化。促进效应从高到低依次为:天门冬酰胺、谷氨酰胺、维生素B1,最优组合中添加的质量浓度分别为:谷氨酰胺200mg/L、天门冬酰胺250mg/L、维生素B15mg/L。因此,在小麦成熟胚愈伤诱导培养基中添加适量的谷氨酰胺,天门冬酰胺及维生素B1可以更好地促进愈伤组织的分化。     

Glycine inhibition of asparaginase

Administration of either Escherichia coli asparaginase or guinea pig serum to C3H/HE mice with the 6C3HED lymphosarcoma is followed by depression of glycine in the tumor. This decrease in cellular glycine concentration does not occur in a tumor resistant to asparaginase. The inhibition of the lymphosarcoma by asparaginase can be reversed by intraperitoneal injection of asparagine or glycine. This reversal appears to be specific because lysine, threonine, serine, and aspartic acid were ineffective. Loss of cellular glycine may be more important than loss of asparagine because of the requirement for glycine in purine synthesis.     

嫁接对烤烟氮代谢关键酶活性及叶绿素含量的影响

以金星6007为砧木,农大202为接穗,比较盆栽试验下不同施氮水平对烤烟嫁接苗和接穗自根苗的烟叶干质量、氮代谢关键酶(硝酸还原酶和谷氨酰胺合成酶)活性和叶绿素含量的影响。结果表明,烟叶干质量、硝酸还原酶活性、谷氨酰胺合成酶活性和叶绿素含量在两种氮水平下均为嫁接苗大于自根苗,且差异极显著。随着施氮量的降低和生育期的推进,嫁接苗和自根苗氮代谢关键酶活性和叶绿素含量都下降,但降幅不同,嫁接苗的降幅较低。以上结果说明,低氮水平下较高的硝酸还原酶活性、谷氨酰胺合成酶活性和叶绿素含量与烤烟嫁接苗的氮效率增强有关。     

不同氨基酸对At.f菌生长情况的影响

通过在不同pH条件下培养At.f菌,并监测其生长情况,以确定At.f菌生长的最适pH,再向所确定的最适pH的9K培养基中添加不同氨基酸,研究不同氨基酸对At.f菌生长情况的影响。结果表明：pH为2.0的培养基中菌种的生长情况最好;另外丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、脯氨酸、酪氨酸、甘氨酸、半胱氨酸对Fe^2＋的利用率较高,而丝氨酸、天冬酰胺、谷氨酰胺、组氨酸作用结果则相反。从而得出结论：At.f菌生长的最适pH为2.0;非极性氨基酸、成环氨基酸对At.f菌生长有促进作用,丝氨酸、天冬酰胺、谷氨酰胺、组氨酸对At.f菌生长有抑制作用。     

Evidence from cassette mutagenesis for a structure-function motif in a protein of unknown structure

The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes. Evidence is presented that an enzyme of unknown structure [Ile-transfer RNA (tRNA) synthetase] may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase). This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis. Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine. Few amino acid substitutions appear to be tolerated in this region. However, one substitution (independently isolated twice) increased the Michaelis constant Km for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the Km for adenosine triphosphate, the apparent Km for tRNA, or the rate constant kcat.     

不同品质类型春玉米子粒灌浆过程中功能叶片蔗糖代谢酶的研究

以不同品质类型春玉米为研究材料,研究其灌浆过程中功能叶片蔗糖代谢酶变化与蔗糖的关系及不同品种之间的区别.结果表明:在整个灌浆期功能叶片的蔗糖代谢酶都呈单峰曲线变化,蔗糖合成酶和磷酸蔗糖合成酶都影响着叶片中蔗糖的积累,使功能叶片中的蔗糖含量也呈单峰曲线变化.但不同品种之间又有着一定的差异:东农早甜中的蔗糖合成与磷酸蔗糖合成酶的关系比东农早粘、四单19要密切,而四单19在灌浆期功能叶片中的蔗糖含量与蔗糖合成酶的关系比东农早甜和东农早粘要密切.东农早粘与蔗糖合成酶和磷酸蔗糖合成酶都关系密切.但总体上,磷酸蔗糖合成酶在光合作用中起着主导作用.