Phytase Activity of Plant Roots

Experiments were conducted to study the phytase activity ofpea, gram, wheat, and barley plant roots grown under sterileand unsterile conditions. The results indicate substantial phytaseactivity of the plant roots studied. The substrate concentrationappeared to affect the activity. Plants grown under sterileconditions exhibited less phytase activity as compared withplants grown in rich garden soil.     

Engineering of phytase for improved activity at low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

植酸酶基因工程研究进展

植酸酶是催化植酸及植酸盐水解成肌醇和无机磷酸的一类酶的总称.添加于食品和饲料中,能消除植酸所引起的抗营养作用,可提高蛋白质和矿物质的生物利用率.对植酸酶的生物学特性、基因结构和基因工程的研究进展做了综述.     

植酸酶及其植物基因工程

综述了植酸酶的来源、其酶学性质及植酸酶的植物摹因工程,并对其存在的问题、应用前景作了展望。     

植酸酶的分子生物学与基因工程

植酸酶是一种新型的、可作为动物饲料添加剂的重要酶制剂,它对提高饲料中磷的利用率,提高动物的生产性能,以及减轻因动物高磷粪便所导致的环境水域的磷污染有着重要意义。本文综述了植酸酶的分子生物学及基因工程研究的最新进展,讨论了其进一步的研究发展方向。     

Lipid Metabolism of Caddisfly Larvae at Low pH

The influence of low pH (5.0 and 4.0) on lipid metabolism of caddisfly larvae Hydropsyche contubernalis L. (Trichoptera) was studied in 48 h toxicity experiments. The results were correlated with lipid composition of caddisfly larvae directly isolated from natural water. Phospholipids, cholesterol, mono-, di-, triacylglycerols, and fatty acids were detected by thin-layer and liquid chromatography. Minimal environmental changes were shown to initiate the biochemical adaptation mechanisms strengthening the cellular membranes through their condensation due to additional phospholipid and cholesterol synthesis. In the natural medium the adaptation processes are more active than in the artificial medium. More serious changes, such as pH decrease to 4.0, suppress the adaptation processes in the first medium and terminate them in the second one.     

植酸酶及其基因工程研究进展

植酸酶可添加于食品与饲料中,能消除因不能降解的植酸所引起的抗营养作用,提高机体对蛋白质及多种微量元素的利用率,降低粪便中磷的含量,从而减少环境中磷的积累污染,有利于保护生态环境。弄清植酸酶的性质、基因结构和功能,了解其基因工程的研究进展,不仅具有重要的理论意义,而且在开发研究植酸酶制剂用于饲料、食品和医药等方面具有重要的实践价值。本文谨对植酸酶的性质、基因结构和功能及其基因工程的研究进展做一综述,并探讨了植酸酶的应用前景。     

Nitrification at Low pH by Aggregated Chemolithotrophic Bacteria

A study was performed to gain insight into the mechanism of acid-tolerant, chemolithotrophic nitrification. Microorganisms that nitrified at pH 4 were enriched from two Dutch acid soils. Nitrate production in the enrichment cultures was indicated to be of a chemolithoautotrophic nature as it was (i) completely inhibited by acetylene at a concentration as low as 1 μmol/liter and (ii) strongly retarded under conditions of carbon dioxide limitation. Electron microscopy of the enrichment cultures showed the presence of bacteria that were morphologically similar to strains of known chemolithotrophic nitrifying genera. Many of the enriched bacteria, in particular those that were identified as ammonium oxidizers, were aggregated. Filtration experiments indicated that aggregated cells were able to nitrify at low pH, whereas single cells were not. It is hypothesized that cells inside the aggregates are protected against the toxicity of nitrous acid. Nitrification by aggregated chemolithoautotrophic bacteria may be the dominating process of nitrate formation in many acid soils as it does not appear to depend on the existence of microsites of high pH (acid-sensitive autotrophic nitrification) or on the availability of organic carbon (heterotrophic nitrification).     

Autotrophic Ammonia Oxidation at Low pH through Urea Hydrolysis

Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.     

来源于Escherichia coli的高比活植酸酶基因的高效表达

高效表达高比活植酸酶是进一步提高植酸酶发酵效价、降低植酸酶生产成本的一个有效途径。对源于Escherichiacoli的高比活植酸酶基因appA ,按照毕赤酵母 (Pichiapastoris)密码子的偏爱进行了密码子优化改造。该改造后的基因appA m按正确的阅读框架融合到毕赤酵母表达载体pPIC9上的α 因子信号肽编码序列 3′端 ,通过电击转化得到重组转化子。对重组毕赤酵母的Southernblotting分析证实植酸酶基因已整合到酵母基因组中 ,并确定了整合基因的拷贝数。Northernblotting分析证实植酸酶基因得到了正常转录。SDS PAGE分析和表达产物的研究表明 ,植酸酶得到了高效分泌表达 ,在 5L发酵罐中植酸酶蛋白表达量达到 2 5mg mL发酵液 ,酶活性 (发酵效价 )达到 7 5× 10 6 IU mL发酵液以上 ,大大高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH

We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus solfataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the beta-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.     

Acetoin Synthesis Acquisition Favors Escherichia coli Growth at Low pH

Some members of the family Enterobacteriaceae ferment sugars via the mixed-acid fermentation pathway. This yields large amounts of acids, causing strong and sometimes even lethal acidification of the environment. Other family members employ the 2,3-butanediol fermentation pathway, which generates comparatively less acidic and more neutral end products, such as acetoin and 2,3-butanediol. In this work, we equipped Escherichia coli MG1655 with the budAB operon, encoding the acetoin pathway, from Serratia plymuthica RVH1 and investigated how this affected the ability of E. coli to cope with acid stress during growth. Acetoin fermentation prevented lethal medium acidification by E. coli in lysogeny broth (LB) supplemented with glucose. It also supported growth and higher stationary-phase cell densities in acidified LB broth with glucose (pH 4.10 to 4.50) and in tomato juice (pH 4.40 to 5.00) and reduced the minimal pH at which growth could be initiated. On the other hand, the acetoin-producing strain was outcompeted by the nonproducer in a mixed-culture experiment at low pH, suggesting a fitness cost associated with acetoin production. Finally, we showed that acetoin production profoundly changes the appearance of E. coli on several diagnostic culture media. Natural E. coli strains that have laterally acquired budAB genes may therefore have escaped detection thus far. This study demonstrates the potential importance of acetoin fermentation in the ecology of E. coli in the food chain and contributes to a better understanding of the microbiological stability and safety of acidic foods.     

High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 ± 0.1) and low bulk ammonium concentrations (9.3 ± 3.3 mg·liter⁻¹), a very high nitrification rate of 5.6 g of N oxidized·liter⁻¹·day⁻¹ was achieved. The specific nitrification rate (0.55 g of N·g of biomass⁻¹·day⁻¹) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 ± 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (¹⁵N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass·g of N oxidized⁻¹. Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.     

Engineering a human IgG2 antibody stable at low pH

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid‐induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti‐2,4‐dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti‐DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain—namely, F300Y, V309L, and T339A (IgG2_YLA)—reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen‐binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.     

激光诱变筛选高产植酸酶黑曲霉菌株的研究

用1.06um,15KHz高重复率声光调QNd：YAG激光以20W和15W的辐照功率分别照射产植酸酶的黑曲霉孢子液和原生质体液不同时间,检定再生菌落数并经透明圈初筛和摇瓶复筛,测定诱变菌株所产植酸酶酶活。结果表明：20W的功率辐照黑曲霉孢子1′以上,致死率近乎100…,15W功率辐照原生质体液20″-4′,存活率在8.571％-117.14％之间,正变率在27.45％-100％之间,正变辐度60.69％-124.96％,说明原生质体比孢子耐受激光诱变的能力强,并且诱变效果好。筛选到了一株酶活提高达3.75倍的单个变异菌株。     

低pH孵放、干热处理冻干静脉注射人免疫球蛋白的实验研究

主要介绍冻干静脉注射人免疫球蛋白 (IVIG)分别通过 60℃ 72h,80℃ 72h ,低pH孵放 2 1d处理后 ,IVIG的各项理化及生物活性的变化 ,以及IVIG经过低pH孵放的灭活病毒情况。实验结果表明 ,处理后的IVIG其IgG各组份相对含量、抗补体活性 (ACA)、前激肽释放酶激活剂 (PKA)、白喉抗体、抗 HBs等结果 ,除ACA ,PKA略有下降 ,其他指标无明显变化 ,各项指标均符合 2 0 0 0版《中国生物制品规程》 ;加热后没有新抗原产生。低pH孵放 2 1d通过加指示病毒的实验 ,病毒灭活效果达到 7.0 0logTCID50 / 0 .1mL以上 ,灭活病毒有效。     

Ionic Conductance Changes in Voltage Clamped Crayfish Axons at Low pH

Giant axons from the crayfish have been voltage clamped with an axial wire system. General characterististics of observed ionic currents under normal conditions are similar to those measured in other giant axons and in nodes of Ranvier. As the pH of the external bath is lowered below 7, a marked, reversible slowing of potassium currents is seen with little effect on sodium currents. The steady-state potassium conductance-voltage curve is shifted along the voltage axis in a manner consistent with the development of a hyperpolarizing surface charge. Results suggest that this potential shift accounts for part, though not all, of the observed increase in τ_n. From the behavior of the kinetics of the delayed current with external pH these alterations in potassium conductance are attributed to the titration of a histidine imidazole residue of a membrane protein. Chemical modification of histidine by carbethoxylation at pH 6 slows and strongly depresses potassium currents. The results suggest that in addition to the introduction of electrostatic forces, possibly resulting from a hyperpolarizing surface charge, protonation of a histidine group at low pH also alters the nonelectrostatic chemical interactions determining the ease with which potassium gates open and close. The evidence indicates that the modified histidine residue is closely associated with the membrane components involved in the control of potassium conductance.     

饲用植酸酶蛋白质工程研究进展*

植酸酶作为一种单胃动物的饲料添加剂,它的推广和应用受到酶性质的局限。本在植酸酶改性策略,以及提高饲用植酸酶的表达量,改善热稳定性,提高催化效率,改良最适pH等方面介绍了植酸酶蛋白质工程方面的研究进展。