D-氨基酸氧化酶性质研究

D-氨基酸氧化酶（DAAO）是一种以黄素腺嘌呤二核苷酸（FAD）为辅基的典型黄素酶。它催化D-氨基酸生成相应的α-酮酸和氨。该文介绍了D-氨基酸氧化酶的研究现状及其特性，包括它的生理功能、基本特性、结构特性、光学动力学特性。     

磁性复合粒子固定化葡萄糖氧化酶的制备

采用化学共沉淀法,制备了Fe3O4粒子,并将带正电荷的PDDA吸附在其表面,形成PDDA-Fe3O4磁性复合体。为提高葡萄糖氧化酶的稳定性和重复利用率,以自制的PDDA-Fe3O4磁性复合体为载体,通过静电引力来固定葡萄糖氧化酶。研究结果表明,PDDA-Fe3O4磁性载体表面的Zeta电位为47.6mV,载体平均粒径为275d.nm。葡萄糖氧化酶固定化最佳条件为：温度15℃,pH=6,酶与载体质量比为15mg/g。固定化酶的热稳定性、pH稳定性及重复使用性能都有很大的提高。     

三角酵母D-氨基酸氧化酶基因在毕赤酵母中的转化

利用跨越内含子的PCR技术,从三角酵母(Trigonopsisvariabilis)中扩增得到D-氨基酸氧化酶基因(DAAO),通过TA克隆的方法将其克隆至pBS-T载体,并转化大肠杆菌TOP10。序列测定结果表明,所得DAAO基因的5’端内含子已被删除,基因总长度为1071bp。之后将该基因与酵母表达载体pPIC3.5K连接,转化巴斯德毕赤酵母(Pichiapastoris)。     

固定化酵母细胞转化肉桂酸生产L-苯丙氨酸

建立了一套较之传统方法更为准确的测定固定化细胞生产L-苯丙氨酸(L-phe)的新方法,采用高效液相色谱法(HPLC)测定反复冲洗凝胶所得冲洗液可以更准确地测定L-phe的生成,并采用新方法研究了固定化黏红酵母细胞的活力与稳定性。复合凝胶聚乙烯醇(PVA)+琼脂糖为最适载体,最佳质量浓度为PVA 120 g/L、琼脂糖10 g/L,反复冻融4次。此法得到的固定化细胞颗粒,机械强度良好,酶活保持时间长达200 h,L-苯丙氨酸产量高。通过流化床反应器测定其半衰期为100 h,200 h后总产量为44.58 mg/10 mL,固定化细胞无崩解现象。     

Toxicological assessment of the yeast Rhodotorula gracilis in experimental animals

 Rhodotorula gracilis is an oleaginous yeast known to synthesize lipids and carotenoid pigments. In this study, the toxicological evaluation of a locally isolated strain, R. gracilis CFR-1, in freeze-dried form has been studied to assess its safety as a source of natural colorant and nutritional additive in experimental animals. Acute and subacute studies on freeze-dried R. gracilis were conducted on both sexes of albino rats. Acute doses of R. gracilis at 0.5–6.0 g/kg body weight (w/w) did not show any symptoms of toxicity nor mortality of the adult rats. Feeding of low concentrations of R. gracilis at the 0.1–2.0% level (w/w) for 14 weeks did not induce any significant change in food intake and gain in body weight of the experimental rats compared to control animals. There were no significant differences in the absolute and relative weights of vital organs, hematological parameters, macroscopic and microscopic changes in vital organs and serum enzyme levels between the experimental and control groups. The fecal matter of R. gracilis-fed rats did not show any viable cells. The results clearly show that acute and subacute oral feeding of freeze-dried whole cells of R. gracilis for 14 weeks did not produce any toxic effects on male and female rats.     

Toxicological assessment of the yeast Rhodotorula gracilis in experimental animals

Rhodotorula gracilis is an oleaginous yeast known to synthesize lipids and carotenoid pigments. In this study, the toxicological evaluation of a locally isolated strain, R. gracilis CFR-1, in freeze-dried form has been studied to assess its safety as a source of natural colorant and nutritional additive in experimental animals. Acute and subacute studies on freeze-dried R. gracilis were conducted on both sexes of albino rats. Acute doses of R. gracilis at 0.5–6.0?g/kg body weight (w/w) did not show any symptoms of toxicity nor mortality of the adult rats. Feeding of low concentrations of R. gracilis at the 0.1–2.0% level (w/w) for 14 weeks did not induce any significant change in food intake and gain in body weight of the experimental rats compared to control animals. There were no significant differences in the absolute and relative weights of vital organs, hematological parameters, macroscopic and microscopic changes in vital organs and serum enzyme levels between the experimental and control groups. The fecal matter of R. gracilis-fed rats did not show any viable cells. The results clearly show that acute and subacute oral feeding of freeze-dried whole cells of R. gracilis for 14 weeks did not produce any toxic effects on male and female rats.     

Synthesis and characterization of Ogataea thermomethanolica alcohol oxidase immobilized on barium ferrite magnetic microparticles

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Optimisation of growth parameters for the production of carotenoids by Rhodotorula gracilis

 Response surface methodological studies of growth parameters for carotenoid production by Rhodotorula gracilis (ATCC 90950) have been carried out for three parameters employed, namely glucose concentration (%), incubation period (days) and volume of inoculum (ml/100 ml). The results indicated maximum carotenogenesis (0.054%) at a 10% glucose level with a 2 ml/100 ml volume of inoculum for an incubation period of 9 days, which was also verified by experimental data. Received: 25 May 1998     

Enzymes acting on peptides containing D-amino acid

Mainly microorganisms but only a few higher organisms are presently known to express enzymes that hydrolyze peptides containing D-amino acids. These enzymes can be involved in proceedings at the bacterial cell wall, in either assembly or modification, and thus cause resistance to glycopeptide antibiotics, or mediate resistance against beta-lactam antibiotics. In other cases the in vivo function is still unknown. New enzymes screened from nature, such as D-aminopeptidase, D-amino acid amidase, alkaline D-peptidase or D-aminoacylase, offer potential application in the production of D-amino acids, the synthesis of D-amino acid oligomers by promoting the reversed reaction under appropriate conditions, or in the field of semi-synthetic antibiotics.     

Selective determination of lactic acid in dry-fermented sausages using a sensor based on immobilized lactate oxidase

Selective determination of lactic acid in dry-fermented sausages is an indicator of quality and its presence prevents the growth of pathogenic bacteria. The analysis of lactic acid represents a high cost for the relevant food industries. The use of an enzymatic sensor would allow to reduce the time and cost of this analysis. An enzymatic sensor employing lactate oxidase (LacOx) with the immobilized enzyme system in combination with an oxygen electrode was optimized to determine the lactic acid content in dry-fermented sausages. In this study using LacOx from Pediococcus sp., a voltage of ?600 mV, low volumes of reaction and the amperometric signal obtained due to the oxygen depletion (consumed oxygen) during the lactic acid oxidation was recorded at 15 s in the immobilized enzyme sensor so that the reaction rates (slope) were related to the lactic acid content. A positive linear relationship between the consumed oxygen as a function of time (mg O2/L*s^?1) and the lactic acid concentration in the range of 250–600 μM, with a coefficient ofR² = 0.9936 for the immobilized enzyme system, was determined. The immobilized enzyme sensor showed a high specificity K_M = 0.865 and sensitivity of 0.25 mM and was stable enough to allow the reutilization of the membranes up to 20 times without loss of activity, where 90% of its initial activity remained after 45 days. The analysis of lactic acid with the immobilized enzyme system in dry-fermented sausages revealed very good agreement with the determination performed through standard HPLC methodology using the same linear range in both methods, which validated the use of this sensor as an alternative technique to evaluate cured meat quality.     

Controlled release of alpha-lipoic acid through incorporation into natural polysaccharide-based gel beads

Alpha-lipoic acid (ALA) is an essential cofactor for mitochondrial multi-enzyme complexes related to energy production. However, it is unstable under light and heat and its biological half-life (t_1/2) is short. In this study, ALA was incorporated into alginic acid (Alg) gel beads to achieve stabilisation and sustained release. The cationic polymer chitosan (CS) was also added to the Alg gel beads to better control ALA release. In artificial gastric juice (pH 1.2), ALA release from the Alg gel beads was controlled through simple diffusion from the gel matrix. However, in artificial intestinal juice (pH 6.8), the Alg gel beads were immediately disintegrated. On the other hand, Alg gel beads with CS were not disintegrated in artificial intestinal juice (pH 6.8), and achieved pH-independent sustained release of ALA through an electrostatic interaction between the carboxyl groups of Alg and the amino groups of CS. ALA contained within the Alg gel beads was protected from light (UV) and heat (65 °C) compared with ALA alone. Addition of CS to the Alg gel beads further increased the stability of ALA. Thus, stabilisation and controlled release of ALA was achieved through incorporation of ALA in Alg gel beads containing CS.     

水滑石固定化木瓜蛋白酶性质的研究

以阴离子型层状材料——水滑石(LDHs)作为载体,经过戊二醛活化处理后采用共价结合的方法固定化木瓜蛋白酶。研究了溶液酶及固定化酶的催化特性,并进行了比较。结果表明:固定化酶的最适反应pH值为8.0~9.0,最适反应温度为65℃;固定化酶的pH稳定性、热稳定性、对尿素和有机溶剂稳定性、贮藏稳定性与溶液酶相比都有显著的提高,并且固定化酶具有较好的操作稳定性,连续反应11批后,酶活未见下降。     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

海藻酸钙法固定化乳酸菌发酵制备L-乳酸的研究

在单因素实验的基础上,利用响应曲面法,对海藻酸钙法固定化植物乳杆菌发酵生产L-乳酸的条件进行优化.建立了显著影响因素和响应值之间的函数关系,得到回归方程.结果表明,最适合乳酸发酵的固定化细胞制作条件为:海藻酸钠浓度2%、包埋量20mL菌悬液/mL凝胶、固定化增殖时间22h、颗粒直径1mm左右、氯化钙浓度2%.     

Comparison of kinetic profile of free and immobilized glucose oxidase, immobilized on low-density polyethylene using UV polymerization

ABSTRACT:  Bioactive packaging is an important area of active packaging in which an active component is incorporated into the food contact surface of the package to interact with the food components without itself migrating into the food. Embedding bioactivity in a UV polymerizable resin is a novel and versatile technique for incorporating bioactive components into food packaging. In a previous article, glucose oxidase was immobilized in a packaging material using a UV curable resin. The relevance of this model system for deoxygenation of fruit juices was discussed. Though the technique efficiently immobilized enzymes in packaging material, during polymerization and immobilization the catalytic ability of the enzyme was not specifically explored. This article compares and contrasts the catalytic ability in terms of the kinetic profile of free and immobilized enzyme for the same model system: deoxygenation of juices. Kinetic behavior of immobilized and free glucose oxidase enzyme was evaluated at both shelf stable (room temperature) and refrigerated storage conditions to simulate the actual package life. It was observed that both the free enzyme and the immobilized enzymes follow the Michaelis–Menten kinetics model. There was no significant difference between the catalytic ability ( k_cat / K_m ) of free and immobilized enzymes at treatment temperatures (30, 25, and 10 °C); however, at refrigeration temperature (5 °C), the values for free enzyme were significantly higher than the immobilized enzyme.     

Lytic antimicrobial activity of hen egg white lysozyme immobilized to polystyrene beads

ABSTRACT:  Lysozyme [EC 3.2.1.17] was covalently attached to polystyrene resin beads by the sole histidine residue (His-15) through peptide spacers of various lengths. The spacers were amino acid chains composed of 6-aminocaproic acid synthesized with the solid phase peptide synthesis method. Immobilized lysozyme with a spacer length of three 6-aminocaproic acid units (2736 U/g resin with a protein load of 2.21 mg/g resin) displayed the greatest degree of hydrolytic activity against lyophilized Micrococcus lysodeikticus cell wall preparations. Enzymatic activity of immobilized lysozyme was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher total activity yet the retained activity was constant at about 14% level. A control that consisted of randomly coupled lysozyme to polystyrene beads without an amino acid spacer gave an enzyme activity of 158 U/g with a protein load of 1.24 mg/g resin which equated to 1.4% retained activity. Properties of the immobilized lysozyme system were studied, including stability and activity against soluble compared with insoluble substrates. A kinetics study of the immobilized lysozyme using Eadie–Hofstee plot parameters suggested significant external diffusion effects indicative of deviation from classic Michaelis–Menten kinetic behavior.     

乳酸菌细胞固定化发酵的研究进展

乳酸菌足能利用糖发酵产生乳酸的一类细菌,在食品、医药、工业、农业和科研等领域均有广泛的应用。本文综述了利用乳酸菌进行细胞固定化发酵生产乳酸、乳制品以及Nisin的国内外研究进展情况。并对乳酸菌固定发酵技术的应用前景进行了简要论述。     

Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii

A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii. The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C. boidinii. We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes.