橡椀单宁降解酶特性的研究

借助于没食子酸丙酯为模型化合物作为底物,对一株能抵抗橡椀单宁(鞣花类单宁)生物毒性并能有效降解这种单宁的菌株的降解特性进行了研究。结果表明这株菌不仅具有单宁酶的活性,而且具有多酚氧化酶（PPO）的活性。通过对不同底物对内胞霉PPO酶促反应速度的影响研究证实橡椀单宁的降解不仅与单宁酶有关还与多酚氧化酶有关。     

Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

Inhibitory Kinetics of p-Substituted Benzaldehydes on Polyphenol Oxidase from the Fifth Instar of Pieris Rapae L.

Polyphenol oxidase (PPO) is the enzyme responsible for enzymatic browning during the growth of insects. It is also involved in defense reactions and is related with immunities in insects. PPO, a metalloenzyme oxidase, catalyzes the oxidation of ο-diphenol to ο-quinone. The present paper de- scribes the effects of benzaldehyde and its p-substituted derivatives on the activity of PPO from the fifth instar of Pieris rapae L. PPO from the fifth instar of Pieris rapae L. was purified using ammonium sulfate fractionation and chromatography on Sephadex G-100. The enzyme kinetics was characterized using L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate. The results show that benzaldehyde, p- hydroxybenzaldehyde, p-chlorobenzaldehyde, and p-cyanobenzaldehyde can inhibit the PPO activity for the oxidation of L-DOPA. The inhibitor concentration leading to 50% activity lost, IC50, was esti- mated to be 5.90, 5.62, 2.83, and 2.91 mmol/L for the four tested inhibitors, respectively. Kinetic analy- ses show that the inhibitory effects of these compounds are reversible. Benzaldehyde, p- hydroxybenzaldehyde, and p-chlorobenzaldehyde are noncompetitive inhibitors while p-cyano- benzaldehyde is a mixed-type inhibitor. The inhibition constants were determined for all four inhibitors. p-chlorobenzaldehyde and p-cyanobenzaldehyde were more potent inhibitors than the other com- pounds. These results provide a basis for developing PPO inhibition-based pesticides.     

真菌诱导物对丹参毛状根氧化酶活性的影响

从一种广谱病原真菌－大丽轮枝菌的菌丝体中制备激发子,用该激发子和商品生物制剂－酵母提取物分别诱导对数生长期的丹参毛状根,发现丹参毛状根过氧化物酶（POD）,过氧化氢酶（CAT）和多酚氧化酶（PPO）活性在诱导后均显著增加,三种酶的活性并随诱导时间和诱导剂的浓度的增加而改变,大丽轮枝菌激发子的作用强于酵母提取物,表现为诱导酶活性峰值较高和峰值时间提前。     

榨菜中多酚氧化酶特性的研究

为探讨榨菜的保鲜及防褐变技术,对榨菜中多酚氧化酶的研究表明,榨菜中多酚氧化酶以邻苯二酚为底物时,产物最大吸收波长是420 nm,酶的最适pH为7.4,最适反应温度为53℃,Km为0.181 9 mol.L-1.该酶在85℃时仍有较强热稳定性,L-半胱氨酸盐酸盐对该酶活性有很强的抑制作用.     

D-Aspartate oxidase activity and D-aspartate content in a mutant mouse strain lacking D-amino acid oxidase

Summary A mutant mouse strain ddY/DAO^– lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant.     

冬果梨黑心病的研究

冬果梨贮藏期间发生黑心病的生理原因正在研究。经测定,多酚氧化酶的活性在果心中含量最高。总的多酚类化合物的含量,在梨果实中不同部位的分布与多酚氧化酶活性相似。通过纸层析的研究,证明绿原酸是主要的酚类化合物。黑心病梨离子的外渗量大于好梨。这些结果证明:引起黑心病的生理原因是由于酶促褐变反应。果心中的主要物质是绿原酸。     

选择性氧化酶基因及其表达的研究进展

综述了选择性氧化酶基因及其表达的研究进展，内容包括一些植物中的Aox多基因家族，以及植物的不同发育阶段、细胞色素途径和ROS对这些基因表达的影响。     

Expression of the mutant gene for L-gulono-γ-lactone oxidase in scurvy-prone rats

A mutant strain of Wistar rats with L-gulono--lactone oxidase deficiency has recently been established. To investigate this deficiency by DNA and RNA blot hybridization analyses, a fragment of a previously cloned cDNA encoding rat L-gulono--lactone oxidase was used as a probe. When genomic DNA of the mutant rat was digested with several restriction enzymes, the probe hybridized to fragments of the same sizes as those produced from DNA of normal rats. Poly(A)⁺RNA from the liver of the mutant rat was found to contain an L-gulono--lactone oxidase-specific mRNA of a normal size at a comparable level to that of normal rats. An in vitro translation experiment revealed that the mRNA programmed the synthesis of an enzyme protein which had the same molecular weight as that of the translational product of the normal mRNA, although the amount synthesized was markedly reduced as compared with that synthesized with the normal mRNA. In accordance with this observation, a very low but definite degree of L-gulono--lactone oxidase activity was detected in the microsomes of the mutant rat by a newly developed, highly sensitive method.Acknowledgments. The authors thank Dr Susumu Makino, Shionogi Research Laboratories, Shionogi & Co., Ltd, Japan, for his kind donation of normal (ODS- +/+) and ODS (ODS-od/od) rats. This work was supported in part by Grant-in-Aid (59570103) for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

水稻乙醇酸氧化酶的纯化和特性分析

从水稻绿叶分离纯化得到一种单来基的乙醇酸氧化酶，其全酶分子量约为２６０ＫＤ，电泳特性表明该酶的ｐＩ〈８．３我们用改进的８％ＰＡＧＥ，结合Ｗｅｓｔｅｒｎ ｂｌｏｔ，测定水稻绍叶中还存在一种ｐＩ〉８．３的全酶Ｍｒ〉６６９ＫＤ的乙醇酸氧化酶。