赤红球菌组氨酸激酶的融合表达和功能分析

对赤红球菌的组氨酸激酶基因进行密码子优化，将优化后的组氨酸激酶基因（rhks）构建重组表达质粒pGEX-4T-2-rhks。将此质粒导入到大肠杆菌BL21（DE3）中进行异源表达。在25 ℃和1 mmol/L异丙基-β-D-硫代吡喃半乳糖苷诱导条件下，组氨酸激酶融合蛋白（GST-RHK）获得成功表达，并具有催化活性。经谷胱甘肽琼脂糖亲和层析纯化，获得电泳纯的GST-RHK，其中纯化倍数为3.1，得率为19.5%。该蛋白大小约为72.75 kDa，Km、Vmax和Kcat值分别为20.92 μmol/L、0.17 μmol/（L·min）和1.4 min－1。野生型赤红球菌、组氨酸激酶基因增强株sdrhkE和组氨酸激酶基因敲减株sdrhkD在分别含有苯酚、甲苯、氯苯、异辛烷4 种有机溶剂的培养基中培养，菌株sdrhkD的生长情况都优于野生型赤红球菌，菌株sdrhkE的生长情况都低于野生型赤红球菌。本研究为进一步揭示赤红球菌SD3中组氨酸激酶涉及的信号转导途径与赤红球菌有机溶剂耐受性的关联机制提供一定参考依据。     

Oxygen transport rate on Rhodococcus erythropolis cultures: Effect on growth and BDS capability

The influence of oxygen transport rate on Rhodococcus erythropolis cultures has been studied in a stirred tank bioreactor under different transport and uptake conditions. Oxygen uptake rate has been measured by applying a modified dynamic method and a kinetic model is proposed, obtaining the kinetic parameter values: specific maintenance and yield coefficients. The volumetric mass transfer coefficients under inert conditions, k_La, and during the bioprocess, K_La, have been determined. The values obtained are different and a biological enhancement factor E, has been considered. These parameters have been predicted by the theoretical model and good agreement with experimental data under the conditions studied has been found. The oxygen limitation has been expressed by a modified dimensionless Damköhler number, Da, the relationship between transport and biological reaction rates. This number decreases with increasing stirrer speed; that is, when mass transport resistance decreases. The efficiency of oxygen utilization can be determined by a film effectiveness factor, η. The effectiveness factor was found to be a strong function of Damköhler number and decrease with increasing Da. Furthermore, oxygen concentration into culture depends on the mass transfer and consumption rates. The theoretical model proposed is able to reasonably predict the evolution of dissolved oxygen concentration with time of cultivation.     

金属离子对红球菌腈水合酶活力的影响

不同金属离子对红球菌（Rhodococcus sp．）TCCC 28001的生长及其腈水合酶的酶活有着不同程度的影响．菌株TCCC 28001产生的腈水合酶是钴型,且Co^2＋对该腈水合酶诱导激活的最适浓度为10×10^-5mol／L．选择酵母粉中含有且含量波动较大的5种金属离子,考察了在添加10×10^-5 mol／L Co^2＋诱导激活下,5种金属离子对酶活的影响．结果表明：Fe^2＋,Mn^2＋,Mo^6＋,Cu^2＋4种金属离子具有促进作用,Zn^2＋产生轻微抑制作用．6×10^-5mol／L Fe^2＋的促进效果显著,使菌株TCCC 28001的酶活从453U／mL增加到1941U／mL,提高了328％．     

Rhodococcus sp-1的Mn~(2+)生物去除能力及诱导特性

利用传统的细菌分离方法,从地下水生物除锰滤池中分离、纯化出1株细菌,经LBB(leucoberbelin blue)法定性检测其具有Mn2+氧化能力,利用Sherlock细菌鉴定系统初步确定其属于红球菌属(Rhodococcus sp).对红球菌Rhodococcus sp-1菌株的生物除锰能力和诱导特性研究结果表明,Rhodococcus sp-1菌株在达到第1个生长稳定期,细菌浓度2.3×108个/mL时,其Mn2+的去除量可达到35 mg/L,具有很强的Mn2+生物去除能力;同时Fe的存在对Rhodococcus sp-1 Mn2+氧化活性的表达具有诱导作用,在相同培养条件下,Fe3+的诱导性强于Fe2+,其诱导速度是Fe2+的3~4倍;有机Fe3+的诱导性强于无机Fe3+,其诱导速度是无机Fe3+的1.6~2倍.     

一株溶藻细菌(P15)的溶藻效应研究

从实验室海绵固定化微生物系统中分离获得一株溶藻细菌——P15，经鉴定其属于红球菌属。研究了该菌对FACHB469、FACHB569、FACHB377、FACHB416四种蓝藻的溶藻效果、溶藻方式及无机磷对该细菌生长的影响。结果表明：液体溶藻现象较固体溶藻现象明显；液体溶藻试验中有藻絮体形成，且初始菌浓度越大，溶藻效果越好。P15菌可能是通过直接接触使藻细胞凝聚并进一步被微生物降解的，同时它还能分泌一种具有热稳定性且可抑制藻细胞生长的胞外物。无机磷对该菌的生长影响显著，当磷浓度（K2HPO4＋KH2PO4，1：1）≤2g／L时，磷加入量越大则其生长情况越好。     

浅谈进口LCD生产线设备维修管理

通过对南京华日公司在2002年从日本引进一条STN-LCD生产线设备维护修理情况进行探讨,为进口LCD生产线设备维护修理管理提供参考。     

A Single Mutation at the Ferredoxin Binding Site of P450 Vdh Enables Efficient Biocatalytic Production of 25‐Hydroxyvitamin D3

Vitamin D₃ hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two‐step hydroxylation of vitamin D₃ (VD₃) to produce 25‐hydroxyvitamin D₃ (25(OH)VD₃) and 1α,25‐dihydroxyvitamin D₃ (1α,25(OH)₂VD₃). These hydroxylated forms of VD₃ are useful as pharmaceuticals for the treatment of conditions associated with VD₃ deficiency and VD₃ metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin‐binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD₃, a promising intermediate for the synthesis of various hydroxylated VD₃ derivatives, by using nisin‐treated Rhodococcus erythropolis cells containing Vdh_T107A. The gene‐expression cassette encoding Bacillus megaterium glucose dehydrogenase‐IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL^?1 25(OH)VD₃ was successfully produced by a 2 h bioconversion.     

工业修补胶在化工设备检修中的应用

介绍了工业修补胶的性能原理及应用实例，提出了工业修补胶在化工设备检修中的应用范围，失效原因及正确应用方法。     

Phylogenetic analysis of Rhodococcus erythropolis based on the variation of ribosomal proteins as observed by matrix-assisted laser desorption ionization-mass spectrometry without using genome information

Rhodococcus erythropolis strains characterized as antibiotic producers can be classified into three groups according to their antibiotic spectrum and growth compatibility. Due to their high genotypic similarity, the taxonomic relationship of these strains has not been elucidated. In this study, ribosomal protein profiling using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was employed to classify twenty-one strains of R. erythropolis (15 antibiotic producers and 6 non-antibiotic producers). In the first step in this method, a total of 30 intense peaks observed for purified ribosomal subunit proteins of the type strain (R. erythropolis JCM 3201^T) were selected as the reference peaks. The mass spectra observed for the cell lysates of each sample strain were then checked as to whether peaks were observed at the same masses of the reference peaks. The results of peak matching were processed by cluster analysis, generating a dendrogram. Four major clusters of the R. erythropolis strains corresponded to three antibiotic groups and the non-antibiotic group. Furthermore, the topology of the dendrogram was highly comparable with the phylogenetic tree based on DNA gyrase subunit B gene (gyrB) sequencing. These results indicate that our proposed ribosomal protein profiling method using MALDI-MS is a potentially reliable and sufficiently high-throughput technique for the taxonomic analysis of closely related bacterial strains without using DNA sequence information.