Purification, identification and characterization of an esterase with high enantioselectivity to (S)-ethyl indoline-2-carboxylate.

OBJECTIVE: To purify an esterase which can selectively hydrolyze (R,S)-ethyl indoline-2-carboxylate to produce (S)-indoline-2-carboxylic acid and characterize its enzymatic properties. RESULTS: An intracellular esterase from Bacillus aryabhattai B8W22 was isolated and the purified protein was identified as a carboxylesterase by MALDI-TOF mass spectrometry. The enzyme (named BaCE) was 59.03-fold purification determined to be of approximately 35 kDa. Its specific activity was 0.574 U/mL with 20% yield. The enzyme showed maximum activity at pH 8.5 and 30 °C and was stable at 20-30 °C using pNPB as the substrate. The Km, Vmax, kcat and kcat/Km of the esterase were 0.52 mM, 6.39 µM/min, 26.87 min-1 and 51.67 mM/min, respectively. The esterase demonstrated high enantioselectivity toward (S)-ethyl indoline-2-carboxylate with 96.55% e.e.p at 44.39% conversion, corresponding to an E value of 133.45. CONCLUSIONS: In this study, a new esterase BaCE with an apparent molecular mass of 35 kDa was purified to homogeneity for the first time. The esterase from Bacillus aryabhattai B8W22 was isolated with a purification more than 59-fold and a yield of 20% by anion exchange chromatography and hydrophobic interaction chromatography. And its biochemical characterization were described in detail with pNPB as substrate. It displayed high enantioselectivity toward (S)-ethyl indoline-2-carboxylate. We next plan to highly express esterase BaCE in Escherichia coli, and apply it to industrial production of (S)-indoline-2-carboxylic acid.

[Mineralogical and spectral characteristics of "Gaozhou stone" from Jiangxi Province].

The seal stone is a kind of artwork with historical and cultural characteristics of China, which has been playing an important role in Chinese traditional culture. "Gaozhou stone", a new kind of the seal stone, has been found in the market recently. To investigate the mineralogical and spectral characterastics of "Gaozhou stone", samples were studied by using XRF, XRD, FTIR, SEM and DTA. Measurements by XRD reveal that kaolin minerals (kaolinite, dickite), pyrophyllite and minor sericite and illite occur in the ores. When kaolinite and dickite are associated, it is not easy to differentiate them from each other. Although some reflections overlap others, kaolin polytypes can be differentiated by XRD patterns in the range 18°-40° (2θ), the reflections at 0. 395, 0. 379, 0. 343, 0. 326, 0. 294, 0. 280, 0. 232 and 0. 221 nm are diagnostic of dickite. The XRD results indicate the presence of transitional mineral of kaolinite and dickite in these samples. The main chemical components of "Gaozhou stone" are SiO2 and Al2O3 with minor Fe2O3, K2O and Na2O, corresponding with that of kaolin minerals. The OH groups in kaolin group minerals have attracted considerable attention as a sensitive indicator of structural disorder. In principle, dickite has three bands, whereas kaolinite has four bands at the OH-stretching region. According to the results of FTIR, transitional mineral of kaolinite and dickite in "Gaozhou stone" has 3 absorption bands of 3 670, 3 650 and 3 620 cm-1 in high frequency region. The intensity of 3 670 cm-1 band that belongs to outer layer hydroxyl vibration is approximately equal to the intensity of 3 620 cm band ascribing to inner layer OH vibration. This value will only have subtle changes due to the different component ratio of kaolinite and dickite layers. Micro-morphology viewed by SEM presents irregular platy or pseudo-hexagonal platy particles with an average diameter of 0. 5-4 µm of "Gaozhou stone". Such morphologies are quite similar to other seal stones of China that the formation environments of all these stones are of the same kind. DTA curves demonstrate that the disparity of dehydroxylation temperature can be seen as a differential feature for identifying kaolin group minerals, but that is not undoubted. And what's more, the size of the mineral grains seems has a greater effect on the disparity of dehydroxylation temperature. This research shows that the mineral type of "Gaozhou stone" is similar to "Four Famous stones of China", and it could be a viable substitute of other famous seal stones. In this point, "Gaozhou stone" has a broad market prospect.

[Study on the effect of enzymatic activity and acute toxicity of three PAEs on Eisenia foetida].

The acute toxic effects of DMP, DEP and DBP to earthworms (Eisenia foetida) were examined in this study. The two pollutants are tested by filter paper contact method and natural soil method, results indicated that they were both poisonous to earthworms. Earthworms were exposed to DMP and DEP for 48 h by filter paper contact method, LC50 (DMP) is 129.603 microg x cm(-2) and LC50 (DEP) is 145.336 microg x cm(-2). Earthworms were exposed to DMP and DEP for 14 d by natural soil method, LC50 (DMP) is 1 560. 120 mg x kg(-1) and LC50 (DEP) is 1516. 186 mg x kg(-1). While non-calculatable for the LC50 of DBP due to its low water solubility, penetrating into the skin and cells of earthworm is not easy. The natural soil test method was used to study the effects of DMP, DEP and DBP on enzyme activities of earthworms, activities of superoxide dismutase (SOD), catalase (CAT) and acetylcholinesterase (AChE) were measured. SOD activity was induced by DMP and DEP, CAT is the most sensitive and the activity was induced by DEP and DBP at the low concentration and inhibited at the high concentration. AChE activity was inhibited by DEP and DBP at the low concentration and induced at the high concentration, finally exhibited a tendency to recover to the control level at 28 d.

Effects of 1,2,4-trichlorobenzene on the enzyme activities and ultrastructure of earthworm Eisenia fetida.

Adult Eisenia fetida earthworms were exposed to sub-lethal concentrations of 1,2,4-trichlorobenzene to assess the toxicity of contaminated soils. The LC50 of 1,2,4-TCB at 7 and 14 d were 945±175 and 890±169 mg kg⁻¹. A lower dose of 1,2,4-TCB (25 mg g⁻¹) had stimulatory effects on SOD and AChE activities, but AChE activities were significantly inhibited at higher treatment levels (100 and 400 mg g⁻¹). SOD activities increased after 2, 7, and 14 d of exposure, but AChE activities were significantly inhibited at 400 mg/kg 1,2,4-TCB after 2, 7, and 14 d of exposure, and the inhibition rates were 25.41%, 28.65%, and 25.05%. Ultrastructural observation of the intestinal epithelium at three concentrations (control, 50, and 400 mg g⁻¹) revealed that the epicuticle, cuticle layer, and microvilli were damaged with increasing 1,2,4-TCB concentration. At 400 mg kg⁻¹, mitochondria were seriously injured and the smooth endoplasmic reticulum was seriously dilated.

[Identification and phylogenetic analysis of an isolated denitrifier from rotating drum biofilter].

A heterotrophic denitrifying strain named ND1 has been isolated from rotating drum biofilter (RDB) which was mainly used for the treatment of exhaust gas such as NOx. It was proved to be Gram-negative. In addition, it was able to form dry and wrinkled colonies on the medium, adhere on the agar surface, produce yellow pigments and be motile with a single polar flagellum. It was indentified as the Pseudomonas stutzeri according to morphological and physiological biochemical characteristics and 16S rDNA sequence analysis. Under the conditions of pH 7.2 and temperature 30 degrees C, the NO3- -N removal efficiency by ND1 was up to 100% in 5 days,while the NO2- -N degradation ratio was only 85% by the strain DN1 when subjected to the same condition as the above.

[Toxic effects of dichloromethane and dichloroethane to Chlorella pyrenoidosa].

The acute toxic and biochemical effects of dichloromethane and 1,2-dichloroethane to Chlorella pyrenoidosa were assessed. The experimental results showed that dichloromethane and 1, 2-dichloroethane had some inhibition to growth of the algae. With increasing concentration of the organic toxicant, the cell density decreased. The 96 h-EC50 of dichloromethane to Chlorella pyrenoidosa was 550.1 mg/L; the 96 h-EC50 of 1,2-dichloroethane to Chlorella pyrenoidosa was 276.0 mg/L, toxicity of 1,2-dichloroethane is a little stronger than that of dichloromethane; the joint toxicity of two organic toxicants was showed as antagonism. Content of chlorophyll a, activities of superoxide dismutase (SOD), catalase (CAT) and content of malondialdehyde (MDA) in algae had been significantly affected under the organic toxicant exposure. The activities of SOD and CAT were increased at first and then decreased remarkably with the increasing concentration of the organic toxicant, showed as "Bell Shaped Curve". The content of malondialdehyde (MDA) flared up and lipid peroxidant aggregated with the increasing concentration of the organic toxicant. The results suggested that the primary mechanism of two organic toxicants on Chlorella pyrenoidosa cells may be related to oxidation damage of lipid and other biological large molecules caused by dichloromethane and 1,2-dichloroethane.

[Isolation, identification and conditions of bacterial strain capable to metabolize high concentrations of formaldehyde].

One bacterial strain capable to degrade and metabolize formaldehyde as a sole carbon source was isolated from soil. Based on the results of standard morphological identification, physiological and biochemical characters, and 16S rDNA sequence analysis, the strain was identified as Pseudomonas putida. After single factor test and orthogonal test, the optimal condition for formaldehyde degradation was determined as the follows: peptone 1.2 g/L, KH2PO4 4 g/L, K2HPO4 3 g/L, MgSO4 x 7H2O 0.2 g/L, trace elements solution 0.1 mL/L, temperature 30 degrees C, pH 8. Under the optimal conditions, the strain tolerance of original formaldehyde concentration was up to 6 g/L and 86% of formaldehyde was consumed after 54 h. It completely consumed 5 g/L formaldehyde after 46 h and degraded 100% of 4 g/L formaldehyde after 35 h.

[Gene cloning and overexpression of dichloromethane dehalogenase from Bacillus circulans WZ-12].

The dcmR gene encoding dichloromethane dehalogenase was amplified by PCR from Bacillus circulans WZ-12 and cloned to expression vector pET28b(+), yielding recombinant plasmid pET28b(+)-dcmR. Then plasmid pET28b(+)-dcmR was introduced into Escherichia. coli BL21(DE3). Expression was induced by IPTG,and the enzyme activity reached 25.78 U/mL, the specific enzyme activity reached 88.86 U/mg protein.The periplasmic and cytoplasmic enzyme activity reached 2.92 U/mL and 22.86 U/mL respectively.All results analysis demonstrated that the E. coli. strain carrying the dcmR gene could produce dichloromethane dehalogenase efficiently. The growth characteristics of dcmR-1 was compared with the original strain, and the result showed that there was no difference, A(600nm) of dcmR-1 in LB medium could reach about 2.4 in logarithmic period,which was the same as that of the original strain. The recombinant strain dcmR-1 showed the higher degrading ability than Bacillus circulans WZ-12 and with more than 90% removal efficiency of 120 mmol/L CH2Cl2 in 25 h. All these results indicated that recombinant strain dcmR-1 was a promising strain in bioremediation of CH2Cl2 contaminated environment.

[Analysis on the characteristics of histological location of bursin in immune organ of chicken and duck].

The distribution of Bursin in major immune organs such as bursa of Fabricius (BF), thymus (Th), Harderian gland(HG) and spleen(Sp) was investigated and compared in both chicken and duck by immuno-histochemical staining method with anti-Bursin monoclonal antibody (McAb)2F9-4. Its distributions in bone marrow(BM) and embryonic organs including BF, Th, BM, HG in chicken were also determined in addition to locating it in germinal center(GC) of Sp and lymphatic nodules of lymphonode in duck. The results showed clearly that Bursin was generally located in immune organs in both chicken and duck. However, its distributive regularity in chick was different from that appeared in duck. In addition, the biological significance on Bursin distribution in immune organs was discussed.