Calcium dependent,alkaline detergent-stable trypsin from the viscera of Goby (Zosterisessor ophiocephalus): Purification and characterization

An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa.The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca²⁺ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, K_m and k_cat, were 0.312 mM and 2.03 s^?1.The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.     

Synthesis of 1-(methanesulfonyl- and aminosulfonylphenyl)acetylenes that possess a 2-(N-difluoromethyl-1,2-dihydropyridin-2-one) pharmacophore: Evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity

A hitherto unknown class of linear acetylene regioisomers were designed such that a SO₂Me or SO₂NH₂ group was located at the ortho-, meta- or para-position of the acetylene C-1 phenyl ring, and a N-difluoromethyl-1,2-dihydropyridin-2-one moiety was attached via its C-5 position to the C-2 position on an acetylene template (scaffold). All three SO₂Me regioisomers, and the 4-SO₂NH₂ analog, were potent inhibitors of 5-lipoxygenase (5-LOX IC₅₀ = 3.2–3.5 μM range) relative to the reference drug caffeic acid (IC₅₀ = 4.0 μM). The SO₂Me regioisomers exhibited weak cyclooxygenease-1 (COX-1) and -2 (COX-2) inhibitory activity with a modest COX-2 selectivity index. The most potent 3-SO₂Me, 4-SO₂Me and 4-SO₂NH₂ compounds, with respective ED₅₀ values of 66.1, 68.5 and 86.5 mg/kg po, exhibited comparable oral anti-inflammatory (AI) activity to that of the reference drug ibuprofen (ED₅₀ = 67.4 mg/kg po). The N-difluoromethyl-1,2-dihydropyridin-2-one moiety provides a novel pharmacophore for the design of cyclic hydroxamic mimetics capable of inhibiting 5-LOX for exploitation in the development of 5-LOX inhibitory AI drugs.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): Purification,primary structure,and inhibitory properties

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K_a = 1.1 × 10⁹ M^?1 and 2.5 × 10⁹ M^?1, respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K_a = 4.5 × 10⁸ M^?1 and 1.2 × 10¹⁰ M^?1). Weak interaction with human plasmin (K_a = 1.2 × 10⁷ M^?1) was also revealed.     

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs)

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC₅₀ = 292.17 ± 27.71 μM and 331.94 ± 21.21 μM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC₅₀ = 275.24 ± 13.15 μM). These values are significantly lower than those of ascorbic acid (EC₅₀ = 1129.32 ± 88.79 μM) and α-tocopherol (EC₅₀ = 944.62 ± 148.00 μM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68 ± 0.27; synthetic (S,S)-SDG-1: 2.09 ± 0.16; synthetic (R,R)-SDG-2: 1.96 ± 0.27], peroxyl [natural (S,S)-SDG-1: 2.55 ± 0.11; synthetic (S,S)-SDG-1: 2.20 ± 0.10; synthetic (R,R)-SDG-2: 3.03 ± 0.04] and DPPH [natural (S,S)-SDG-1: EC₅₀ = 83.94 ± 2.80 μM; synthetic (S,S)-SDG-1: EC₅₀ = 157.54 ± 21.30 μM; synthetic (R,R)-SDG-2: EC₅₀ = 123.63 ± 8.67 μM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.     

The D-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding

The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH₄)₂SO₄ and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH₄)₂SO₄. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH₄)₂SO₄ with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The K_a’s for Gal, Lac and GalNAc in the presence of (NH₄)₂SO₄ were 0.16 ± 0.002, 0.21 ± 0.004 and 8.45 ± 0.78 (×10^?3) M^?1 respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding.     

Toxicity of Acalypha indica (Euphorbiaceae) and Achyranthes aspera (Amaranthaceae) leaf extracts to Aedes aegypti (Diptera: Culicidae)

Alternative control strategies for the dengue vector Aedes aegypti L. (Diptera: Culicidae) include botanical insecticides. They are believed to pose little threat to the environment or to human health and may provide practical substitutes for synthetic insecticides. In this study, we determined the biological activities of methanol extracts of Acalypha indica L. (Euphorbiaceae) and Achyranthes aspera L (Amaranthaceae) leaves individually and in combination as botanical insecticides against Ae. aegypti. Based on LC₅₀ values for 4th instar Ae. aegypti, the combined extracts showed the strongest larvicidal activity (277 ppm). A. aspera and A. indica extracts individually gave similar results (409 and 420 ppm, respectively). Respective LC₅₀ values for pupae were 326 ppm, 456 ppm, and 467 ppm. In studies of smoke toxicity, 64% of females exposed to negative control smoke (no extract) blood fed on chicken, whereas 17% blood fed when exposed to smoke containing A. aspera extract and to positive control smoke (0.2% d-allethrin). In the field, treatment of water storage tanks (≈ 0.5 m³) with combined plant extract reduced larval and pupal populations by 97% and 81%, respectively, after 5 days. Given the results of this study, further evaluation of the combined (A. indica + A. aspera) extract as a mosquito larvicide is warranted. Mosquito coils with A. aspera extract also show promise as a practical and potentially economical means for mitigating mosquito blood feeding.     

Pectinase and cellulase activity in the digestive system of the elm leaf beetle,Xanthogaleruca luteola Muller (Coleoptera: Chrysomelidae)

The elm leaf beetle, Xanthogaleruca luteola, is a serious pest of elm trees in urban areas. Partial biochemical characterization of pectinases and cellulases was conducted using the larval digestive system of the pest. Midgut extracts from larvae showed optimum activity for pectinase and cellulase against pectin and carboxymethyl cellulose, respectively, under acidic conditions (pH 6). Pectinases and cellulases were respectively more stable under acidic conditions (pH 4–7) and slightly acidic conditions (pH 5–7) than under highly acidic and alkaline conditions. However, the enzymes were more stable in slightly acidic conditions (pH 6) when incubation time was increased. Maximum activity for the pectinases and cellulases incubated at different temperatures was observed at 45 and 50 °C, respectively. Mg²⁺ remarkably increased pectinase activity, and cellulase activity increased significantly in the presence of Ca²⁺ and Mg²⁺. Sodium dodecyl sulfate significantly decreased pectinase and cellulase activity. The Michaelis–Menten constant (K_M) and the maximal reaction velocity (V_max) values for pectinase were 2 mg·mL^? 1 and 0.017 mmol·min^? 1·mg^? 1 protein toward pectin, respectively. Zymogram analyses revealed the presence of one and five bands of pectinase and cellulase activity, respectively, in the larval midgut extract.     

A convenient preparation of pentaaquanitrosylchromium(2+) sulfate: The crystal structure revisited

The complex pentaaquanitrolsylchromium(2+) sulfate, [Cr(OH₂)₅(NO)]SO₄ has been prepared in a high yield by the hydrolysis of [Cr(NCCH₃)₅(NO)](BF₄)₂ in dilute sulfuric acid. Crystals of [Cr(OH₂)₅(NO)]SO₄ · H₂O have been grown and characterized by X-ray crystallography. Continuous photolysis of [Cr(NCCH₃)₅(NO)]²⁺ in acetonitrile solution with 404 nm light results in a release of NO with the quantum yield Φ = 0.55 mol einstein⁻¹ at 298 K with the resulting solvated Cr²⁺ ion being trapped by molecular dioxygen present in the solution.     

Local control rates in stereotactic body radiotherapy (SBRT) of lung metastases associated with the biologically effective dose

AimTo evaluate dose differences in lung metastases treated with stereotactic body radiotherapy (SBRT), and the correlation with local control, regarding the dose algorithm, target volume and tissue density.BackgroundSeveral studies showed excellent local control rates in SBRT for lung metastases, with different fractionation schemes depending on the tumour location or size. These results depend on the dose distributions received by the lesions in terms of the tissue heterogeneity corrections performed by the dose algorithms.Materials and methodsForty-seven lung metastases treated with SBRT, using intrafraction control and respiratory gating with internal fiducial markers as surrogates (ExacTrac, BrainLAB AG), were calculated using Pencil Beam (PB) and Monte Carlo (MC) (iPlan, BrainLAB AG).Dose differences between both algorithms were obtained for the dose received by 99% (D_99%) and 50% (D_50%) of the planning treatment volume (PTV). The biologically effective dose delivered to 99% (BED_99%) and 50% (BED_50%) of the PTV were estimated from the MC results. Local control was evaluated after 24 months of median follow-up (range: 3–52 months).ResultsThe greatest variations (40.0% in ΔD_99% and 38.4% in ΔD_50%) were found for the lower volume and density cases. The BED_99% and BED_50% were strongly correlated with observed local control rates: 100% and 61.5% for BED_99% > 85 Gy and <85 Gy (p < 0.0001), respectively, and 100% and 58.3% for BED_50% > 100 Gy and <100 Gy (p < 0.0001), respectively.ConclusionsLung metastases treated with SBRT, with delivered BED_99% > 85 Gy and BED_50% > 100 Gy, present better local control rates than those treated with lower BED values (p = 0.001).     

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (t_f) and the number of successive repeated fed-batch cycles on the maximum cell concentration (X_m), cell productivity (P_x), nitrogen-to-cell conversion yield (Y_x/n), maximum specific growth rate (μ_m) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and t_f = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X_m = 2101 ± 113 mg L^?1, P_x = 219 ± 13 mg L^?1 d^?1 and Y_x/n = 10.3 ± 0.8 g g^?1).     

Flavonoids and other bioactive constituents from Ficus thonningii Blume (Moraceae)

Continued interest in the chemistry of Ficus spp. led to the investigation of the figs and the roots of Ficus thonningii Blume. Two new flavonoids, thonningiol (1) and thonningiisoflavone (2) along with nineteen known compounds were isolated. β-Isoluteone (13) was isolated here for the first time from a natural source. Their structures were elucidated on the basis of spectroscopic evidence. Interestingly, thonningiisoflavone (2) and hydroxyalpinumisoflavone (21) showed strong DPPH radical scavenging activity with IC₅₀ = 65.50 μM and 68.20 μM respectively compared to the standard BHA with IC₅₀ = 44.20 μM. The methanolic extract of figs, taxifolin (14), conrauiflavonol (17) and shuterin (19) exhibited moderate antimicrobial activity against six micro-organisms with MIC below 1.5 mg/mL.     

Insecticidal and developmental inhibitory properties of monoterpenes on Culex pipiens L. (Diptera: Culicidae)

Twelve monoterpenes were evaluated for larvicidal and adulticidal activities towards Culex pipiens. Geraniol and cuminaldehyde were the most toxic monoterpenes to larvae, with LC₅₀ values of 38.6 and 38.9 mg/l after 24 h of treatment, respectively, whereas cuminaldehyde was the most potent compound after 48 h of treatment, followed by geraniol and thymol. In fumigant toxicity experiments, (R)-carvone and geraniol were the most toxic monoterpenes against the adults at all three tested concentrations and after both 24 and 48 h. When tested at sublethal concentrations (0.5 LC₅₀), (R)-carvone, (S)-limonene and cuminaldehyde decreased hatchability, pupation and adult emergence and induced high larval mortality. Our results suggest that geraniol, cuminaldehyde and (R)-carvone are promising toxicants against Culex pipiens and could be useful in the search for new natural insecticides.     

Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis-epoxysuccinic acid to generate l(+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus, and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH₄)₂SO₄ were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL⁻¹.     

Removal of Zn(II) ions from aqueous solution using Moringa oleifera Lam. (horseradish tree) biomass

The removal of Zn(II) ions from aqueous solution using pure and chemically pretreated biomass of Moringa oleifera was investigated at 30 ± 1 °C in this study. The experimental results explored that the maximum pH (pH_max) for efficient sorption of Zn(II) was 7 ± 0.1 at which evaluated biosorbent dosage and biosorbent particle size, were 0.5 g/L, <0.255 mm, respectively. The cellular Zn(II) concentration increased with the concentrations of Zn(II) in solution. Pretreatment of M. oleifera biomass affected the sorption process and the uptake capacity (mg/g) of biomass for Zn(II) uptake was in following order: NaOH (45.76) > H₂SO₄ (45.00) > CTAB (42.80) > Ca(OH)₂ (42.60) > Triton X-100 (42.06) > H₃PO₄ (41.22) > Al(OH)₃ (41.06) > SDS (40.41) > HCl (37.00) > non-treated biomass (36.07). There was significant increase in uptake capacity of M. oleifera biomass, which suggested that affinity between metal and sorbent can be increased after some sort of pretreatment. Both Langmuir and Freundlich isotherm model fitted well to data of Zn(II) biosorption as represented by high value of their correlation coefficient (i.e. R² ≈ 1). Kinetic studies revealed that Zn(II) uptake was fast with 90% or more of uptake occurring with in 40 min of contact time and the equilibrium was reached in 50 min of contact time. The sorption rates were better described by a second order expression than by a more commonly applied Lagergren equation. Finally it was concluded that pretreatment of M. oleifera biomass can achieve superior Zn(II) uptake capacity in comparison to non-pretreated biomass.     

Synthesis,spectral and magnetical characterization of monomeric [Cu(2-NO2bz)2(3-pyme)2(H2O)2] and polymeric [Cu{3,5-(NO2)2bz}2(3-pyme)2]n

Two new complexes of composition [Cu(2-NO₂bz)₂(3-pyme)₂(H₂O)₂] (1) and/or [Cu{3,5-(NO₂)₂bz}₂(3-pyme)₂] (2) (3-pyme = 3-pyridylmethanol, ronicol or 3-pyridylcarbinol, 2-NO₂bz = 2-nitrobenzoate and 3,5-(NO₂)₂bz = 3,5-dinitrobenzoate) have been prepared and studied by elemental analysis, electronic, infrared and EPR spectroscopy, magnetic susceptibility measurements and the structure of both complexes has been solved. Complex (1) shows an unusual molecular type of structure consisting of the [Cu(2-NO₂bz)₂(3-pyme)₂(H₂O)₂] molecules held together by hydrogen bonds and van der Waals interactions. Complex (2) exhibits a polymeric chain-like structure [Cu{3,5-(NO₂)₂bz}₂(3-pyme)₂]_n with copper atoms doubly bridged by two 3-pyridylmethanol molecules and the polymeric molecules are held together by van der Waals interactions. Complex (1) exhibits a magnetic moment μ_eff = 1.84 B.M. at 300 K that remains nearly constant within the temperature region (5–300 K). Further cooling results in lowering the magnetic moment to μ_eff = 1.82 B.M. at 1.8 K. The magnetic susceptibility temperature dependence obeys Curie–Weiss law with Curie constant of 0.423 cm³ K mol⁻¹ and with Weiss constant of −0.06 K. The magnetic moment of (2) exhibits a small increase with a decrease in the temperature (μ_eff = 1.80 B.M. at 300 K and μ_eff = 1.85 B.M. at 1.8 K) with Curie constant of 0.409 cm³ K mol⁻¹ and with Weiss constant of +1.1 K, which can indicate a very weak ferromagnetic interaction between the copper atoms within the chain. Applying the molecular field model resulted in obtaining zJ′ values −0.08 cm⁻¹ for complex (1), and −0.07 cm⁻¹ for complex (2), respectively, that could characterize intermolecular and interchain interactions transmitted through π–π stacking.     

Two-sex life table and predation rate of Cybocephalus flavocapitis Smith (Coleoptera: Cybocephalidae) reared on Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae), in Taiwan

Development, survival, and fecundity of the predator, Cybocephalus flavocapitis Smith, reared on its host Aulacaspis yasumatsui Takagi, were studied at 24 °C, 70 ± 10% RH, and photoperiod of 12:12 (L:D) h under laboratory conditions. Developmental periods for egg, larval, and pupal stages were 6.4, 12.1 and 14.3 d, respectively. The mean longevity of adults was 85.1 d. Raw life history data of all individuals were pooled and analyzed according to the age-stage, two-sex life table theory. The intrinsic rate of increase (r) was 0.05 d^? 1, the finite rate of increase (λ) was 1.05 d^? 1, the net reproductive rate (R₀) was 29.1 offspring, and the mean generation time (T) was 63.3 d. When we incorporated the age-stage variability of predation rate with the two-sex life table, our results indicated that the predation rates of larvae and adults were 743 eggs and 1595 scales, respectively. The net predation rate of C. flavocapitis is 1874 scales. Additionally, we compared life table results of C. flavocapitis with those of a previous life table study on Cybocephalus nipponicus because both species are currently used for the biological control of A. yasumatsui in Taiwan.     

Design and synthesis of novel chloramphenicol amine derivatives as potent aminopeptidase N (APN/CD13) inhibitors

Herein we report a series of novel chloramphenicol amine derivatives as aminopeptidase N (APN)/CD13 inhibitors. All compounds were synthesized starting from commercially available (1S,2S)-2-amino-1-(4-nitrophenyl) propane-1,3-diol. The preliminary biological screening showed that some compounds exhibited potent inhibitory activity against APN. It should be noted that one compound, 13b (IC₅₀ = 7.1 μM), possess similar APN inhibitory activity compared with Bestatin (IC₅₀ = 3.0 μM).     

Immobilization of Bacillus subtilis esterase by simple cross-linking for enzymatic resolution of dl-menthyl acetate

A recombinant esterase (EC 3.1.1.1) cloned from Bacillus subtilis 0554 (BSE) was carrier-freely immobilized with the method of cross-linked enzyme aggregates. The conditions for preparing the cross-linked aggregates of BSE (CLA-BSE) were optimized, including the type and concentration of precipitants, and the concentration of cross-linker, and a simple and efficient procedure for preparing CLA-BSE was developed, consisting of a precipitation step with 0.5 g mL⁻¹ (NH₄)₂SO₄ and a cross-linking step with 60 mM glutaraldehyde for a period of 3 h as the cross-linking time. As a result, about 70% of the initial free BSE activity was incorporated into the CLA-BSE. The thermal stabilities of the immobilized enzyme at 30 °C and 50 °C were >360 and 14 times those of free BSE, respectively. More importantly, the operational stability of CLA-BSE was also considerably improved. In the kinetic resolution of dl-menthyl acetate to produce l-menthol with CLA-BSE gave ee_p > 94% at conversion of >40% and the CLA-BSE could be reused for 10 times with only about 8% reduction in activity. Therefore, the new biocatalyst immobilized through the methodology of CLEAs could significantly decrease the manufacturing cost of l-menthol and would be more beneficial for its practical applications.