The biotransformation of astragalosides by a novel acetyl esterase from Absidia corymbifera AS2

Absidia corymbifera AS2 has been previously screened for effective biotransformation of astragalosides since it is able to catalyze the hydrolysis of acetyl ester moieties. In this study, an acetyl esterase from A. corymbifera AS2 was purified and its catalytic pathways were investigated. The purified enzyme was monomeric, with a molecular mass of 36 kDa, and with optimal activity observed at pH 8.0 and 35 °C. It was stable within pH 7.0–9.5 and at temperatures lower than 45 °C. The K_m and V_max values for p-nitrophenyl acetate was estimated to be 3.76 and 17.64 mmol (min mg)⁻¹, respectively. We found that this enzyme can hydrolyze the acetyl groups at positions O-2 or O-3 of xylopyranosyl residue at the C-3 position of AS-I, isoAS-I, AS-II and isoAS-II, and convert these all to ASI. The pathways of deacetylation catalyzed by this enzyme were also clarified for the first time: AS-II→ASI, isoAS-II→AS-II→ASI, AS-I→(AS-II, isoAS-II)→ASI and isoAS-I→AS-II→ASI. In summary, an acetyl esterase from A. corymbifera AS2 was extracted, which showed unique enzymatic characteristics and enabled clarification of the biotransformation pathways of astragalosides. This enzyme has potential industrial applications, especially for utilizing abundant astragaloside precursors for the production of rare ASI.     

Pathways and kinetics analysis of biotransformation of Dioscorea zingiberensis by Aspergillus oryzae

In this paper, the pathways and kinetics for the production of diosgenin via biotransformation of Dioscorea zingiberensis C.H. Wright by Aspergillus oryzae CICC 2436 were analyzed. After 120 h of biotransformation at 30 °C, the concentration of diosgenin in the culture reached 36.87 ± 1.27 μmol/g raw herb, which was 21.2 times its initial concentration. A number of steroidal compounds were also isolated as minor products from the biotransformation, and one of these was identified as a novel compound named 3-O-β-d-glucopyranosyl (1 → 3) – β-d-glucopyranosyl (1 → 4) – β-d-glucopyranosyl-diosgenin (diosgenin-triglucoside). The biotransformation consisted of two stages: the release of steroids from the herb (accompanied by fungal growth) and hydrolysis of the steroids by glycosidases. Kinetic analysis and mathematical modelling showed that the process of biotransformation could be described by first-order kinetics under the condition of high K_m/[S] values. It consisted of a cascade of consecutive and parallel reactions involving three kinds of enzymes, five steroid saponins and their sapogenin. The main hydrolysis reactions that led to the production of diosgenin were also discussed.     

The endophytic fungal entomopathogens Beauveria bassiana and Purpureocillium lilacinum enhance the growth of cultivated cotton (Gossypium hirsutum) and negatively affect survival of the cotton bollworm (Helicoverpa zea)

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum, were assessed on the growth of cultivated cotton (Gossypium hirsutum) and development of the cotton bollworm (Helicoverpa zea). In two replicate greenhouse trials, cotton plants were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia and evaluated for effects on both plant dry biomass, number of nodes and number of developing flowers (squares). We similarly treated cotton plants and evaluated H. zea performance using no-choice in planta assays starting at the 2nd larval instar. Treatment with both fungal endophytes resulted in a significant increases in plant dry biomass (ANOVA, P = 0.024). Plant developmental stage and number of squares were also significantly enhanced in the endophyte treated plants (ANOVA, P = 0.005 and P = 0.027, respectively). The survivorship of H. zea was significantly different among the endophyte treatment groups (Kaplan–Meier, P = 0.02), where insects feeding on control plants exhibited higher survival than insects on the endophyte treated plants. There were no significant endophyte treatment effects on larval or pupal weights of H. zea individuals. There was no endophyte effect on days to pupation among treatments, but there was a marginal effect on days to eclosion (Kaplan–Meier, P = 0.07). Overall, our results demonstrate (i) the positive plant growth enhancing effects of the target endophyes on cultivated cotton under greenhouse conditions and (ii) the negative effects of endophytic P. lilacinum and B. bassiana on H. zea survivorship and development using whole plant assays.     

Sex-specific light acclimation of Chara canescens (Charophyta)

Bisexual populations of the charophyte Chara canescens (Desv. et Loisel. in Loisel., 1810) containing male and female individuals are rarely found. Two experiments were carried out to study whether male and female algae from the same site exhibit different physiological capacities, especially with respect to light acclimation.Algae from two different shore levels and from laboratory cultures acclimated to six irradiance conditions (35–500 μmol photons m⁻² s⁻¹) were compared. Field measurements showed that both female and male algae of C. canescens are able to acclimate to daily changes in solar irradiance. The quantum yield of Photosystem II (PSII) decreased with increasing irradiance in the morning and increased with decreasing irradiance in the afternoon. Growth experiments showed increasing growth rates from 35 μmol photons m⁻² s⁻¹ (∼7 mg FW) up to 500 μmol photons m⁻² s⁻¹ (∼27 mg FW) in female and male C. canescens. The irradiance saturation point for photosynthesis (E_k) was about 140 μmol m⁻² s⁻¹ for both sexes within the whole range of acclimation irradiances. The maximum photosynthesis rate at saturating irradiances (P_max) of male algae was highest at E_k, whereas P_max of female algae was highest at 500 μmol photons m⁻² s⁻¹. The photosynthetic efficiency in the light-limited range (α) increased in female C. canescens and decreased in male C. canescens. The ratio of the non-photochemical quenching parameter (NPQ) to the relative electron transport rates rETR_(MT) increased in both sexes with irradiance, but showed a steeper increase in male than in female algae. Pigment analysis showed similar acclimation pattern for male and female C. canescens. Chl a/Chl b ratios of both sexes were constant over the whole range of E_g, whereas Chl a/carotenoid ratios in male and female C. canescens decreased from 70 μmol photons m⁻² s⁻¹ upwards. Pigment analysis pointed out that the carotenes α-, β- and γ-carotene were more prominent in male than in female algae.Our results indicate that female C. canescens are more efficient in light acclimation than male algae from the same site. Nevertheless, further investigations of bisexual C. canescens populations resolving CO₂-uptake mechanisms and/or genetic differences are needed.     

Fungal endophyte–grass symbioses are rare in the California floristic province and other regions with Mediterranean-influenced climates

Research on agronomic grasses has shown that Class 1 fungal endophytes (Neotyphodium/Epichloë; Clavicipitaceae) can have profound effects on host plant fitness. However, in natural systems, even basic ecological knowledge of most endophyte symbioses is lacking. Here, I describe the distribution and abundance of endophytes across 36 native (or naturalized) grasses in a previously unsurveyed region, the California Floristic Province. Symbiosis was generally low: 8.33 % of species and 18.75 % of genera hosted endophytes. I then compared the proportions of symbiotic species and genera found in California and other Mediterranean regions to the proportions found in non-Mediterranean regions. Surveys of Mediterranean-influenced regions showed significantly lower proportions of species (～66 % lower) and genera (～65 % lower) hosting endophyte than surveys of non-Mediterranean regions. This pattern suggests that selection in Mediterranean climates may not favor endophyte symbioses.     

Light acclimation of photosynthesis in three charophyte species

The main aim of this study was to investigate if the charophyte species Chara baltica, Chara canescens (two populations from the Baltic Sea (BS) and the Gulf of Korinth, Greece (GK)), and Lamprothamnium papulosum exhibit different acclimation capacities to irradiance. Growth, photosynthesis and pigment content were examined in the laboratory under six irradiance conditions (35–500 μmol photons m⁻² s⁻¹). Growth experiments showed increasing growth rates from 35 μmol photons m⁻² s⁻¹ (∼10 mg fresh weight (FW)) up to 70 μmol photons m⁻² s⁻¹ (∼20 mg FW) in C. baltica, from 35 μmol photons m⁻² s⁻¹ (∼15 mg FW) up to 380 μmol photons m⁻² s⁻¹ (∼145 mg FW) in C. canescens (BS), and up to the highest growth irradiance in algae of L. papulosum (35 μmol: ∼5 mg FW; 500 μmol: ∼20 mg FW). The species were tested for their ability to acclimate to different growth irradiances (E_g) by calculating P_max (maximum photosynthesis rate at saturating irradiances), α (the efficiency of light utilization at limiting irradiance), and E_k (the light saturation point of photosynthesis, P_max/α). All species exhibited increasing P_max with increasing E_g. Whereas both populations of C. canescens increased α with increasing E_g, L. papulosum and C. baltica did not acclimate α at all. E_k, the irradiance at which photosynthesis ceased to be light-limited, was constant for all Chara species within the range of irradiances tested. Chl a/Chl b ratios of all species were constant over the whole range of E_g. Chl a/carotenoid ratios were constant in C. baltica, whereas Chl a/carotenoid ratios in L. papulosum and C. canescens (BS) decreased from 250 and 70 μmol photons m⁻² s⁻¹ upwards, respectively. Pigmentation analysis showed that Chl a/carotenoid acclimation was mainly caused by species-specific capacity to raise the content of lutein and carotene (C. canescens (BS), C. canescens (GK)) and xanthophyll cycle pigments (XCP; L. papulosum). The non-photochemical quenching (NPQ) capacities of L. papulosum, C. canescens (BS), and C. canescens (GK) were dependent from preacclimation status of algae, whereas NPQ of C. baltica was independent from growth irradiance.Our results indicate that C. baltica and C. canescens (BS) were light saturated within the chosen irradiances, whereas C. canescens (GK) and L. papulosum did not reach their limits of high-light acclimation. The photosynthetic pigments lutein, α- and β-carotene are suggested to act as photo-protective pigments in L. papulosum and C. canescens.     

Novel biotransformation process of podophyllotoxin to produce podophyllic acid and picropodophyllotoxin by Pseudomonas aeruginosa CCTCC AB93066, Part II: Process optimization

This work optimized the novel biotransformation process of podophyllotoxin to produce podophyllic acid by Pseudomonas aeruginosa CCTCC AB93066. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/l of yeast extract and 5 g/l of peptone were favorable for podophyllic acid production (i.e. 25.3 ± 3.7 mg/l), while not beneficial for the cell growth of P. aeruginosa. This indicated that the accumulation of podophyllic acid was not corresponded well to the cell growth of P. aeruginosa. 0 g/l of sucrose was beneficial for podophyllic acid production (i.e. 34.3 ± 3.9 mg/l), which led to high podophyllotoxin conversion (i.e. 98.2 ± 0.1%). 1 g/l of NaCl was the best for podophyllic acid production (i.e. 47.6 ± 4.0 mg/l). Secondly, the production of podophyllic acid was significantly enhanced by fed-batch biotransformation. When each 100 mg/l of podophyllotoxin was added to the biotransformation system after 4, 10 and 25 h of culture, respectively, podophyllic acid concentration reached 99.9 ± 12.3 mg/l, enhanced by 284% comparing to one-time addition (i.e. 26.0 ± 2.1 mg/l). The fundamental information obtained in this study provides a simple and efficient way to produce podophyllic acid.     

Triterpenoid saponins from Astragalus wiedemannianus Fischer

Three cycloartane-type triterpene glycosides were isolated from Astragalus wiedemannianus together with eight known secondary metabolites namely cycloastragenol, cycloascauloside B, astragaloside IV, astragaloside VIII, brachyoside B, astragaloside II, astrachrysoside A, and astrasieversianin X. The structures were established mainly by a combination of 1D and 2D-NMR techniques as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-25-O-β-D-glucopyranosyl-20(R),24(S)-epoxy-3β,6α,16β,25-tetrahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-(4’-O-acetoxy)-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane. To the best of our knowledge, the presence of an arabinose moiety on the acyclic side chain of cycloartanes is reported for the first time.     

Evaluation of new fermentation and formulation strategies for a high endophytic establishment of Beauveria bassiana in oilseed rape plants

Since several years it is known that strains of the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) are able to colonize plants as a true endophyte. However, so far no integrated bioprocess engineering approaches have been published where fermentation and formulation strategies are combined to optimize colonization of oilseed rape plant tissues. We therefore aimed at investigating whether and how blastospore (BS) formation can be shifted to resilient submerged conidiospores (SCS) by introducing osmotic stress in different growth phases.When 50 g/L NaCl was added after 48 h to a culture of B. bassiana a yield of 1.4 ± 0.1 × 10¹⁰ SCS/g sucrose in shake flasks and 1.8 ± 0.1 × 10¹⁰ SCS/g sucrose in a stirred tank reactor were obtained. In a bioreactor, 24 h after the addition of NaCl, the formation of BS slowed down, the respiratory quotient decreased and a shift from BS to SCS set in.Following these steps, different formulation strategies, namely encapsulation, film coating and liquid formulation were evaluated. B. bassiana grew out of beads as well as on commercial fungicide-coated seeds. Due to the complete suppression of fungal growth on non-sterile soil, the most suitable option was a foliar application. A liquid formulation consisting of 0.1% Triton X-114, 1% molasses, 1% titanium dioxide and 10⁶ spores/mL was applied on leaf tips. After 14 days, the endophyte was detected by PCR and microscopic analysis in the leaves.Further research should focus on formation of SCS and protection of plants colonized by B. bassiana against herbivorous insects.     

Impact of fluctuating temperatures on development of the koinobiont endoparasitoid Venturia canescens

The effect of temperature on the biology of Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae) is well understood under constant temperature conditions, but less so under more natural, fluctuating conditions. Herein we studied the influence of fluctuating temperatures on biological parameters of V. canescens. Parasitized fifth-instar larvae of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) were reared individually in incubators at six fluctuating temperature regimes (15–19.5 °C with a mean of 17.6 °C, 17.5–22.5 °C with a mean of 19.8 °C, 20–30 °C with a mean of 22.7 °C, 22.5–27.5 °C with a mean of 25 °C, 25.5-32.5 °C with a mean of 28.3 °C and 28.5–33 °C with a mean of 30 °C) until emergence and death of V. canescens adults. Developmental time from parasitism to adult eclosion, adult longevity and survival were recorded at each fluctuating temperature regime. In principle, developmental time decreased with an increase of the mean temperature of the fluctuating temperature regime. Upper and lower threshold temperatures for total development were estimated at 34.9 and 6.7 °C, respectively. Optimum temperature for development and thermal constant were 28.6 °C and 526.3 degree days, respectively. Adult longevity was also affected by fluctuating temperature, as it was significantly reduced at the highest mean temperature (7.0 days at 30 °C) compared to the lowest one (29.4 days at 17.6 °C). Survival was low at all tested fluctuating temperatures, apart from mean fluctuating temperature of 25 °C (37%). Understanding the thermal biology of V. canescens under more natural conditions is of critical importance in applied contexts. Thus, predictions of biological responses to fluctuating temperatures may be used in population forecasting models which potentially influence decision-making in IPM programs.     

Enhanced biotransformation of l-phenylalanine to 2-phenylethanol using an in situ product adsorption technique

An in situ product adsorption technique was used to enhance the biotransformation of l-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae BD. As a suitable adsorbent, the non-polar macroporous resin D101, selected from several resins tested, showed high adsorption capacity for 2-phenylethanol but not l-phenylalanine. Product inhibition was effectively alleviated by the addition of macroporous resin D101 to the biotransformation medium. When 2 g of hydrated resin D101 was added to 30 mL of the biotransformation medium, the total 2-phenylethanol concentration achieved was 6.17 g/L, of which 3.15 g/L remained in the aqueous phase and 3.02 g/L was adsorbed onto the resin. The molar yield of 2-phenylethanol reached 0.70 after 24 h cultivation. Addition of the macroporous resin greatly increased the volumetric productivity of 2-phenylethanol, and made the downstream processing more feasible and easier to perform in an industrial application.     

HPLC-DAD analysis of arbutin produced from hydroquinone in a biotransformation process in Origanum majorana L. shoot culture

The hydroquinone glucoside arbutin is a plant derived compound medically applied due to its uroantiseptic activity. It also has skin whitening properties and thus is widely used in dermatology and cosmetology. Origanum majorana L. (Lamiaceae) is known to produce arbutin, however the content of the compound in cultivated plants is very variable and low. Since plant cell and tissue cultures are capable to perform specific biotransformation reactions including glucosylation, this investigation targeted the formation of arbutin from hydroquinone in agitated O. majorana shoot cultures. For this purpose different doses of hydroquinone (96, 144, 192, 288 and 384 mg/L of medium) were added to the culture flasks in one, two or three portions. Arbutin was qualitatively and quantitatively determined in methanol extracts from dry biomass and lyophilized media using HPLC-DAD. Cells of O. majorana shoot cultures efficiently converted hydroquinone into arbutin. The product was accumulated in the biomass and was not observed (or in trace amounts) in the medium samples. Different doses as well as portioning of the precursor had a significant impact on the biotransformation process. Arbutin accumulation increased from 0.23 ± 0.03 mg/g DW up to 52.6 ± 4.8 mg/g DW in the biomass. The highest product content was observed after the addition of 192 mg/L hydroquinone in three portions. The highest efficiency of the biotransformation process, i.e. 67.5 ± 5.2% was calculated for a dose of 96 mg/L precursor divided into three portions. After further optimization of the biotransformation process, O. majorana shoot cultures could serve as a rich source of arbutin.     

Diversity and heavy metal tolerance of endophytic fungi from six dominant plant species in a Pb–Zn mine wasteland in China

The diversity and metal tolerance of endophytic fungi from six dominant plant species in a Pb–Zn mine wasteland in Yunnan, China were investigated. Four hundred and ninety-five endophytic fungi were isolated from 690 tissue segments. The endophytic fungal colonization extent and isolation extent ranged from 59 % to 75 %, and 0.42–0.93, respectively, and a positive correlation was detected between them. Stems harboured more endophytic fungi than leaves in each plant species, and the average colonization extent of stems was 82 %, being significantly higher than that of leaves (47 %) (P ≤ 0.001, chi-square test). The fungi were identified to 20 taxa in which Phoma, Alternaria and Peyronellaea were the dominant genera and the relative frequencies of them were 39.6 %, 19.0 % and 20.4 %, respectively. Metal tolerance test showed that 3.6 mM Pb²⁺ or 11.5 mM Zn²⁺ exhibited the greatest toxicity to some isolates and they did not grow on the metal-amended media. In contrast, some isolates were growth stimulated in the presence of tested metals. The isolates of Phoma were more sensitive to Zn²⁺ than the isolates of Alternaria and Peyronellaea. However, the sensitivity of isolates to Pb²⁺ was not significantly different among Phoma, Alternaria, Peyronellaea and other taxa (P > 0.05, chi-square test). Our results suggested that fungal endophyte colonization in Pb–Zn polluted plants is moderately abundant and some isolates have a marked adaptation to Pb²⁺ and Zn²⁺ metals, which has a potential application in phytoremediation in this area.     

Oral morphology and dietary choices of goats on rangeland

The morphometrics of mandible and teeth of mixed-breed goats on rangeland were analyzed to investigate the association between the oral apparatus and diet type. Canonical relationship between sets of mandible measurements (7) and main plants ingested (17) indicated that, overall, oral characteristics tended to modify ingestive behavior (canonical correlation = 0.81). In general, incisor length had a greater effect (absolute value of standardized coefficient: AVSC = 0.56) on ingestion of Nolina cespitifera than did other mandible variables. Incisor width influenced the ingestion of Parthenium incanum (AVSC = 0.47) and Flourensia cernua (AVSC = 0.43). Distance between the bases of the last most lateral incisors (I₄ or corners) was associated with the ingestion of Atriplex canescens (AVSC = 0.54) and Florensia cernua (AVSC = 0.69). Incisor breath was important for the consumption of Larrea tridentata (AVSC = 0.68) and Cowania plicata (AVSC = 0.50). The ingestion of Agave lechuguilla (AVSC = 0.51) and Sphaeralcea angustifolia (AVSC = 0.43) were influenced principally by muzzle width. Distance between first molars was the variable most closely associated to the ingestion of Buchloe dactyloides (AVSC = 0.51) and Croton dioicus (AVSC = 0.62). Mandible length had a greater effect on the ingestion of F. cernua and Atriplex canescens than did other oral traits. Variation in the oral apparatus did not affect percentage of grasses, forbs and shrubs of goat diets. Fecal nitrogen, fecal phosphorous and number of species consumed were not related to oral traits. These results suggest that different dimensions in the oral apparatus of grazing goats lead to divergent selection of range plants, without altering the quality of their diet.     

Biotransformation of betulin to betulone by growing and resting cells of the actinobacterium Rhodococcus rhodochrous IEGM 66

The ability of Rhodococcus actinobacteria to transform betulin to betulone was proved and reported for the first time. Betulone, the product of regioselective oxidation of a 3β-hydroxyl group of betulin, is a useful intermediate in the synthesis of novel biologically active compounds. Of 56 strains of Rhodococcus tested, Rhodococcus rhodochrous IEGM 66 was selected because it had the highest betulin-transforming ability. It was shown that R. rhodochrous IEGM 66 growing cells transformed 0.5 g/L betulin to betulone with 45% conversion rate within 240 h. A substantial reduction in the time of betulin (0.5 g/L) biotransformation was achieved by using resting cells, which catalyzed the production of 75% betulone after 96 h. At higher initial betulin concentrations (1.0–3.0 g/L), resting cells catalyzed 40–60% betulone production within 24 h.     

Biotransformation of 2-phenylethanol to phenylacetaldehyde in a two-phase fed-batch system

Phenylacetaldehyde (PA) can be produced by the oxidation of 2-phenylethanol (PE) through biotransformation. In order to prevent substrate and product inhibitions and the transformation of the PA to phenylacetic acid (PAA), utilization of a two-phase system is very attractive. Gluconobacter oxydans B-72 was used as the microorganism and iso-octane as the solvent. The effect of initial substrate concentration on the PA production was investigated in single- and two-phase systems. In the single-phase system, substrate inhibition occurred above 5 g/l, and in the two-phase system, above 7.5 g/l. Substrate inhibition kinetics were also studied in the two-phase system and kinetic constants were determined as r_max=0.64 g/l min, K_M=8.15 g/l, K_PA=2.5 g/l. Because it was observed that two-phase system is insufficient to remove the substrate inhibition effect, fed-batch operation was utilised in this study. For 7.5 g/l of PE, 1.65, 3.85, and 7.35 g/l of PA were obtained in the single-phase, two-phase, and two-phase three fed-batch systems, respectively. Effect of biotransformation time, initial substrate concentration, agitation speed, and fed-batch number on the PA production was investigated in a two-phase fed-batch system by the response surface methodology (RSM). The optimum values were found as 3 fed-batch number, 2.75 g/l initial substrate concentration, 150 rpm agitation speed, and 65 min of one batch biotransformation time. In order to verify these results, an experiment was performed at these optimum conditions and 7.10 g/l of PA concentration was obtained.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

In vitro metabolism study of a new nitrosyl ruthenium complex [Ru(NH·NHq)(terpy)NO]3+ nitric oxide donor using rat microsomes

A new nitrosyl ruthenium complex [Ru(NH·NHq)(terpy)NO]³⁺ nitric oxide donor was recently developed and due to its excellent vasodilator activity, it has been considered as a potential drug candidate. Drug metabolism is one of the main parameters that should be evaluated in the early drug development, so the biotransformation of this complex by rat hepatic microsomes was investigated. In order to perform the biotransformation study, a simple, sensitive and selective HPLC method was developed and carefully validated. The parameters evaluated in the validation procedure were: linearity, recovery, precision, accuracy, selectivity and stability. Except for the stability study, all the parameters evaluated presented values below the recommended by FDA guidelines. The stability study showed a time-dependent degradation profile. After method validation, the biotransformation study was accomplished and the kinetic parameters were determined. The biotransformation study obeyed the Michaelis–Menten kinetics. The V_max and K_m were, respectively, 0.1625 ± 0.010 μmol/mg protein/min and 79.97 ± 11.52 μM. These results indicate that the nitrosyl complex is metabolized by CYP450.     

Discovery of potent dipeptidyl peptidase IV inhibitors through pharmacophore hybridization and hit-to-lead optimization

A novel dipeptidyl peptidase IV inhibitor hit (5, IC₅₀ = 0.86 μM) was structurally derived from our recently disclosed preclinical candidate 4 by replacing the cyanobenzyl with a butynyl based on pharmacophore hybridization. A hit-to-lead optimization effort was then initiated to improve its potency. Most N-substituted analogs exhibited good in vitro activity, and compound 18o (IC₅₀ = 1.55 nM) was identified to be a potent dipeptidyl peptidase IV inhibitor with a significantly improved pharmacokinetic properties (bioavailablity: 41% vs 82.9%; T_1/2: 2 h vs 4.9 h).     

Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM

d-Lactic acid and pyruvic acid are two important building block intermediates. Production of d-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l^?1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l^?1 dl-lactic acid into 25.23 g l^?1 d-lactic acid and 19.70 g l^?1 pyruvic acid in 10 h. Using a simple ion exchange process, d-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.