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d-Allulose, a rare sugar and functional sweetener, can be biosynthesized by d-allulose 3-isomerase (DAE). However, most of the reported DAEs exhibit poor resistance under acidic conditions, which severely limited their application. Here, surface charge engineering and random mutagenesis were used to construct a mutant library of CcDAE from

Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising production yield of 9OHAD (9α-hydroxy4-androstene-3,17-dione) from AD, and further proved the importance of [2Fe–2S] clusters of ferredoxin-oxidoreductase in transferring electrons in steroidal conversion. The results of truncated Fdx domain in all oxidoreductases and mutagenesis data elucidated the indispensable role of [2Fe–2S] clusters in the electron transfer process. By adding the independent plant-type Fdx to the reaction system, the AD (4-androstene-3,17-dione) conversion rate have been significantly improved. A novel efficient electron transfer pathway of PRF + Fdx + KshA (KshA, Rieske-type oxygenase of 3-ketosteroid-9-hydroxylase) in the reaction system rather than KshAB complex system was proposed based on analysis of protein–protein interactions and redox potential measurement. Adding free Fdx created a new conduit for electrons to travel from reductase to oxygenase. This electron transfer pathway provides new insight for the development of efficient exogenous Fdx as an electron carrier. Graphical Abstract

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T5060 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.

Salicylic acid (SA) decarboxylase from Trichosporon moniliiforme (TmSdc), which reversibly catalyses the decarboxylation of SA to yield phenol, is of significant interest because of its potential for the production of benzoic acid derivatives under environmentally friendly reaction conditions. TmSdc showed a preference for C2 hydroxybenzoate derivatives, with kcat/Km of SA being 3.2 × 103 M-1 s-1. Here, we presented the first crystal structures of TmSdc, including a complex with SA. The three conserved residues of Glu8, His169, and Asp298 are the catalytic residues within the TIM-barrel domain of TmSdc. Trp239 forms a unique hydrophobic recognition site by interacting with the phenyl ring of SA, while Arg235 is responsible for recognizing the hydroxyl group at the C2 of SA via hydrogen bond interactions. Using a semi-rational combinatorial active-site saturation test, we obtained the TmSdc mutant MT3 (Y64T/P191G/F195V/E302D), which exhibited a 26.4-fold increase in kcat/Km with SA, reaching 8.4 × 104 M-1 s-1. Steered molecular dynamics simulations suggested that the structural changes in MT3 relieved the steric hindrance within the substrate access channel and enlarged the substrate-binding pocket, leading to the increased activity by improving substrate access. Our data elucidate the unique substrate recognition mode and the substrate entrance tunnel of SA decarboxylase.

Cytochrome P450 enzyme CYP68J5 from filamentous fungus Aspergillus ochraceus is industrially used for selective C11α-hydroxylation of canrenone and progesterone. To improve its selectivity of C11α-hydroxylation for relevant steroid substrates, a sequence-based targeted mutagenesis combined with saturation mutagenesis was conducted to search for variants with improved hydroxylation reaction specificity toward progesterone and D-ethylgonendione. Recombinant yeast expressing triple mutant V64F/E65G/N66T showed significantly increased C11α-hydroxylation selectivity (85 % VS WT 69.7 %). Saturation mutagenesis of V64, E65 and N66 resulted in the identification of single mutant V64K with greatly enhanced 11α-hydroxylation specificity toward progesterone (90.6 % VS WT 69.7 %). Furthermore, mutant N66D showed significant enhanced selectivity of C11α-hydroxylation toward D-ethylgonendione (70.8 % VS WT 58 %). Evaluation of recombinant yeast over-expressing V64K for progesterone transformation in 50 mL scale resulted in product 11α-OH progesterone concentrations of 432.5 mg/L, a 30.2 % increase compared with the CYP68J5 control. Our results also reveal that V64, E65 and N66 are key residues of CYP68J5 influencing its selectivity of C11α-hydroxylation, thus offering opportunities for further engineering of CYP68J5 for expanded industrial applications.

Fructosyltransferases have been used in the industrial production of fructooligosaccharides (FOS). However, it is still not possible to explain the difference in FOS production based on the variations observed in the FOS synthesizing enzymes of A. niger. In the present study, a novel fructosyltransferase (FT-A) from A. niger TCCC41686 with high FOS synthesis ability was characterized. The FT-A gene was obtained and expressed in Pichia pastoris. A homology model of FT-A showed that the changes of residues identified outside the conserved domains were found to have an effect on its characteristics and its FOS-synthesis capacity. The optimal activity of the recombinant FT-A was observed at 50 °C and pH 6.0, and it was stable below 50 °C and over the range of pH 3.0–11.0. The Km and Vmax values of FT-A were 151.13 g L−1 and 6.55 g L−1 min−1, respectively. The production of FOS using the recombinant FT-A remained above 60% during 50–80 min of synthesis based on sucrose as substrate. The novel fructosyltransferase (FT-A) investigated in this study can potentially be applied for the efficient industrial production of FOS. The results also provide more valuable information for explaining the relationship between the structure and function of the FT-A.

The rare sugar d-allulose is an attractive sucrose substitute due to its sweetness and ultra-low caloric value. It can be produced from D-fructose using d-allulose 3-epimerase (DAE) as the biocatalyst. However, most of the reported DAEs show low catalytic efficiency and poor thermostability, which limited their further use in food industrial. Here, a putative d-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense (HcDAE) was characterized, showing optimal activity at pH 8.0 and 70 °C in the presence of Mg2+. Saturation mutagenesis of Y7, C66, and I108, the putative residues responsible for substrate recognition at the O-4, -5, and -6 atoms of D-fructose was performed, and it yielded the triple mutant Y7H/C66L/I108A with improved activity toward D-fructose (345 % of wild-type enzyme). The combined mutant Y7H/C66L/I108A/R156C/K260C exhibited a half-half (t1/2) of 5.2 h at 70 °C and an increase of the Tm value by 6.5 °C due to the introduction of disulfide bridges between intersubunit with increased interface interactions. The results indicate that mutants could be used as industrial biocatalysts for d-allulose production.

The Δ1-dehydrogenation of 3-ketosteroid substrates is a crucial reaction in the production of steroids. Although 3-ketosteroid Δ1-dehydrogenase (KsdD) catalyzes this reaction with high efficiency and selectivity, the low stability and high cost of the purified enzyme catalyst have limited its industrial application. In this study, an epoxy support was used to evaluate the covalent immobilization of KsdD from Pimelobacter simplex, and the best androsta-1,4-diene-317-dione (ADD) production was achieved after optimized immobilization of KsdD enzyme in 1.5 M NaH2PO4- Na2HPO4 buffer (pH 6.5) for 12 h at 25 °C. The immobilized KsdD exhibited higher tolerance toward 20 % methanol. The dehydrogenation reaction reached a conversion efficiency of up to 90.0 % in 2 h when using 0.6 mg/mL of 4-androstene-317-dione (AD). The W299A and W299 G mutants of KsdD were also immobilized, and both showed the better catalytic performance with higher kcat/KM values compared with the wild type (WT). The immobilized W299A, W299 G and WT KsdD respectively maintained 70.5, 65.7 and 38.7 % of their initial activity at the end of 15 reaction cycles. Furthermore, the W299A retained 66.3 % of the initial activity after 30 days of incubation at 4 °C, and was more stable than free KsdD, Thus, the immobilized W299A is a promising biocatalyst for steroid dehydrogenation. In this study, we investigated the application of immobilized enzymes for the dehydrogenation of steroids, which will be of great importance for improving the development of green technology and sustainable use of biocatalysts in the steroid manufacturing industry.

Transphosphatidylation catalyzed by phospholipase D has gained increasing attention for producing phosphatidylserine (PS), which can be used in functional food and medicine. In this study, we investigated the effects of six signal peptides on the secretion of PLD (PLDsa) from Streptomyces antibioticus TCCC 21059 in the food-grade GRAS bacterium Bacillus subtilis. It indicated that the optimal signal peptide DacB with an Ala-X-Ala sequence motif at the C-terminus showed the highest secretory expression ability, resulting in increased production of 2.84 U/mL PLDsa. Then PLDsa was immobilized on the epoxy-based carriers, and one of these carriers allowed PLDsa loading of up to 2.7 mg/g. The immobilized PLDsa was more stable over a wide range of pH value (4.5–7.5) and temperature (16 °C–60 °C) than free PLDsa. Subsequently, the synthesis of PS from soybean phosphatidylcholine (PC) was carried out in purely aqueous solution using immobilized PLDsa, leading to a high yield of 65%. The immobilized PLDsa catalyst maintained a relative PS production of 60% after 5 recycles. Notably, the use of toxic solvent was completely eliminated in the whole process, which would be more profitable for the application of PS.

Corynebacterium glutamicum, an important microorganism to produce amino acids and organic acids, has been widely applied in food and medicine fields. Therefore, using editing tools to study the function of unknown genes in C. glutamicum has great significance for systematic development of industrial strain with efficient and novel production capability. Recently, gene editing has been greatly developed. Traditional gene editing based on homologous recombination and gene editing mediated by nuclease are successfully applied in C. glutamicum. Among these, the CRISPR system has been developed to be a main tool used for gene knockout of C. glutamicum due to its advantages of efficiency, simplicity and good target specificity. However, more efficient and reliable knockout system is still urgently demanded, to help develop high-performing strains in industrial application.谷氨酸棒杆菌是生产氨基酸、有机酸等的重要菌株，广泛应用于食品、医药领域。利用基因编辑技术对谷氨酸棒杆菌进行基因功能研究，在提高目的产物产量、发现新的基因功能等方面有重要意义。近年来，基因编辑技术发展日新月异，从基于同源重组的传统基因编辑技术到以人工核酸酶介导的基因编辑均在谷氨酸棒杆菌中得到合理应用。其中，CRISPR 技术以其快速、简便、编辑效率高等优点成为现阶段研究者用于改造谷氨酸棒杆菌的主要技术，但是更为简单、高效的编辑手段依旧需要进一步研究开发，以获得优良菌株应用于工业生产中。.

Aldonic acids are receiving increased interest due to their applications in nanotechnology, food, pharmaceutical and chemical industries. Microbes with aldose-oxidizing activity, rather than purified enzymes, are used for commercial production with limited success. Thus it is still very important to develop new processes using strains with more efficient and novel biocatalytic activities for the production of adonic acids. In the present study, Pseudomonas fragi TCCC11892 was found to be an efficient producer of aldonic acids, with the production of galactonic and L-rhamnonic acid by P. fragi reported for the first time. The semi-continuous production of maltobionic acid and lactobionic acid was developed for P. fragi TCCC11892, achieving a yield of over 90 g L−1 for the first 7 cycles. The excellent performance of P. fragi in the production of lactobionic acid (119 g L−1) was also observed when using waste cheese whey as an inexpensive fermentation medium. Scaling up of the above process for production of aldonic acids with P. fragi TCCC11892 cells should facilitate their commercial applications.

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.

D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from Rhodopirellula baltica (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a Km of 58.57 mM and kcat of 1849.43 min−1. It showed a melting temperature (Tm) of 45.7 °C and half-life (t1/2) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn2+. The Site-directed mutation L144 F strengthened the thermostability to a Δt1/2 of 50.4 min, ΔTm of 12.6 °C, and ΔT5060 of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.

The C-7 cholesterol dehydrogenase (NVD), which converts cholesterol to 7-dehydrocholesterol (7-DHC) by 7,8-dehydrogenation, plays a pivotal role in the metabolism of cholesterol and steroid intermediates. The NVD protein was successfully expressed in insect Sf9 cells. To reduce the production cost for industrial application, the NVD gene was cloned into E. coli BL21(DE3). However, the His-tagged NVD protein showed poor binding to Ni-NTA resin, mainly due to the formation of inclusion bodies. Consequently, the expression and solubility of NVD were optimized by respectively fusing it with maltose-binding protein (MBP), glutathione S-transferase (GST), and the nonspecific DNA binding protein from Sulfolobus solfataricus (Sso7d) as solubility tags. The NVD fusion with MBP at the N-terminus and His-tag at the C-terminus achieved a high yield of the soluble enzyme. It was further purified by ion-exchange chromatography with 95.4% purity and with a 10.4% yield. The product 7-DHC, which is produced in a reaction catalyzed by NVD and ferredoxin reductase KshB, was initially identified by GC–MS. An analysis of the amino acid sequence alignment revealed a distinct Rieske-type iron-sulfur cluster and non-heme Fe2+-binding domain, which are evolutionarily conserved among NVD family enzymes.

Biotransformation of steroid compounds: androst-4-ene-3, 17-dione (1), 19-nor-4-androstene-3, 17-dione (2), 13β-ethyl-4-gonene-3, 17-dione (3) using Penicillium decumbens as biocatalyst was investigated, and selective hydroxylation of 1–3 was observed. Biotransformation of 1 afforded 1α-hydroxy-androst-4-ene-3, 17-dione (4) and 1α, 6β-dihydroxy-androst-4-ene-3, 17-dione (5). Incubation of 19-nor-4-androstene-3, 17-dione (2) with P. decumbens culture resulted in the isolation of compounds 1α-hydroxy-19-nor-4-androstene-3, 17-dione (6) and 1α, 15β-dihydroxy-19-nor-4-androstene-3, 17-dione (7). Biotransformation of 3 gave one product 1α-hydroxy-13β-ethyl-4-gonene-3, 17-dione (8) at the same condition. The four metabolic products 5–8 were reported for the first time and the identified C1-α hydroxylation reaction of steroid by P. decumbens provides an another possibility to perform chemical modifications on C-1.

3-Hydroxyarginine (3-OH-Arg) is an important intermediate for the synthesis of viomycin, an important antibiotic for the clinical treatment of tuberculosis. An efficient strategy for 3-OH-Arg production based on protein engineering and recombinant whole-cell biocatalysis was demonstrated for the first time. To avoid challenging product separation due to the generation of α-ketoglutarate (α-KG) in the system, the molar ratio of the substrates L-Arg and L-Glu was optimized to ensure the efficient production of 3-OH-Arg as well as the complete consumption of α-KG. Through the establishment of a fed-batch process, 3-OH-Arg and succinic acid (SA) production reached to 9.9 g/L and 5.98 g/L after 36 h of reaction under the optimized conditions. This is the highest biosynthetic yield of 3-OH-Arg achieved to date, potentially offering a promising strategy for commercial production of hydroxylated amino acids.

Additional file 1: Table S1. Primers used for the construction of KsdD3 WT and mutants. Table S2. Energy minimization of KsdD3 with and without substrate for the wild-type and mutants. Table S3. Productivity of steroid bioconversion in 48 h (g L−1 h−1). Fig. S1. Purification of KsdD3. Fig. S2. The relative activity of KsdD3 mutants towards steroidal substrates. Fig. S3. Products analysis of steroidal substrates by GC–MS. Fig. S4. A Spectral data for hydrocarbons analysis of 16α,17α-epoxypregna-11α-hydroxy-1,4-diene-3,20-dione by NMR. B Spectral data for hydrocarbons analysis of 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione by NMR. C Spectral data for hydrocarbons analysis of 17α-hydroxypregna-1,4-diene-3,11,20-trione by NMR. Fig. S5. Amino acid sequence alignments of 3-ketosteroid-∆1-dehydrogenase from different strains. Fig. S6. The relative catalytic activity of KsdD3 mutants toward AD. Fig. S7. The relative catalytic activity of saturation mutagenesis on W299 toward AD. Fig. S8. The relative catalytic activity of KsdD WT and mutants toward various steroidal substrates. Fig. S9. Michaelis–Menten plots of KsdD3 WT and W299A, W299G mutants toward nine steroidal substrates. Fig. S10. Potential of mean force (PMF) profiles of the KsdD3 wild type and mutants over the distance along the substrate channel.

BACKGROUND As a low-cost alternative to the traditional ionic liquids (ILs), deep eutectic solvents (DESs) have currently attracted increasing attention for their various applications including biocatalysis. This study aimed at analyzing the effect of DESs on Δ1, 2-dehydrogeneration of cortisone acetate(CA) to prednisone acetate (PA) by Arthrobacter simplex. RESULTS Of the three DESs examined, ChCl:U was successfully employed as co-solvent to improve the bioconversion efficiency. For substrate feeding concentration of 5 g L−1, substrate conversion reached 93% by immobilized A. simplex cells (4 gDW L−1) with ChCl:U content of 6%, which is more efficient than conversions without DESs. Importantly, it has been shown that DESs and the immobilized cells can be easily recovered and then reused for five batches of bioconversion with a final conversion above 80%. CONCLUSION Our results show the potential use of DESs-based systems in industrial steroid biodehydrogenation by A. simplex, and provide an environmentally benign means of steroid biotransformation. © 2015 Society of Chemical Industry

An experiment was designed to assess the effects of a commercial β-mannanase on performance and immunity of tilapia fed plant-based diets. A basal diet was supplemented with 0.0 (control), 0.5 and 1.0 g β-mannase kg−1 to formulate three experimental diets. Each treatment contained 4 tanks with 30 fish per tank. Trial lasted 8 weeks. Our results demonstrated that β-mannanase addition (0.5 and 1.0 g kg−1) improved significantly (P 0.05). β-mannanase supplementation also led to an increase (P 0.05). Moreover, the dietary β-mannanase supplementation groups exhibited an increase in the total leukocyte counts (WBC), differential leukocyte counts, respiratory burst activity, lysozyme activity and superoxide dismutase (SOD) activity compared to the controls (P

Cholesterol oxidase catalyzes the oxidation and isomerization of the cholestane substrates leading to the addition of a hydroxyl group at the C3 position. Rational engineering of the cholesterol oxidase from Pimelobacter simplex (PsChO) was performed. Mutagenesis of V64 and F70 improved the catalytic activities toward cholestane substrates. Molecular dynamics simulations, together with structure-activity relationship analysis, revealed that both V64C and F70V increased the binding free energy between PsChO mutants and cholesterol. F70V and V64C mutations might cause the movement of loops L56-P77, K45-P49 and L350-E354 at active site. They enlarged the substrate-binding cavity and relieved the steric interference with substrates facilitating recognition of C17 hydrophobic substrates with long side chain substrates.

Carbohydrate is one kind of raw materials available in large scale, and they are also important renewable resources for industry. Their oxidation products have many applications in different fields. In this paper, biotransformation of L-rhamnose using Pseudomonas taetrolens resting cells was investigated, and the product was separated and indentified to be L-rhamnonic acid by high-performance liquid chromatography (HPLC) and NMR. P. taetrolens capable of transforming L-rhamnose to L-rhamnonic acid was reported for the first time. The influence of temperature, cell density and shaker speed on L-rhamnonic acid production from L-rhamnose by P. taetrolens was also investigated, and the final production rate reached 90% when the transformation was carried out for about 48 h under the condition of temperature 37 °C, cell concentration, OD600 = 40 and 180 rpm.

BACKGROUND: The improved efficiency of steroid biotransformation using the biphasic system is generally attributed to the positive effect on the solubility of substrate in aqueous media. A promising alternative for the application of organic solvents in biphasic systems is the use of ionic liquids (ILs). This study aims to investigate the applicability of the biphasic ILs/water system for 11α hydroxylation of 16α, 17-epoxyprogesterone (HEP) by Aspergillus ochraceus. RESULTS: Of the seven ILs tested, [C3mim][PF6] exhibited the best biocompatibility, with markedly improved biotransformation efficiency. In the [C3mim][PF6]-based biphasic system, substrate conversion reached 90% under the condition in which buffer pH, volume ratio of buffer to ILs, cell concentration, and substrate concentration were 4.8, 10/1, 165 g L−1 and 20 g L−1, respectively. This is more efficient than that of the monophasic aqueous system. The effects of the cations and anions of these ILs on the 11α hydroxylation of 16α, 17-epoxyprogesterone (HEP) by A. ochraceus is also discussed. CONCLUSION: The above results showed that IL/water biphasic system improved the efficiency of 11α hydroxylation of 16α, 17-epoxyprogesterone (HEP) by A. ochraceus, thus suggesting the potential industrial application of ILs-based biphasic systems for steroid biotransformation. © 2012 Society of Chemical Industry

The effects of oral administration of chitosan oligosaccharides (COS) and Bacillus coagulans , single or combined, on the growth performance, immunity and disease resistance of Cyprinus carpio koi were investigated. The koi (24.9 ± 0.52 g) were divided into 4 groups and each group was fed with diets supplemented with or without immunostimulant for 8 weeks. After 8 weeks of feeding trial, five koi per tank were sampled for immunity determination, ten koi per tank were challenged by Aeromonas veronii . The results showed that the koi fed with diets supplemented with a combination of the COS and B. coagulans had the highest final weight, specific growth rate (SGR), total leukocyte count (WBC), respiratory burst activity, phagocytic activity, lysozyme activity, SOD activity of koi and disease resistance to A. veronii ( P B. coagulans and COS, and the lowest in koi fed with the control diet. However, there were no significant differences ( P A. veronii between dietary B. coagulans and dietary COS. Under the experimental conditions, dietary B. coagulans and COS had a synergistic effect on enhancing immunity and disease resistance of koi ( P

In order to discover the steroid biotransformation ability of filamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1 per thousand, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids

Microbial transformation of 13β-alkyl-3-methoxy-8, 14, secogona-1, 3, 5(10), 9-tetraen-14, 17-dion with Saccharomyces cerevisiae resulted in two metabolites: product 1 (P1) and product 2 (P2). Among these, P1 was identified to be 13β alkyl-3-methoxy-8, 14-seco-1, 3, 5(10), 9(11)-estratetraene-17β-ol, 14-one, which has been studied extensively. P2 was purified, crystallized, and identified as 13β-alkyl-3-methoxy-8, 14, secogona-1, 3, 5(10), 9-tetraen-14, 17-diol by X-ray single crystal diffraction method. It was first reported in fermentation medium and the dihydroxyl reduction is a new reaction in biotransformation of steroid.

Screening of strains for improvement in the yields of the desired steroid as well as production of novel steroid is highly demanded. In this study, a filamentous fungus Aspergillus oryzae capable of transforming steroid efficiently was screened from the collection of fresh bark of south China. A novel transformation product was purified, crystallized, and determined as [13, 17β-]furan-17-hydroxyl-4-gonene-3one by single-crystal X-ray diffraction. At the end of the transformation, the yields of the product reached to 95 %, which indicated A. oryzae can be efficiently applied in the field of steroid biotransformation with the novel biocatalytic ability.

Improved efficiency of steroid microbial transformation by co-solvents is generally attributed to their positive effects on the solubility of substrate in aqueous media. Ionic liquids have been widely researched as possible ‘green’ replacements for organic solvents. In this study, the effect of water miscible ionic liquids on the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii ATCC 10490 was investigated. The results indicated that the striking improvement of biotransformation of substrates up to 94.46 % were obtained after addition of [BMIM]BF4 under the optimized conditions. This also resulted in morphological changes of P. raistrickii mycelia in the fermentation broth with or without [BMIM]BF4. Based on the above observations, we therefore conclude that [BMIM]BF4-induced changes of mycelial morphology of P. raistrickii have positive effects on the production of the desired product, thus most likely contributing to the observed higher yield of steroid biotransformation in addition to [BMIM]BF4 known effects on substrate solubility.

Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.

Biphasic processes are used in whole-cell biotransformation to overcome the low water solubility of substrates and products as well as their inhibitory effects on the biocatalyst. Commercially available [NTf(2)]- and [PF(6)]-based ionic liquids (ILs) were used in a biphasic system for the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii. With the substrate at 5 g l(-1) and a volume ratio of IL to buffer, buffer pH and cell density at, 1:9, 6.5, 16.8 g(DW) l(-1), respectively, the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione was achieved with a yield of 70 % after 72 h using [BMIm][NTf(2)] in a 50 ml biphasic system. This is compared to a 30 % yield in a monophasic aqueous system. This suggests the potential industrial application of ILs-based biphasic systems for steroid biotransformation.

The present study was conducted to investigate the effects of dietary chitooligosaccharides (COS) supplementation on the innate immune response and protection against Vibrio harveyi infection in Trachinotus ovatus. A basal diet was supplemented with 0.0 (control), 2.0, 4.0 and 6.0 g COS kg(-1) to formulate four experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 × 1.0 × 2.0 m), and each cage was stocked with 80 fish (initial average weight 10.8 ± 0.05 g). After 8 weeks of feeding trial, Both the final weight and specific growth rate (SGR) significantly increased with increasing dietary COS levels up to 4.0 g kg(-1), whereas there were no significant differences for COS levels from 4.0 to 6.0 g kg(-1). A decreased feed conversion ratio (FCR) was observed with increasing dietary COS levels. The total leukocyte counts (WBC), differential leukocyte counts, respiratory burst activity, lysozyme and superoxide dismutase (SOD) activity were significantly increased with the increased levels of dietary COS (P0.05), and reached a maximum at level of 4.0 g kg(-1) COS. There were no significant differences in those immunological parameters between 4.0 and 6.0 g kg(-1) COS. Moreover, the dietary COS supplementation groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with V. harveyi. These results indicated that dietary intake containing COS could enhance the immune responses of fish and improve its resistance to infection by V. harveyi. Especially supplementation with 4.0 g kg(-1) COS to the fish for 56 days showed considerable improvement in the growth, survival and immune response of the fish.

Group II introns, found in bacterial genones and organelles of eukaryotes, are potential tools in genetic engineering and genomic analysis. The new properties of group II introns were first explored by ionizing radiation giving rise to multiple cellular and biological effects. Experimental results showed that group II introns can be effective vectors in gene therapy.