Arthrobacter Simplex Research Articles

ITRAQ-Based Quantitative Proteomic Analysis of Arthrobacter simplex in Response to Cortisone Acetate and Its Mutants with Improved Δ1-Dehydrogenation Efficiency.

Arthrobacter simplex is extensively used for cortisone acetate (CA) biotransformation in industry, but the Δ1-dehydrogenation molecular fundamental remains unclear. Herein, the comparative proteome revealed several proteins with the potential role in this reaction, which were mainly involved in lipid or amino acid transport and metabolism, energy production and conversion, steroid degradation, and transporter. The influences of six proteins were further confirmed, where pps, MceGA, yrbE4AA, yrbE4BA, and hyp2 showed positive impacts, while hyp1 exhibited a negative effect. Additionally, KsdD5 behaved as the best catalytic enzyme. By the combined manipulation in multiple genes under the control of a stronger promoter, an optimal strain with better catalytic enzyme activity, substrate transportation, and cell stress tolerance was created. After biotechnology optimization, the production peak and productivity were, respectively, boosted by 4.1- and 4.0-fold relative to the initial level. Our work broadens the understanding of the Δ1-dehydrogenation mechanism, also providing effective strategies for excellent steroid-transforming strains.

Construction of automated high-throughput screening method for finding efficient 3-ketosteroid 1,2-dehydrogenating strains.

Dehydrogenation reaction at C1(2) positions is typical and representative of industrial production of steroid drugs. Anti-inflammatory activity can be doubled when the nucleus of the anti-inflammatory steroid hormone drug introduces double bonds at the C1(2) positions. Arthrobacter simplex is currently the most widely studied and used strain for C1(2) dehydrogenation. Therefore, breeding Arthrobacter simplex with high-efficiency dehydrogenation ability is of great significance. In order to obtain high-efficiency strains, the research proposed a new screening strategy based on image process technique: firstly, a color reaction between 2,4-dinitrophenylhydrazine (DNPH) and 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) was established to characterize the dehydrogenation ability of the strain; secondly, the color data of strains mutated by atmospheric and room temperature plasma (ARTP) in the "color reaction" were automated and analyzed for dehydrogenation ability prediction using optimized support vector machine model. Result showed that the prediction accuracy reached as high as 96% in verification experiments. After a series of mutagenesis, including breaking the bottleneck of a single mutation in ARTP, the dominant strain ARLU-146 was finally obtained from 5168 strains. Its initial conversion rate was 0.8059g/L/h, with a conversion of 94.41% at 24h, compared to the original strain ASP which increased the transformation rate by more than 10%. By further process optimization, a high conversion (94.34% within 20h) with high substrate (85g/L cortisone acetate) was achieved. According to literature research, it is the highest conversion at this substrate concentration. KEY POINTS: • A high-throughput screening method was developed by using image processing and machine learning technique. • "Mutation bottleneck" of single ARTP mutagenesis was surpassed by complex mutagenesis. • A high substrate (85g/L CA) and high transformation rate craft (94.34% within 20h) were built.

Mining and characterization of 3-ketosteroid-∆1-dehydrogenases from Arthrobacter simplex genome and applications for steroid dehydrogenation

Steroid ∆1-dehydrogenation catalyzed by 3-ketosteroid-∆1-dehydrogenase (KsdD) plays key roles in steroid biodegradation and industrial steroid modification. In this study, five KsdDs from Arthrobacter simplex were heterogeneously expressed, purified and characterized. Of the five KsdDs, KsdD1 and KsdD2 showed the highest catalytic activity toward androstenedione (AD), cortisone and cortisone acetate (CA) except for KsdD4. Molecular dynamics (MD) simulations indicated that KsdD1 and KsdD2 have relatively higher binding energy in substrate recognition. Furthermore, Compared with KsdD1, KsdD2 showed higher stability according to root mean square fluctuations (RMSF) and root mean square deviations (RMSD) analysis. To further increase the ∆1-dehydrogenation efficiency, a novel deep eutectic solvent (DES) was used in the reaction system. The highest conversion was obtained using KsdD2 as the biocatalyst in a choline chloride:glycerol (ChCl:Gly) containing system. To our best knowledge, this is the first report on the purification and characterization of KsdD1 and KsdD2 from Arthrobacter simplex. KsdD2 has been shown to be a promising candidate for developing efficient process for industrial production of steroid-based pharmaceuticals.

Genomewide Transcriptome Responses of Arthrobacter simplex to Cortisone Acetate and its Mutants with Enhanced Δ1-Dehydrogenation Efficiency.

Due to its superior Δ1-dehydrogenation ability, Arthrobacter simplex has been widely used for the biotransformation of cortisone acetate (CA) into prednisone acetate (PA) in the steroid industry. However, its molecular fundamentals are still unclear. Herein, the genome organization, gene regulation, and previously unreported genes involved in Δ1-dehydrogenation are revealed through genome and transcriptome analysis. A comparative study of transcriptomes of an industrial strain induced by CA or at different biotransformation periods was performed. By overexpression, the roles of six genes in CA conversion were confirmed, among which sufC and hsaA behaved better by reinforcing catalytic enzyme activity and substrate transmembrane transport. Additionally, GroEL endowed cells with the strongest stress tolerance by alleviating oxidative damage and enhancing energy levels. Finally, an optimal strain was created by coexpressing three genes, achieving 46.8 and 70.6% increase in PA amount and productivity compared to the initial values, respectively. Our study expanded the understanding of the Δ1-dehydrogenation mechanism and offered an effective approach for excellent steroid-transforming strains.

Improving Biotransformation Efficiency of Arthrobacter simplex by Enhancement of Cell Stress Tolerance and Enzyme Activity.

Arthrobacter simplex exhibits excellent Δ1-dehydrogenation ability, but the acquisition of the desirable strain is limited due to lacking of comprehensive genetic manipulation. Herein, a promoter collection for fine-tuning gene expression was achieved. Next, the expression level was enhanced and directed evolution of the global transcriptional factor (IrrE) was applied to enhance cell viability in systems containing more substrate and ethanol, thus contributing to higher production. IrrE promotes a stronger antioxidant defense system, more energy generation, and changed signal transduction. Using a stronger promoter, the enzyme activities were boosted but their positive effects on biotransformation performance were inferior to cell stress tolerance when exposed to challenging systems. Finally, an optimal strain was created by collectively reinforcing cell stress tolerance and catalytic enzyme activity, achieving a yield 261.8% higher relative to the initial situation. Our study provided effective methods for steroid-transforming strains with high efficiency.

Biotechnological Transformation of Hydrocortisone into 16α-Hydroxyprednisolone by Coupling Arthrobacter simplex and Streptomyces roseochromogenes.

16α-Hydroxyprednisolone, an anti-inflammatory drug, could be potentially obtained from hydrocortisone bioconversion by combining a 1,2-dehydrogenation reaction performed by Arthrobacter simplex ATCC31652 with a 16α-hydroxylation reaction by Streptomyces roseochromogenes ATCC13400. In this study we tested, for the first time, potential approaches to couple the two reactions using similar pH and temperature conditions for hydrocortisone bioconversion by the two strains. The A. simplex capability to 1,2-dehydrogenate the 16α-hydroxyhydrocortisone, the product of S. roseochromogenes transformation of hydrocortisone, and vice versa the capability of S. roseochromogenes to 16α-hydroxylate the prednisolone were assessed. Bioconversions were studied in shake flasks and strain morphology changes were observed by SEM. Whole cell experiments were set up to perform the two reactions in a sequential mode in alternate order or contemporarily at diverse temperature conditions. A. simplex catalyzed either the dehydrogenation of hydrocortisone into prednisolone efficiently or of 16α-hydroxyhydrocortisone into 16α-hydroxyprednisolone in 24 h (up to 93.9%). Surprisingly S. roseochromogenes partially converted prednisolone back to hydrocortisone. A 68.8% maximum of 16α-hydroxyprednisolone was obtained in 120-h bioconversion by coupling whole cells of the two strains at pH 6.0 and 26 °C. High bioconversion of hydrocortisone into 16α-hydroxyprednisolone was obtained for the first time by coupling A. simplex and S. roseochromogenes.

Identification, Biological Characteristics, and Active Site Residues of 3-Ketosteroid Δ1-Dehydrogenase Homologues from Arthrobacter simplex.

3-Ketosteroid Δ1-dehydrogenase (KsdD) is the key enzyme responsible for Δ1-dehydrogenation, which is one of the most valuable reactions for steroid catabolism. Arthrobacter simplex has been widely used in the industry due to its superior bioconversion efficiency, but KsdD information is not yet fully clear. Here, five KsdD homologues were identified in A. simplex CGMCC 14539. Bioinformatic analysis indicated their distinct properties and structures. Each KsdD was functionally confirmed by transcriptional response, overexpression, and heterologous expression. The substantial difference in substrate profiles might be related to the enzyme loop structure. Two promising enzymes (KsdD3 and KsdD5) were purified and characterized, exhibiting strong organic solvent tolerance and clear preference for 4-ene-3-oxosteroids. KsdD5 seemed to be more versatile due to good activity on substrates with or without a substituent at C11 and high optimal temperature and also possessed unique residues. It is the first time that KsdDs have been comprehensively disclosed in the A. simplex industrial strain.

Compatible solutes adaptive alterations in Arthrobacter simplex during exposure to ethanol, and the effect of trehalose on the stress resistance and biotransformation performance.

Ethanol-tolerant Arthrobacter simplex is desirable since ethanol facilitates hydrophobic substrates dissolution on an industrial scale. Herein, alterations in compatible solutes were investigated under ethanol stress. The results showed that the amount of trehalose and glycerol increased while that of glutamate and proline decreased. The trehalose protectant role was verified and its concentration was positively related to the degree of cell tolerance. otsA, otsB and treS, three trehalose biosynthesis genes in A. simplex, also enhanced Escherichia coli stress tolerance, but the increased tolerance was dependent on the type and level of the stress. A. simplex strains accumulating trehalose showed a higher productivity in systems containing more ethanol and substrate because of better viability. The underlying mechanisms of trehalose were involved in better cell integrity, higher membrane stability, stronger reactive oxygen species scavenging capacity and higher energy level. Therefore, trehalose was a general protectant and the upregulation of its biosynthesis by genetic modification enhanced cell stress tolerance, consequently promoted productivity.

Highly efficient synthesis of boldenone from androst-4-ene-3,17-dione by Arthrobacter simplex and Pichia pastoris ordered biotransformation.

Boldenone (BD) is an important steroid hormone drug which is the derivative of testosterone. In this study, an ordered biotransformation method was proposed employing Arthrobacter simplex and recombinant Pichia pastoris with 17β-hydroxysteroid dehydrogenase from Saccharomyces cerevisiae to produce BD from androst-4-ene-3,17-dione (AD) efficiently. To lower the oxidation towards BD in A. simplex, the transformation was conducted sequentially by C1,2 dehydrogenation in A. simplex and 17β-carbonyl reduction in recombinant P. pastoris GS115. Moreover, the reaction system was inactivated before recombinant P. pastoris GS115 was added to further inhibit the oxidation of BD by A. simplex, and the productivity of BD was improved 10.6% compared with the control. Furthermore, by optimizing the conditions of transformation from AD to BD, 4.2g/L BD was generated with 83% productivity from 5.0g/L AD, which was the highest productivity reported by biological method. This study offers a promising method to produce BD by ordered biotransformation system, which can also be used to manufacture other steroidal compounds that are difficult to acquire directly.

Engineering of 3-ketosteroid-\u22061-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates

BackgroundBiosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.ResultsRecombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).ConclusionsThe successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.

The ethanol-induced global alteration in Arthrobacter simplex and its mutants with enhanced ethanol tolerance.

Arthrobacter simplex has received considerable interests due to its superior Δ1-dehydrogenation ability. Ethanol used as co-solvent is a stress commonly encountered during biotransformation. Therefore, studies of ethanol tolerance of A. simplex are of great importance to improve the biotransformation efficiency. In this paper, the combined analysis of physiological properties, cell compositions, stress-responsive metabolites, and proteome profiles was carried out to achieve a global view of ethanol tolerance of A. simplex. Under sublethal conditions, cell permeability and membrane fluidity exhibited concentration-dependent increase by affecting the contents or compositions of cell peptidoglycan, lipids, phospholipids, and fatty acids. Among them, cis-trans isomerization of unsaturated fatty acids was a short-term and reversible process, while the changes in phospholipid headgroups and increase in saturation degree of fatty acids were long-term and irreversible processes, which collectively counteracted the elevated membrane fluidity caused by ethanol and maintained the membrane stability. The decreased intracellular ATP content was observed at high ethanol concentration since proton motive force responsible for driving ATP synthesis was dissipated. The involvement of trehalose and glycerol, oxidative response, and DNA damage were implicated due to their changes in positive proportion to ethanol concentration. Proteomic data supported that ethanol invoked a global alteration, among which, the change patterns of proteins participated in the biosynthesis of cell wall and membrane, energy metabolism, compatible solute metabolism, and general stress response were consistent with observations from cell compositions and stress-responsive metabolites. The protective role of proteins participated in DNA repair and antioxidant system under ethanol stress was validated by overexpression of the related genes. This is the first demonstration on ethanol tolerance mechanism of A. simplex, and the current studies also provide targets to engineer ethanol tolerance of A. simplex.

Biochemical and structural characterization of 3‐ketosteroid‐Δ1‐dehydrogenase, a candidate enzyme for efficient bioconversion of steroids

AbstractBackground3‐ketosteroid‐Δ1‐dehydrogenase, a key enzyme involved in microbial catabolism of sterols, catalyzes the 1, 2‐desaturation of steroidal substrates using FAD as a cofactor. Recombinant 3‐ketosteroid‐Δ1‐dehydrogenase from Arthrobacter simplex (KsdD4) was expressed in Escherichia coli BL21 (DE3).ResultsKsdD4 exhibited optimal activity at pH 7.0 and 35°C. KsdD4 had the highest activity toward 4‐androstene‐3,17‐dione (AD) and a moderate activity toward cortisone acetate and hydrocortisone. Structure‐based homology modeling revealed that Y118, Y355, Y528 and G532 are located in the active site, thus the most likely catalytic residues. The substrate‐binding cavity was composed of hydrophobic residues with small side chains, including A49, V328, A390 and A531, which facilitate the recognition of steroidal substrates with large C17 side‐chains. E332 forms a unique hydrogen bond with substrates. Recombinant E. coli resting cells expressing KsdD4 showed high catalytic activity in the presence of various organic solvents. A fed‐batch bioconversion using resting cells resulted in efficient production of 2.66 g L−1 androst‐1,4‐diene‐3,17‐dione (ADD).ConclusionBiochemical data and structural analysis greatly advanced our understanding of the KsdD family enzymes, and the fed‐batch strategy improved the bioconversion of steroids. © 2018 Society of Chemical Industry

IrrE Improves Organic Solvent Tolerance and Δ1-Dehydrogenation Productivity of Arthrobacter simplex.

During steroid bioconversion, organic solvents are widely used for facilitating hydrophobic substrate dissolution in industry. Thus, strains that tolerate organic solvents are highly desirable. IrrE, a global transcriptional factor, was introduced into Arthrobacter simplex with Δ1-dehydrogenation ability. The results evidenced that IrrE did not affect cell biological traits and biotransformation performance under non-stress conditions. However, the recombinant strain achieved a productivity higher than that of the control strain in systems containing more ethanol and substrate, which coincided with cell viability under ethanol stress, the major stress factor during biotransformation. It also demonstrated that IrrE caused genome-wide transcriptional perturbation, and several defense proteins or systems were linked with higher organic solvent tolerance. IrrE simultaneously enhanced cell resistance to various stresses, and its horizontal impacts showed strain and stress dependence. In conclusion, the introduction of exogenous global regulators is an efficient approach to enhance organic solvent tolerance in steroid-transforming strains, resulting in higher productivity.

Synergistic effects of components in deep eutectic solvents relieve toxicity and improve the performance of steroid biotransformation catalyzed by Arthrobacter simplex

AbstractBACKGROUNDThe discovery of deep eutectic solvents (DESs) has introduced a new class of promising green solvents to overcome many drawbacks associated with organic solvents. In this study, the synergistic effects of the components in the form of DESs on bacterial viability, membrane integrity, and retention of metabolic activity as well as the efficiency of Δ1,2‐dehydrogeneration of cortisone acetate (CA) using whole cells of Arthrobacter simplex were investigated.RESULTSThe agar disc diffusion method, viability assays and the retention of sugar metabolic activity were used to investigate their toxicity to A. simplex. The results indicated that colinium salts were more toxic than the corresponding hydrogen bond donor (HBDs), but their deleterious effect could be weakened by incorporating them into DESs. Moreover, the best catalytic performance was found in the ChCl:U(DES)‐containing system when Δ1,2‐dehydrogeneration using A. simplex was carried out in three different systems followed by the addition of DES, their components and their mixtures, respectively. In addition, metabolites with significantly changed concentration were identified by means of gas chromatography–mass spectrometry (GC–MS) and principal component analysis, which revealed five compounds that may be used as potential indicators to discriminate the effects of different concentrations of DESs on A. simplex.CONCLUSIONThe cholinium salts used in this study were toxic to A. simplex to some extent, while synergistic effects were observed after DES formation. The efficient biotransformation in DESs especially in the ChCl:U(DES)‐containing system potentially offers a promising strategy for the production of many different steroids. © 2018 Society of Chemical Industry

Sodium alginate-grafted β-cyclodextrins as a matrix for immobilized Arthrobacter simplex for cortisone acetate biotransfromation

Cyclodextrins (CDs) are used to resolve the low aqueous solubility of steroids, but the high cost of CDs is still a limiting factor in biotransformation process. This study, which is based on grafting and immobilization techniques, focused on synthesizing for the first time sodium alginate (SA)-grafted β-CD (SA-β-CD) and alginate-grafted β-CD for the immobilization of Arthrobacter simplex (ASP) cells (SA-β-CD-cells) and subsequent recycling of CDs and cells. FTIR spectium and X-ray diffraction proved that β-CD was successfully grafted with SA, whereas the grafting yield of β-CD was 10.3 μmol g−1. SA-β-CD could increase the solubility of CA by 3.5-fold, whereas the transformation rate was enhanced by 10%. The conversion ratio of CA was over 92% after the SA-β-CD recycling for nine cycles. In addition, after SA-β-CD-cells were applied in biocatalytic reactions for eight cycles, the conversion ratio of CA was over 90%. These advantages suggest great potential for using both grafting and immobilized techniques in steroid transformation.

Effect of β-cyclodextrins Derivatives on Steroids Biotransformation by Arthrobacter simplex.

β-cyclodextrins derivatives (CDs) have applied in steroids biotransformation industry because of their unique properties. However, the effect of β-CDs on the growth rate, activity, conversion, and characters of whole cells has not been concerned. In this study, the growth rate and cellular morphology of Arthrobacter simplex (ASP) pretreated by six kinds of β-CDs were measured. The results showed that most β-CDs inhibited the growth of ASP, among which randomly methylated-β-CDs has the most serve inhibition; however, sulfonic acid-β-CD promoted the growth of cells. The morphology size and the surface of all β-CDs-pretreated cells were changed compared with the control group. Besides, the conversion of cortisone acetate (CA) increased in β-CDs-pretreatment system and β-CDs-containing system, which reached 97.98% in hydroxypropyl-β-cyclodextrin (HP-β-CD) containing-system and 78.69% in HP-β-CD-pretreatment-system, but the dehydrogenase activity of all β-CDs-pretreated cells decreased. β-CDs with higher K value have stronger inclusion ability with CA, and along with the membrane permeability of β-CDs-pretreated cells increased more, but they also have more serve damage on the ASP cells, which is negative to increase the conversion of CA. This study improved our understanding of the effect of β-CDs when they were used in the steroids biotransformation by ASP whole cells, and provided data basis for the selection of suitable CDs for application.

Influence of imidazolium‐based ionic liquids on steroid biotransformation by Arthrobacter simplex

AbstractBACKGROUNDIonic liquids (ILs) have become increasingly attractive as alternatives for organic solvents. In this study, the influences of ILs on the cellular growth rate, cellular activity, cortisone acetate (CA) conversion, CA solubility, and cell membrane permeability of Arthrobacter simplex CPCC 140451 (ASP) were examined.RESULTSResults showed that ILs with ≥3 carbon atoms in their side chains increased CA conversion, CA solubility, and cell membrane permeability of ASP and universally inhibited the cellular growth rate and activity, with ILs with [PF6]‐anion exhibiting the most severe inhibition activity. Inhibition was also more serious using ILs with longer alkyl side chains and at higher concentrations. CA conversion, CA solubility, and leakage of cell inclusions showed higher increase under ILs with [PF6]‐anion than those of ILs with [BF4]‐anion and lower increase for ILs with longer side chains.CONCLUSIONThis study improved the understanding of the influence and primary selection of imidazolium‐based ILs when applied in steroid biotransformation with Arthrobacter simplex whole cells. The data provided a foundation for the selection of suitable ILs for various applications. © 2017 Society of Chemical Industry

QUANTIFICATION OF EXEMESTANE ACCUMULATION DURING MICROBIAL BIOCONVERSION BY TLC IMAGE ANALYSIS

Objective: The present study was aimed at developing a rapid, cost effective and accurate method for quantification of exemestane using thin layer chromatography (TLC) separation followed by image analysis and to test it for monitoring the accumulation of exemestane during microbial bioconversion.Methods: After microbial bioconversion and TLC separation of products formed, exemestane was quantified using ImageQuant TL v2003 image analysis software and the results were compared with high performance liquid chromatography (HPLC) analysis.Results: The percentage error between TLC and HPLC analyses was ranged from (-) 5.18 to (+) 5.51. Bacterial strains Arthrobacter simplex IAM 1660, Nocardia sp. MTCC 1534, Pseudomonas putida MTCC 1194 and Rhodococcus rhodochorus MTCC 291 respectively yielded 79.7 (72 h), 63.9 (72 h), 69.8 (96 h) and 83.2 (96 h) mole percent bioconversion of 6-methylene androstenedione to exemestane. Conclusion: Rhodococcus rhodochorus MTCC 291 was found to be the most suitable organism for the bioconversion and may be used to develop an eco-friendly route to replace chemical synthesis that eliminates the use of toxic chemicals and side products.

SUBSTRATE CARRIERS FOR C-1(2)-DEHYDROGENATION OF 6-METHYLENE ANDROSTENEDIONE TO EXEMESTANE BY GROWING AND IMMOBILIZED ARTHROBACTER SIMPLEX NCIM 2449

Objective: Permeability of hydrophobic steroid substrates across cell membrane is a critical factor during microbial bioconversion. To increase substrate intake, the feasibility of some organic solvents and emulsifiers as substrate carrier on the bioconversion of 6-methylene androstenedione to exemestane was assessed.Methods: Androstenedione, a commonly available steroid precursor, was chemically converted 6-methylene androstenedione. The time course of exemestane accumulation was estimated after addition of 6-methylene androstenedione dissolved in some organic solvents or dispersed with emulsifiers by growing and immobilized cells of Arthrobacter simplex NCIM 2449 in shake flask cultures. Results: The use of substrate carriers for addition of 6-methylene androstenedione enhanced the bioconversion several folds. With growing bacterium in triplicate flasks, a peak mol % bioconversion recorded was- ethanol (67.25, 72 h); soybean oil + tween 80 (50.37, 48 h); acetone (38.84, 48 h); soybean oil (38.36, 48 h); lecithin (32.73, 48 h), methanol (32.71, 48 h) and tween 80 (10.37, 48 h). As compared to the growing cells, the bioconversion with Ca-alginate immobilized cells was delayed and peak mol % bioconversion was recorded as ethanol (60.78, 120 h); soybean oil + tween 80 (42.98, 120 h); methanol (40.50, 72 h); soybean oil (38.36, 48 h); acetone (31.18, 72h ) and lecithin (33.67, 120 h); tween 80 (13.87, 120 h).Conclusion: The use of substrate carriers for addition of 6-methylene androstenedione increased the permeability of substrate and may be used to increase the yield of exemestane and reduce incubation time.

A new technique for promoting cyclic utilization of cyclodextrins in biotransformation.

Cyclodextrins (CDs) can improve the productivity of steroid biotransformation by enhancing substrate solubility. CDs can be recycled by grafting them with appropriate carriers. Loofah fiber is an excellent grafting material for CDs, and can be applied to the biotransformation and recycling of β-cyclodextrin (β-CD). In this work, a technique for recycling β-CD in cortisone acetate (CA) biotransformation by Arthrobacter simplex CPCC 140451 was studied. Loofah fiber-grafted β-CD (LF-β-CD) was prepared using epichlorohydrin, which is a cross-linking agent. The grafting yield of β-CD was 74.8mgg-1 dried fibers. LF-β-CD could increase the solubility of CA and enhance biotransformation. The initial conversion rate of CA was 1.5-fold higher than that of the blank group. LF-β-CD was also used in biocatalytic reactions for eight cycles, and it maintained the conversion ratio of CA at approximately 90%. Given the above positive results, LF-β-CD can be utilized in biotechnological recycling applications. This method can also be applied to CD derivatives and hydrophobic compounds.