Biotransformation Conditions Research Articles

Production of isofloridoside from galactose and glycerol using α-galactosidase from Alicyclobacillus hesperidum

Isofloridoside (D-isofloridoside and L-isofloridoside) is the main photosynthetic product in red algae. Here, given the importance of isofloridoside, a potentially effective method to produce isofloridoside from galactose and glycerol using whole-cell biocatalysts harboring α-galactosidase was developed. α-Galactosidase-encoding genes from Alicyclobacillus hesperidum, Lactobacillus plantarum, and Bifidobacterium adolescentis were cloned and the proteins were overproduced in Escherichia coli. The α-galactosidase from A. hesperidum (AHGLA) was chosen to synthesize isofloridoside. The effects of reaction pH, temperature, and substrate concentration were investigated. In the optimum biotransformation conditions, the final isofloridoside concentration reached 0.45 M (galactose conversion 23 %). The reaction mixtures were purified using activated charcoal and calcined Celite, and the purified product was identified as a mixture of D- and L-isofloridoside by liquid chromatography-mass spectrometry and nuclear magnetic resonance. This study provides a possible feasible method for the biosynthesis of isofloridoside from low-cost glycerol and galactose.

Biotransformation using resting cells of Rhodococcus UKMP-5M for phenol degradation

Abstract Phenol is a toxic compound that may be transformed into non-toxic compounds by the activity of microbial cells. The possibility of using biotransformation method for the degradation of phenol was studied using the whole cells of Rhodococcus UKMP-5M suspended in 250 mL shake flask with buffered liquid containing phenol. The cells of Rhodococcus UKMP-5M were produced by cultivation in Minimal Salt Medium (MSM) with the addition of phenol and/or glucose as carbon source. The biotransformation conditions to obtain the highest percentage of phenol degradation were as follows; pH 7.4, 0.5 g/L phenol in MSM as biotransformation medium, cells were produced by cultivation in MSM supplemented with 0.5 g/L phenol and the optimal cell concentration was 10%. The phenol degradation rate obtained in biotransformation using Rhodococcus UKMP-5M cells correlated well with phenol hydroxylase activity. The highest percentage of phenol degradation in biotransformation using suspended cells of Rhodococcus UKMP-5M was only up to 89%, which was slightly lower than those obtained in growing cell system (98%).

A Biotransformation Process for Production of Genistein from Sophoricoside by a Strain of Rhizopus oryza

Genistein is known to have multiple biological activities and has great potential for use as a preventative medicine and in disease treatment. Genistein can be extracted from plants, but also can be obtained from its glycoside form, sophoricoside, which is more abundant in some plants. Biotransformation by unpurified microbial enzymes has the advantage of low cost and is a preferred method for production of natural compounds. This study isolated a strain of Rhizopus oryzae that could produce β-glucosidase, which efficiently hydrolyzes sophoricoside into genistein, from an enrichment culture of the dried fruits of Sophora japonica. After the composition of enzyme-producing medium and biotransformation conditions were optimized, a genistein yield of 85.6% was obtained after 24 h in a shake-flask biotransformation at pH 7.0 using an initial substrate concentration of 1 g/L. The developed process provides an alternative method for production of genistein, and would be suitable for scale-up production in the pharmaceutical industry.

Co-biosynthesis of 3-hydroxypropionic acid and 1,3-propanediol by a newly isolated Lactobacillus reuteri strain during whole cell biotransformation of glycerol

The probiotic bacterium, Lactobacillus reuteri, possesses a unique pathway for metabolizing glycerol into 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PD), which are two important platform chemicals. In this work, a potent L. reuteri strain, designated as FXZ014, was isolated and identified from healthy human feces for co-biosynthesis of 3-HP and 1,3-PD during whole cell biotransformation of glycerol. Initially, biotransformation conditions were optimized for this new strain using single factor optimization and response surface methodology (RSM). Under the optimum batch conditions, FXZ014 produced 8.74 ± 0.24 g/L of 3-HP and 9.94 ± 0.36 g/L of 1,3-PD with a corresponding net yield of 0.67 mol/mol. Finally, efficiency of the isolated strain in the co-production of these two metabolites was compared with the same of a reference strain, namely L. reuteri CICC 6118. Compared to the reference strain, the amounts of 3-HP and 1,3-PD produced by the isolated strain were significantly higher that corresponded to 38.4% and 21.14%, respectively. Therefore, FXZ014 could be a promising natural strain for co-biosynthesis of 3-HP and 1,3-PD from glycerol.

Phenol biodegradation by Candida tropicalis ATCC 750 immobilized on cashew apple bagasse

Phenol biodegradation by cells of Candida tropicalis ATCC 750, both free and immobilized onto cashew apple bagasse, an alternative support widely acknowledged as cheap and abundant, was studied. Microscopy analyses revealed that the yeast was successfully immobilized by adsorption with an efficiency of 45.4%. The best conditions for phenol biotransformation were found to be at 30 °C, pH 6.0, 150 RPM and with 1100 mg L−1 of initial phenol concentration, for both free and immobilized cells. The adapted and immobilized strain was more resistant to higher phenol concentrations. The metabolic pathway of the phenol biotransformation was the ortho-cleavage of the aromatic ring by the enzyme 1,2-dioxygenase. The biocatalyst showed a high operational stability and the storage studies revealed that 85% of the immobilized cells-CAB remained effectively retained during storage at 4 °C up to 30 days. The results obtained confirm the applicability of cashew apple bagasse as a support matrix for the immobilization of C. tropicalis ATCC 750 and endorses the protocol proposed as a potential biotechnological process for phenolic degradation in industrial wastewater.

Biotransformation of ginsenoside using covalently immobilized snailase enzyme onto activated carrageenan gel beads

Ginsenoside transformation has received significant attention from scientists. The main objective of this study is to use immobilized enzymes in ginsenoside transformation. Factors affecting immobilization process were studied; carrageenan beads treated with polyethyleneimine and then activated using glutaraldehyde (GA) were used for snailase enzyme immobilization. The functionalized gel beads were characterized using Fourier transform infrared spectroscopy to verify the modification process. Furthermore, the optimum conditions for biotransformation of ginsenoside were also deliberated and showed that optimum biotransformation pH is 4.5 and 5–5.5 and temperature 50 and \(60^{\circ }\hbox {C}\) for free and immobilized snailase, respectively. Michaelis constants, \(K_{\mathrm{m}}\) and \(V_{\max }\), were also studied. The immobilized enzyme retains 96% of its initial activity after being used 10 consecutive times. The results clearly suggested that ginsenoside transformation was performed using immobilized snailase; this process can reduce the transformation cost as the enzyme can be reused many times.

Establishment of culture conditions for bio-transformation of R-(+)-limonene to limonene-1,2-diol by Colletotrichum nymphaeae CBMAI 0864

Abstract Before reaching commercial scale, a biotechnological process must be well characterized in terms of process conditions. Thus, this study aimed to investigate the culture conditions in limonene bio-transformation to limonene-1,2-diol by Colletotrichum nymphaeae. The results revealed that this bio-process was aerobic, suggesting that aeration is an important parameter to be considered on a larger scale (e.g. bio-reactors). Moreover, a single feeding of R-(+)-limonene (15 g.L−1) at the start of the bio-transformation resulted in the highest concentration (4.19 g.L−1) and yield (27.9%, w. w−1) of limonene-1,2-diol, indicating that fed-batch operation was not a good choice. In terms of biomass age, 72 h-old biomass performed better when compared to 24 or 48 h-old biomasses. Substrate induction test suggested that this bio-transformation is carried out by non-inducible enzymes. Three successive freeze-thawing processes did not present any observable change in the production, indicating that the biomass could be stored frozen. It was also evidenced that the use of resting cells was as efficient as growing cells, and, therefore, biomass recovery/re-suspension was unnecessary. Although feasible, the bio-transformation in an aqueous-hexadecane biphasic system was not indicated for this process, since lower concentration of diol was obtained. These results are very important to guide scaled-up studies of this bio-transformation process.

Direct biodegradation of eugenol to coniferyl aldehyde and other higher value-added products by Gibberella fujikuroi ZH-34

Abstract Background Eugenol is an economically favorable substrate for the microbial biotransformation of aromatic compounds. Coniferyl aldehyde is one kind of aromatic compound that is widely used in condiment and medical industries; it is also an important raw material for producing other valuable products such as vanillin and protocatechuic acid. However, in most eugenol biotransformation processes, only a trace amount of coniferyl aldehyde is detected, thus making these processes economically unattractive. As a result, an investigation of new strains with the capability of producing more coniferyl aldehyde from eugenol is required. Results We screened a novel strain of Gibberella fujikuroi, labeled as ZH-34, which was capable of transforming eugenol to coniferyl aldehyde. The metabolic pathway was analyzed by high-performance liquid chromatography–mass spectrometry and transformation kinetics. The culture medium and biotransformation conditions were optimized. At a 6 h time interval of eugenol fed-batch strategy, 3.76 ± 0.22 g/L coniferyl aldehyde was obtained, with the corresponding yield of 57.3%. Conclusions This work improves the yield of coniferyl aldehyde with a biotechnological approach. Moreover, the fed-batch strategy offers possibility for controlling the target product and accumulating different metabolites. How to cite: Chen P-C, Zhang H, Zheng P. Direct biodegradation of eugenol into coniferyl aldehyde and other higher value-added products by Gibberella fujikuroi ZH-34. Electron J Biotechnol 2019;38. https://doi.org/10.1016/j.ejbt.2018.12.001 .

BIOTRANSFORMATION OF GINSENOSIDE Rb1 BY BACTERIAL CRUDE ENZYME OF Paenibacillus spp. strain E3 ISOLATED FROM VIETNAMESE GINSENG SOIL

In ginseng, minor ginsenosides were more effective pharmacological properties than major ginsenosides. Therefore, finding bacteria that can convert major ginsenosides has been paying attention. Ginsenoside Rb1 is one of major ginsenosides of ginseng and its biotransformation produces pharmacologically active compounds such as compound K. In this study, the isolation of bacterial strains from Vietnamese ginseng cultivated soil was carried out with the objective of evaluating their hydrolytic capacity for use in biotransformation of ginsenosides Rb1. In the screening of β-glucosidase producing bacteria, seven isolates exhibited black color zones on R2A agar medium containing esculine. Among seven isolates, strain E3 showed the highest ability biotransformation of ginsenosides Rb1 in Luria-Bertani broth. Therefore, strain E3 was selected for further research. Biotransformation of Rb1 was observed by using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) with ginsenoside standards for comparison. We determined the optimal conditions for biotransformation of ginsenoside Rb1 into the compound K of crude enzymes of strain E3 were at 30°C, pH 7,0 and 3 days. Ginsenoside Rb1 was converted into compound K via ginsenoside Rd and F2. The analyses of 16S rRNA gene and phylogenetic tree indicated that strain E3 was closely related to Paenibacillus terrigena with 99,4% identity and formed a discrete cluster with type strain Paenibacillus terrigena A35T with high bootstrap support (99%), supporting strain E3 belonging to the species Paenibacillus terrigena. Physiological characteristics also supported strain E3 belonging to the genus Peanibacillus. This is the first report of biotransformation of ginsenosides using bacterial strains isolated from Vietnamese ginseng cultivated soil in Vietnam. Based on our the obtained results, strain E3 could be applied for the preparation of ginsenoside compound K for use in the cosmetic and pharmaceutical industries.

Enhanced estrogenic effects of biotransformed soy extracts

Soy isoflavones have been associated with several beneficial effects to human health including estrogenic action. Due to the risks associated with pharmaceutical hormone replacement therapy, soy extracts rich in isoflavones and metabolites appear as a safe alternative for menopausal women. This study aimed to improve the process conditions for biotransformation of soymilk polyphenols by tannase to obtain higher amounts of isoflavone aglycones and metabolites such as equol. The estrogenic potential of the extracts was evaluated in in vitro assays. The results showed that the aglycones increased 36–46 times after reaction with tannase and proved that it is possible to produce equol in soymilk using an enzymatic bioprocess, without gut microbial intervention. Moreover, the soymilk biotransformed by tannase presented higher estrogenic action in the MCF-7 BUS cell line assay, making it a promising nutraceutical with possible effects in the attenuation or treatment of menopause symptoms.

Optimizational production of phenyllactic acid by a Lactobacillus buchneri strain via uniform design with overlay sampling methodology

Abstract A Lactobacillus buchneri GBS3 strain isolated from the traditional Chinese pickles was used for the production of 3-phenyllactic acid (PLA), an important compound with antimicrobial activities against a wide species of gram-positive and gram-negative bacteria and some fungi. The growth performance of this strain in the de Man, Rogosa and Sharpe (MRS) medium, the production of metabolites of valuable organic acids, and the biosynthesis of PLA using this strain as the whole-cell biocatalyst and phenylpyruvic acid (PPA) as the precursor, were investigated experimentally. The uniform design method with overlay sampling was developed for the optimization of the biotransformation conditions. The results showed that although it produced naturally lactic acid with the maximum concentration of 1.84 g·L− 1 and PLA with the concentration of 0.015 g·L− 1 after 66 to 72 h cultivation in MRS broth by fermentation, the present strain displayed an effective utilization ability by transforming PPA to PLA. By the uniform design method with overlay sampling for the design and optimization of transformation conditions, a maximum yield of 10.93 g·L− 1 PLA with the mole conversion ratio of 83.07% from PPA to PLA was achieved under the optimized condition, i.e., 20 g·L− 1 glucose, 270 g·L− 1 cells, 13 g·L− 1 PPA, pH 8.0 and the reaction time of 15 h, indicating that Lactobacillus buchneri GBS3 was an interesting strain for the biosynthesis of PLA via the microbial transformation. The prediction of PLA yield under different conditions was achieved successfully based on the limited information of only a small number of experiments by the uniform design with overlay sampling. Therefore, the present methodology is effective and helpful for the optimization of the biosynthesis processes of PLA.

Mimicking a New 2-Phenylethanol Production Pathway from Proteus mirabilis JN458 in Escherichia coli

Bacteria rarely produce natural 2-phenylethanol. We verified a new pathway from Proteus mirabilis JN458 to produce 2-phenylethanol using Escherichia coli to coexpress l-amino acid deaminase, α-keto acid decarboxylase, and alcohol dehydrogenase from P. mirabilis. Based on this pathway, a glucose dehydrogenase coenzyme regeneration system was constructed. The optimal conditions of biotransformation by the recombinant strain E-pAEAKaG were at 40 °C and pH 7.0. Finally, the recombinant strain E-pAEAKaG produced 3.21 ± 0.10 g/L 2-phenylethanol in M9 medium containing 10 g/L l-phenylalanine after a 16 h transformation. Furthermore, when the concentration of l-phenylalanine was 4 g/L (24 mM), the production of 2-phenylethanol reached 2.88 ± 0.18 g/L and displayed a higher conversion rate of 97.38 mol %.

Nanosilica synthesis mediated by Aspergillus parasiticus strain

Rice husks (RHs) are plant waste materials abundant in phytoliths silica bodies. These were used as starting material for fungal-mediated biotransformation leading to the synthesis of a high-value added product. A strain of Aspergillus parasiticus was capable of transforming the amorphous silica conglomerates into structured nanoparticles (NPs) in the process of RHs biotransformation. Silica NPs were produced extracellularly and their size ranged from 3 to 400 nm depending on the biotransformation conditions and the post-biotransformation supernatant processing. To characterize the NP's structure and dimension, SEM, STEM, EDX and FTIR technics were applied. These demonstrated and confirmed that pyramid (400 nm), cubical (85 nm) and spherical (3 nm and 24 ± 8 nm) forms of silica NPs were obtained.

Characterization of a Whole-Cell Biotransformation Using a Constitutive Lysine Decarboxylase from Escherichia coli for the High-Level Production of Cadaverine from Industrial Grade L-Lysine.

Cadaverine is used for the synthesis of the novel bio-polyamides 54, 56, and 510. Here, we examine the feasibility of using a lysine decarboxylase (LdcC) from Escherichia coli for high-level production of cadaverine. After sequential optimization of whole-cell biotransformation conditions, recombinant E. coli-overexpressing LdcC (EcLdcC) could produce 1.0M cadaverine from 1.2M crude L-lysine solution after 9h. EcLdcC retained a higher cadaverine yield after being reused 10 times at acidic and alkaline pH values than that of a recombinant E. coli strain overexpressing an inducible lysine decarboxylase (CadA), a conventional cadaverine producer (90 vs. 51% at pH6 and 55 vs. 15% at pH8). This study reveals that EcLdcC is a promising whole-cell biocatalyst for the bio-based production of cadaverine from industrial grade L-lysine in comparison to EcCadA.

Microbial synthesis of a useful optically active (+)-isomer of lactone with bicyclo[4.3.0]nonane structure

Lactone 2a of a bicyclo[4.3.0]nonane structure is a good starting material for synthesis of many attractive compounds. Enantiomerically enriched (−)-(3aR,7aS)-lactone 2a is produced by whole cells of bacteria. In order to examine the impact of the absolute configuration on biological activity we evaluated the process affording the opposite isomer. To this purpose Candida pelliculosa ZP22 characterized by high dehydrogenase activity was used. The goal of presented work was to perform bioreactor scale microbial one-pot oxidation of diol with selected yeast strain C. pelliculosa ZP22 to obtain chiral (+)-(3aS,7aR)-lactone 2a. The idea was to influence on alcohol dehydrogenase activity by increasing the activity of pro-(+)-ADH and simultanously diminishing the activity of pro-(−)-ADH. The optimization of biotransformation conditions involved the manipulation of the nutritional and physical parameters. Selection of the optimal medium in order to improve yield and process enantioselectivity was based on a two-level factorial design methodology. We have also studied the relationship between microbial growth and biosynthesis of lactone 2a. Preparative oxidation of diol 3a (400 mg/L, 2.9 mM) catalyzed by C. pelliculosa ZP22 in an optimized conditions afforded enantiomerically enriched (+)-(3aS,7aR)-isomer of lactone 2a with the isolated yield (30%).

Enzymatic synthesis of d-alanine from a renewable starting material by co-immobilized dehydrogenases

d-alanine is an important pharmaceutical intermediate. In this study, an effective method was developed to prepare optically pure d-alanine from sodium pyruvate with co-immobilized meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum and formate dehydrogenase from Candida Boidinii onto a resin. After optimizing the conditions of immobilization and biotransformation, the co-immobilized enzymes exhibited excellent stereoselectivity, yield and reusability. The co-immobilized enzymes kept 80% of its activity after 12 reuses, and d-alanine was isolated in 75.7% average yield and 99.5% ee for the first 20 cycles in the batch reactions. This suggests the co-immobilized enzyme system as a promising biocatalyst for the green production of d-alanine from pyruvic acid, a bio-based starting material.

Surfactant-mediated permeabilization of Pseudomonas putida KT2440 and use of the immobilized permeabilized cells in biotransformation

Abstract Surfactants were used to permeabilize cells of Pseudomonas putida KT2440 so as to maximize retention of the arginine deiminase (ADI) activity within the treated cells. The surfactants cetyltrimethylammoniumbromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X100 were tested separately. Statistical models were developed for the effects on the ADI activity of the following factors: the concentration of the surfactant, the length of the treatment period and the concentration of the cells. For all surfactants, the concentration of cells was the most significant factor in influencing permeabilization. All permeabilization treatments used mild conditions (pH 7, 37 °C). The permeabilized cells were immobilized in alginate beads for the biotransformation of arginine to citrulline. The optimal conditions for immobilization and biotransformation were as follows: 2% (w/v, g/100 mL) sodium alginate, 100 g/L of treated cells, 40 mM arginine, pH 6.0, a temperature of 35 °C and an agitation speed of 150 rpm. The immobilized biocatalyst retained nearly 90% of its initial activity after nine cycles of repeated use in batch operations. In contrast, the freely suspended cells were barely active after the second use cycle.

Application of magnetically immobilized edible fungus for the biotransformation of panax notoginseng saponin Rb1 to Rd and Rg3

In this study, we developed a new magnetically immobilized edible fungus for the biotransformation of panax notoginseng saponins Rb1 to Rd and Rg3. The optimum biotransformation conditions were as follows: temperature 32°C, pH 6.5, time 48h and liquid-solid ratio 25:1(mL/g). The yields of Rd and Rg3 reached 41.35±0.12mg/g and 6.35±0.08mg/g which increased 6.97-fold and 3.23-fold to that of untreated control, respectively. Additionally, SEM demonstrated that vestured pits and cell walls of samples were destroyed obviously which was beneficial to the Rb1 biotransformation and target Rd and Rg3 release. Meanwhile, the reusability of magnetically immobilized microorganism was tested and the activity of the magnetically immobilized microorganism remained 83.8% after 15 runs. The recycling experiments demonstrated that magnetically immobilized fungus exhibited higher efficiency than the non-magnetical one. These results proved that this new magnetically immobilized microorganism could be applied for industrial production and pharmaceutical industry with good efficiency.

Improvement of resveratrol production from waste residue of grape seed by biotransformation of edible immobilized Aspergillus oryzae cells and negative pressure cavitation bioreactor using biphasic ionic liquid aqueous system pretreatment

A biotransformation method of producing resveratrol with immobilized Aspergillus oryzae negative pressure cavitation bioreactor (ICNPCB) with biphasic ionic liquid aqueous system pretreatment was investigated. Subsequently, the biotransformation conditions were optimized for the resveratrol production under types of ILs [C4MIM]Br, concentration of ILs 0.75M, temperature 31°C, ratios of solid to liquid 1:15 (mg/L), time 60h and pH 6.0, the yield of resveratrol reached 224.61±0.35μg/g, which increased 4.36-fold to that of untreated one. The conversion rate of piceid reached 85.21%. The residual activity of immobilized microorganism was 82.26% after used for 15 runs. The developed method could be an effectively alternative biotransformation method for producing resveratrol from the waste residue of wine.

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling.

Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L−1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L−1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L−1 (99.3%) and 75.1 ± 2.5 g·L−1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.