Whole-cell biocatalysts offer a highly enantioselective, minimally polluting route to optically active alcohols. Currently, most of the whole-cell catalytic performance involves resting cells rather than growing cell biotransformation, which is one-step process that benefits from the simultaneous growth and biotransformation, eliminating the need for catalysts preparation. In this paper, asymmetric reduction of 14 aromatic ketones to the corresponding enantiomerically pure alcohols was successfully conducted using the growing and resting cells of marine-derived fungi under optimized conditions. Good yields and excellent enantioselectivities were achieved with both methods. Although substrate inhibition might be a limiting factor for growing cell biotransformation, the selected strain can still completely convert 10-mM substrates into the desired products. The resting cell biotransformation showed a capacity to be recycled nine times without a significant decrease in the activity. This is the first study to perform asymmetric reduction of ketones by one-step growing cell biotransformation.
In our previous study, Rhodotorula mucilaginosa (R. mucilaginosa) was selected via high throughput screening as a very active and selective whole-cell biocatalyst for the asymmetric reduction of ketones. In this study, the reduction of ketones to the desired chiral alcohols by immobilized cells of this strain was investigated. Characterization with Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) showed that whole R. mucilaginosa cells were successfully immobilized on support matrices composed of agar, calcium alginate, PVA-alginate and chitosan. The immobilized cells were applied to the enantioselective reduction of fourteen different aromatic ketones. Good to excellent results were achieved with R. mucilaginosa cells immobilized on agar and calcium alginate. The immobilized cells on the selected support matrix composed of agar exhibited a significant increase in pH tolerance at pH 3.5–9 and demonstrated highly improved thermal stability compared to free cells. The cells immobilized on agar retained 90% activity after 60 days storage at 4 °C and retained almost 100% activity after 6 reuse cycles. In addition, the immobilization procedures are very simple and cause minimal pollution. These results suggest that the application of immobilized R. mucilaginosa can be practical on an industrial scale to produce chiral alcohols.
Alkaloids are a large and structurally diverse group of marine-derived natural products. Most marine-derived alkaloids are biologically active and show promising applications in modern (agro)chemical, pharmaceutical, and fine chemical industries. Different approaches have been established to access these marine-derived alkaloids. Among these employed methods, biotechnological approaches, namely, (chemo)enzymatic synthesis, have significant potential for playing a central role in alkaloid production on an industrial scale. In this review, we discuss research progress on marine-derived alkaloid synthesis via enzymatic reactions and note the advantages and disadvantages of their applications for industrial production, as well as green chemistry for marine natural product research.
Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.
Marine-derived fungus Geotrichum candidum AS 2.361 was previously reported by our group as an active strain for the enantioselective reduction of ketones. Although some other Geotrichum strains were also found from the terrestrial sources, information on their stability and reusability is scarce. Herein, the stabilities—in terms of pH tolerance, thermostability, and storage stability, and reusability—of G. candidum AS 2.361 were described for the asymmetric reduction of a series of aromatic ketones. Two differently immobilized cells (agar immobilization and calcium alginate immobilization) as well as free cells were prepared. For three substrates (1-(3-bromophenyl) ethan-1-one (1b), 1-(2-chlorophenyl) ethan-1-one (1d), and acetophenone (1g)) immobilized cells on agar showed a great improvement in the bioreduction activities compared to the free cells, increasing yields up to 97% with ee values of 99%. Cells immobilized on agar/calcium alginate could maintain more than 90% of the original activities within the assayed pH ranges of 3.5–11, while free cells were highly sensitive to alkaline and acidic conditions. Concerning thermostability, immobilized cells on agar kept 99% of their original activities after incubation at 60 °C for 1 h, while almost no activity was detected for the free cells under the same condition. Immobilized cells were stable at 4 °C for 80 days without any activity loss, while free cells started to decrease the activity after storage at 4 °C for six days. The immobilized cells retained almost 99% activity after four reuse cycles, while free cells lost almost all the activities at on the third cycle.