1,4-α-Glucosidase from Fusarium solani for Controllable Biosynthesis of Silver Nanoparticles and Their Multifunctional Applications.

In the study, monodispersed silver nanoparticles (AgNPs) with an average diameter of 9.57 nm were efficiently and controllably biosynthesized by a reductase from Fusarium solani DO7 only in the presence of ß-NADPH and polyvinyl pyrrolidone (PVP). The reductase responsible for AgNP formation in F. solani DO7 was further confirmed as 1,4-α-glucosidase. Meanwhile, based on the debate on the antibacterial mechanism of AgNPs, this study elucidated in further depth that antibacterial action of AgNPs was achieved by absorbing to the cell membrane and destabilizing the membrane, leading to cell death. Moreover, AgNPs could accelerate the catalytic reaction of 4-nitroaniline, and 86.9% of 4-nitroaniline was converted to p-phenylene diamine in only 20 min by AgNPs of controllable size and morphology. Our study highlights a simple, green, and cost-effective process for biosynthesizing AgNPs with uniform sizes and excellent antibacterial activity and catalytic reduction of 4-nitroaniline.

A Versatile Competitive Coordination Strategy for Tailoring Bioactive Zeolitic Imidazolate Framework Composites.

Zeolitic imidazolate frameworks (ZIFs) serving as platforms for bioactive guest encapsulation have attracted growing attention, yet the tailoring of its architectures and bioactivity remains a major challenge. Herein, a versatile competitive coordination strategy is proposed by using amorphous zinc nucleotide gel as template for step-by-step growth of ZIFs, which enables the tailoring of bioactive ZIF composites under facile conditions. Mechanism investigation reveals that introduced nucleotide determines the hierarchical pore structure and hydrophilicity, leading to customized activity retention and stability of the resultant bioactive ZIF composites. Furthermore, nucleoside monophosphate enhances the acidic tolerance of ZIFs. To the authors' knowledge, this is the first example showing the dynamic evolution of amorphous gels to crystalline ZIFs for in situ encapsulation of enzymes with tailored catalytic performance. This study provides insights for rational design of ZIF-based biocomposites and broadens the application of bioactive metal-organic frameworks.

Biotechnology and bioengineering of pullulanase: state of the art and perspectives.

Pullulanase (EC 3.2.1.41) is a starch-debranching enzyme in the α-amylase family and specifically cleaves α-1,6-glycosidic linkages in starch-type polysaccharides, such as pullulan, ß-limited dextrin, glycogen, and amylopectin. It plays a key role in debranching and hydrolyzing starch completely, thus bring improved product quality, increased productivity, and reduced production cost in producing resistant starch, sugar syrup, and beer. Plenty of researches have been made with respects to the discovery of either thermophilic or mesophilic pullulanases, however, few examples meet the demand of industrial application. This review presents the progress made in the recent years from the first aspect of characteristics of pullulanases. The heterologous expression of pullulanases in different microbial hosts and the methods used to improve the expression effectiveness and the regulation of enzyme production are also described. Then, the function evolution of pullulanases from a protein engineering view is discussed. In addition, the immobilization strategy using novel materials is introduced to improve the recyclability of pullulanases. At the same time, we indicate the trends in the future research to facilitate the industrial application of pullulanases.

Microbial synthesis of functional odd-chain fatty acids: a review.

Odd-chain fatty acids (OCFAs) naturally occur in bacteria, higher animals, and in plants. During recent years, they have received increasing attention due to their unique pharmacological properties and usefulness for agricultural and industrial applications. Recently, OCFAs have been identified and quantified in a few organisms, and new pharmacological functions of OCFAs have been reported. Some of the publications are related to the optimization of OCFA production through fermentation and genetic engineering. The present review aims to provide a summary on the recent progress in the field of microbial-derived OCFAs. More specifically, we outline the publications of OCFAs related to (i) different sources of OCFAs; (ii) endogenous synthesis of OCFAs; (iii) production of OCFAs through fermentation; (iv) genetic engineering related to OCFA; and (v) role of OCFAs in human health and disease. Finally, some areas that require further research are discussed.

Metabolic engineering of a robust Escherichia coli strain with a dual protection system.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 107 plaque-forming units/ml and produce GFP with a yield of up to 30 µg/109 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.

Using 1-propanol to significantly enhance the production of valuable odd-chain fatty acids by Rhodococcus opacus PD630.

Odd-chain fatty acids (OCFAs) have been reported to possess pharmacological activity and have been used in the manufacture of agricultural and industrial chemicals. We here provided a new method to increase the OCFAs content in oil produced by Rhodococcus opacus PD630 through addition of 1-propanol to the fermentation media. The OCFAs in oil of R. opacus PD630 are primarily pentadecanoic acid (C15:0), heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1). After adding 0.5-1.5% (v/v) 1-propanol, the production of oil increased from 1.27 g/L to 1.31-1.61 g/L, and the OCFAs content in oil increased by 46.7-55.1%. Metabolic intermediates determination and transcriptome analysis revealed that R. opacus assimilated 1-propanol through methylmalonyl-CoA pathway. When the nitrogen source was limited, propionyl-CoA was converted to propionyl-acyl carrier protein (ACP) which could be used as primer during the elongation of fatty acid synthesis. Then OCFAs were produced when odd number of propionyl-ACP was incorporated in the cycles of fatty acid synthesis.

The application of deep eutectic solvent on the extraction and in vitro antioxidant activity of rutin from Sophora japonica bud.

The extraction conditions and antioxidant activities of rutin from Sophora japonica bud by deep eutectic solvents were investigated. Box-Behnken design was used to optimize the extraction conditions and the scavenging activities of DPPH, O2- and ·OH of purified rutin were evaluated. The highest yield of 279.8 mg/g was achieved in the extraction medium of choline chloride/triethlene glycol (1/4) under the optimum conditions: water content of the DES 18.1%, extraction time 28.3 min, extraction temperature 70 °C and liquid-solid ratio 10 mg/1 g. The highest extraction amount was slightly different from the predicted value of the established second-order polynomial equation. In addition, The EC50 of DPPH scavenging, O2- scavenging and ·OH scavenging of rutin were 5.68 µg/mL, 0.19 and 0.28 mg/mL, respectively. The above results indicate rutin extracted by the choline chloride/triethylene glycol has excellent antioxidant activity and was an admirable free radical scavenger.

Markedly improving asymmetric oxidation of 1-(4-methoxyphenyl) ethanol with Acetobacter sp. CCTCC M209061 cells by adding deep eutectic solvent in a two-phase system.

BACKGROUND: Enantiopure (S)-1-(4-methoxyphenyl) ethanol {(S)-MOPE} can be employed as an important synthon for the synthesis of cycloalkyl [b] indoles with the treatment function for general allergic response. To date, the biocatalytic resolution of racemic MOPE through asymmetric oxidation in the biphasic system has remained largely unexplored. Additionally, deep eutectic solvents (DESs), as a new class of promising green solvents, have recently gained increasing attention in biocatalysis for their excellent properties and many successful examples in biocatalytic processes. In this study, the biocatalytic asymmetric oxidation of MOPE to get (S)-MOPE using Acetobacter sp. CCTCC M209061 cells was investigated in different two-phase systems, and adding DES in a biphasic system was also explored to further improve the reaction efficiency of the biocatalytic oxidation. RESULTS: Of all the examined water-immiscible organic solvents and ionic liquids (ILs), 1-butyl-3-methylimidazolium hexafluorophoshpate ([C4MIM][PF6]) afforded the best results, and consequently was selected as the second phase of a two-phase system for the asymmetric oxidation of MOPE with immobilized Acetobacter sp. CCTCC M209061 cells. For the reaction performed in the [C4MIM][PF6]/buffer biphasic system, under the optimized conditions, the initial reaction rate, the maximum conversion and the residual substrate e.e. recorded 97.8 µmol/min, 50.5 and >99.9 % after 10 h reaction. Furthermore, adding the DES [ChCl][Gly] (10 %, v/v) to the aqueous phase, the efficiency of the biocatalytic oxidation was rose markedly. The optimal substrate concentration and the initial reaction rate were significantly increased to 80 mmol/L and 124.0 µmol/min, respectively, and the reaction time was shortened to 7 h with 51.3 % conversion. The immobilized cell still retained over 72 % of its initial activity after 9 batches of successive reuse in the [C4MIM][PF6]/[ChCl][Gly]-containing buffer system. Additionally, the efficient biocatalytic process was feasible up to a 500-mL preparative scale. CONCLUSION: The biocatalytic asymmetric oxidation of MOPE with Acetobacter sp. CCTCC M209061 cells was successfully conducted in the [C4MIM][PF6]-containing biphasic system with high conversion and enantioselectivity, and the reaction efficiency was further enhanced by adding [ChCl][Gly] to the reaction system. The efficient biocatalytic process was promising for the preparation of enantiopure (S)-MOPE.

Biocatalytic anti-Prelog reduction of prochiral ketones with whole cells of Acetobacter pasteurianus GIM1.158.

BACKGROUND: Enantiomerically pure alcohols are important building blocks for production of chiral pharmaceuticals, flavors, agrochemicals and functional materials and appropriate whole-cell biocatalysts offer a highly enantioselective, minimally polluting route to these valuable compounds. At present, most of these biocatalysts follow Prelog's rule, and thus the (S)-alcohols are usually obtained when the smaller substituent of the ketone has the lower CIP priority. Only a few anti-Prelog (R)-specific whole cell biocatalysts have been reported. In this paper, the biocatalytic anti-Prelog reduction of 2-octanone to (R)-2-octanol was successfully conducted with high enantioselectivity using whole cells of Acetobacter pasteurianus GIM1.158. RESULTS: Compared with other microorganisms investigated, Acetobacter pasteurianus GIM1.158 was shown to be more effective for the reduction reaction, affording much higher yield, product enantiomeric excess (e.e.) and initial reaction rate. The optimal temperature, buffer pH, co-substrate and its concentration, substrate concentration, cell concentration and shaking rate were 35°C, 5.0, 500 mmol/L isopropanol, 40 mmol/L, 25 mg/mL and 120 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 89.5% and >99.9%, respectively, in 70 minutes. Compared with the best available data in aqueous system (yield of 55%), the yield of (R)-2-octanol was greatly increased. Additionally, the efficient whole-cell biocatalytic process was feasible on a 200-mL preparative scale and the chemical yield increased to 95.0% with the product e.e. being >99.9%. Moreover, Acetobacter pasteurianus GIM1.158 cells were proved to be capable of catalyzing the anti-Prelog bioreduction of other prochiral carbonyl compounds with high efficiency. CONCLUSIONS: Via an effective increase in the maximum yield and the product e.e. with Acetobacter pasteurianus GIM1.158 cells, these results open the way to use of whole cells of this microorganism for challenging enantioselective reduction reactions on laboratory and commercial scales.

Structural and interfacial properties of acetylated Millettia speciosa Champ polysaccharide and stability evaluation of the resultant O/W emulsion containing ß-carotene.

The aim of this study was to investigate the effects of acetylation modification on the structural, interfacial and emulsifying properties of Millettia speciosa Champ polysaccharide (MSCP). Besides, the influence of acetylation modification on the encapsulation properties of polysaccharide-based emulsion was also explored. Results indicated that modification resulted in a prominent reduction in molecular weight of MSCP and the interfacial layer thickness formed by acetylated MSCP (AC-MSCP) was also decreased, but the adsorption rate and ability of AC-MSCP to reduce interfacial tension were improved. AC-MSCP formulated emulsion possessed smaller droplet size (6.8 µm) and exhibited better physical stability under stressful conditions. The chemical stability of ß-carotene was also profoundly enhanced by AC-MSCP fabricated emulsion. Moreover, AC-MSCP improved lipids digestion extent, thus facilitating the formation of micelle and increasing bioaccessibility of ß-carotene. This study provided insights for rational modification of polysaccharide-based emulsifier and designing delivery system for chemically labile hydrophobic bioactive components.

Novel Multitarget ACE Inhibitory Peptides from Bovine Colostrum Immunoglobulin G: Cellular Transport, Efficacy in Regulating Endothelial Dysfunction, and Network Pharmacology Studies.

In this study, we used traditional laboratory methods, bioinformatics, and cellular models to screen novel ACE inhibitory (ACEI) peptides with strong ACEI activity, moderate absorption rates, and multiple targets from bovine colostrum immunoglobulin G (IgG). The purified fraction of the compound proteinase hydrolysate of IgG showed good ACEI activity. After nano-UPLC-MS/MS identification and in silico analysis, eight peptides were synthesized and verified. Among them, SFYPDY, TSFYPDY, FSWF, WYQQVPGSGL, and GVHTFP were identified as ACEI peptides, as they exhibited strong ACEI activity (with IC50 values of 104.7, 80.0, 121.2, 39.8, and 86.3 µM, respectively). They displayed good stability in an in vitro simulated gastrointestinal digestion assay. In a Caco-2 monolayer model, SFYPDY, FSWF, and WYQQVPGSGL exhibited better absorption rates and lower IC50 values than the other peptides and were thereby identified as novel ACEI peptides. Subsequently, in a H2O2-induced endothelial dysfunction (ED) model based on HUVECs, SFYPDY, FSWF, and WYQQVPGSGL regulated ED by reducing apoptosis and ROS accumulation while upregulating NOS3 mRNA expression. Network pharmacology analysis and RT-qPCR confirmed that they regulated multiple targets. Overall, our results suggest that SFYPDY, FSWF, and WYQQVPGSGL can serve as novel multitarget ACEI peptides.

Millettia speciosa Champ cellulose-based hydrogel as a novel delivery system for Lactobacillus paracasei: Its relationship to structure, encapsulation and controlled release.

We report for the first time the usage of Millettia speciosa Champ cellulose (MSCC) and carboxymethylcellulose (MSCCMC) for the fabrication of 3D-network hydrogel as delivery system for probiotics. The structural features, swelling behavior and pH-responsiveness of MSCC-MSCCMC hydrogels and their encapsulation and controlled-release behavior for Lactobacillus paracasei BY2 (L. paracasei BY2) were mainly studied. Structural analyses demonstrated that MSCC-MSCCMC hydrogels with porous and network structures were successfully synthesized through the crosslinking of -OH groups between MSCC and MSCCMC molecules. An increasing concentration of MSCCMC significantly improved the pH-responsiveness and swelling ability of the MSCC-MSCCMC hydrogel toward neutral solvent. Besides, the encapsulation efficiency (50.38-88.91 %) and release (42.88-92.86 %) of L. paracasei BY2 were positively correlated with the concentration of MSCCMC. The higher the encapsulation efficiency was, the higher the release in the target intestine. However, due to the existence of bile salts, controlled-release behavior decreased the survivor rate and physiological state (degrading cholesterol) of encapsulating L. paracasei BY2. Even so, the number of viable cells encapsulated by hydrogels still reached the minimum effective concentration in the target intestine. This study provides an available reference for the practical application of hydrogels fabricated from the cellulose of the Millettia speciosa Champ plant for probiotic delivery.

Iron-incorporated metal-organic frameworks for oxidative cleavage of trans-anethole to p-anisaldehyde.

An iron-incorporated Zn-MOF catalyst Zn-bpydc·Fe was fabricated for the oxidative cleavage of trans-anethole to p-anisaldehyde under facile conditions, under 1 atm of O2. The Fe coordinated bipyridine serves as the catalytically active center inside the structural skeleton of Zn-MOFs. This work affords a new avenue for the mild oxidation of olefins.

Extraction and characterization of a functional protein from Millettia speciosa Champ. leaf.

Natural plant-derived protein with excellent bioactivities has attracted much attention so a functional protein with molecular weight of 15.2 kDa was extracted from Millettia speciosa Champ. leaf for the first time. Under the pH of 12.0, solid-liquid ratio of 1:40 (w/v), extraction time of 2.0 h, and extraction temperature of 50 °C, the highest extracting efficiency (79.25 ± 0.78%) of the Millettia speciosa Champ. leaf protein (MLP) was achieved. The main structure of MLP contained ß-fold and ß-corner by Fourier transform infrared spectroscopy (FTIR) and Circular dichroism (CD) spectra analysis. Additionally, MLP was predominant with glutamic acid, aspartic acid, and leucine, which could be considered as a high quality natural protein. MLP showed great water holding capacity (WHC), oil absorption capacity (OAC), as well as emulsifying and foaming properties. Simultaneously, MLP exhibited considerable antioxidant activity. These results suggested that MLP could be utilised as a promising ingredient of functional foods.

Purification, structural elucidation and biological activities of exopolysaccharide produced by the endophytic Penicillium javanicum from Millettia speciosa Champ.

An acid polysaccharide, named HP, was produced by endophytic Penicillium javanicum MSC-R1 isolated from southern medicine Millettia speciosa Champ. The molecular weight of HP was 37.8 kDa and consisted of Ara f, Galр, Glcр, Manр, and GlcрA with a molar ratio of 1.09: 3.47: 68.48: 16.59: 8.85. The glycosidic linkage of HP was proven to be →3, 4)-α-D-Glcр-(1→6)-α-D-Manр-(1→, →3, 4)-α-D-Glcр-(1→4)-α-D-Glcр-(1→, →3), →6)-α-D-Manр-(1→4)-α-D-Glcр-(1→, →3), ß-D-Galр-(1→3)-α-D-Glcр-(1→, →4), →5)-α-L-Ara f -(1→3)-α-D-Glcр-(1→, →4), →6)-α-D-Manр-(1→4)-α-D-GlcAр-(1→ and →4)-α-D-GlcAр-(1→4)-α-D-Glcр-(1→, →3). Additionally, 250 µg/mL of HP possessed nontoxicity to RAW 264.7 cells and exhibited anti-inflammation activity. HP could significantly restrain the amount of tumor necrosis factor-α, interleukin-6 and NO release in RAW264.7, which property is possibly associated with its abundant glucosidic linkage. These results indicated that HP could be regarded as a ponderable ingredient for the health-beneficial functional foods.

Efficient extraction, excellent activity, and microencapsulation of flavonoids from Moringa oleifera leaves extracted by deep eutectic solvent.

A deep eutectic solvent (choline chloride (ChCl)-urea) was chosen to extract flavonoids from Moringa oleifera leaves (FMOL), the condition of extraction was tailor-made, under the optimal extraction conditions (material-to-liquid ratio of 1:60 g/mL, extraction time of 80 min, extraction temperature of 80 °C), the highest extraction efficiency reached 63.2 ± 0.3 mg R/g DW, and nine flavonoids were identified. Then, the biological activities including antioxidant activities, antibacterial activities, and anti-tumor activities were systematically studied. FMOL was superior to positive drugs in terms of antioxidant activity. As to DPPH investigation, the IC50 of FMOL and Vc were 64.1 ± 0.7 and 176.1 ± 2.0 µg/mL; for the ABTS, the IC50 of FMOL and Vc were 9.5 ± 0.3 and 38.2 ± 1.2 µg/mL, the FRAP value of FMOL and Vc were 15.5 ± 0.6 and 10.2 ± 0.4 mg TE/g, and ORAC value of FMOL and Vc were 4687.2 ± 102.8 and 3881.6 ± 98.6 µmol TE/g. The bacteriostatic (MICs were ≤ 1.25 mg/mL) activities of FMOL were much better than propyl p-hydroxybenzoate. Meanwhile, FMOL had comparable inhibitory activity with genistein on tumor cells, IC50 was 307.8 µg/mL, and could effectively induce apoptosis in HCT116. Microcapsules were prepared with xylose-modified soybean protein isolate and gelatin as wall materials; after that, the intestinal release of modified FMOL microcapsules was 86 times of free FMOL. Therefore, this study confirmed that FMOL extracted with ChCl/urea has rich bioactive components, and microencapsulated FMOL has potential application in food industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s13399-023-03877-8.

Glycolic acid-based deep eutectic solvents boosting co-production of xylo-oligomers and fermentable sugars from corncob and the related kinetic mechanism.

BACKGROUND: Xylo-oligomers are a kind of high value-added products in biomass fractionation. Although there are several chemical methods to obtain xylo-oligomers from biomass, the reports about the deep eutectic solvents (DESs)-mediated co-production of xylo-oligomers and fermentable sugars and the related kinetic mechanism are limited. RESULTS: In this work, glycolic acid-based DESs were used to obtain xylo-oligomers from corncob. The highest xylo-oligomers yield of 65.9% was achieved at 120 °C for 20 min, of which the functional xylo-oligosaccharides (XOSs, DP 2-5) accounted for up to 31.8%. Meanwhile, the enzymatic digestion of cellulose and xylan in residues reached 81.0% and 95.5%, respectively. Moreover, the addition of metal inorganic salts significantly accelerated the hydrolysis of xylan and even the degradation of xylo-oligomers in DES, thus resulting in higher selectivity of xylan removal. AlCl3 showed the strongest synergistic effect with DES on accelerating the processes, while FeCl2 is best one for xylo-oligomers accumulation, affording the highest xylo-oligomers yield of 66.1% for only 10 min. Furthermore, the kinetic study indicates that the 'potential hydrolysis degree' model could well describe the xylan hydrolysis processes and glycolic acid/lactic acid (3:1) is a promising solvent for xylo-oligomers production, in particular, it worked well with FeCl2 for the excellent accumulation of xylo-oligomers. CONCLUSIONS: Glycolic acid-based deep eutectic solvents can be successfully applied in corncob fractionation with excellent xylo-oligomers and fermentable sugars yields on mild conditions, and the large amount of xylo-oligosaccharides accumulation could be achieved by specific process controlling. The strategies established here can be useful for developing high-valued products from biomass.

Efficient anti-Prelog enantioselective reduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol by immobilized Candida parapsilosis CCTCC M203011 cells in ionic liquid-based biphasic systems.

BACKGROUND: Biocatalytic asymmetric reductions with whole cells can offer high enantioselectivity, environmentally benign processes and energy-effective operations and thus are of great interest. The application of whole cell-mediated bioreduction is often restricted if substrate and product have low water solubility and/or high toxicity to the biocatalyst. Many studies have shown that a biphasic system is often useful in this instance. Hence, we developed efficient biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs), to improve the biocatalytic anti-Prelog enantioselective reduction of acetyltrimethylsilane (ATMS) to (R)-1-trimethylsilylethanol {(R)-1-TMSE}, which is key synthon for a large number of silicon-containing drugs, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst. RESULTS: It was found that the substrate ATMS and the product 1-TMSE exerted pronounced toxicity to immobilized Candida parapsilosis CCTCC M203011 cells. The biocompatible water-immiscible ILs can be applied as a substrate reservoir and in situ extractant for the product, thus greatly enhancing the efficiency of the biocatalytic process and the operational stability of the cells as compared to the IL-free aqueous system. Various ILs exerted significant but different effects on the bioreduction and the performances of biocatalysts were closely related to the kinds and combination of cation and anion of ILs. Among all the water-immiscible ILs investigated, the best results were observed in 1-butyl-3-methylimidazolium hexafluorophosphate (C(4)mim·PF(6))/buffer biphasic system. Furthermore, it was shown that the optimum substrate concentration, volume ratio of buffer to IL, buffer pH, reaction temperature and shaking rate for the bioreduction were 120 mM, 8/1 (v/v), 6.0, 30°C and 180 r/min, respectively. Under these optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 8.1 µmol/min g(cwm), 98.6% and >99%, respectively. The efficient whole-cell biocatalytic process was shown to be feasible on a 450-mL scale. Moreover, the immobilized cells remained around 87% of their initial activity even after being used repeatedly for 8 batches in the C(4)mim·PF(6)/buffer biphasic system, exhibiting excellent operational stability. CONCLUSIONS: For the first time, we have successfully utilized immobilized Candida parapsilosis CCTCC M203011 cells, for efficiently catalyzing anti-Prelog enantioselective reduction of ATMS to enantiopure (R)-1-TMSE in the C(4)mim·PF(6)/buffer biphasic system. The substantially improved biocatalytic process appears to be effective and competitive on a preparative scale.

Immobilization of Acetobacter sp. CCTCC M209061 for efficient asymmetric reduction of ketones and biocatalyst recycling.

BACKGROUND: The bacterium Acetobacter sp. CCTCC M209061 is a promising whole-cell biocatalyst with exclusive anti-Prelog stereoselectivity for the reduction of prochiral ketones that can be used to make valuable chiral alcohols such as (R)-4-(trimethylsilyl)-3-butyn-2-ol. Although it has promising catalytic properties, its stability and reusability are relatively poor compared to other biocatalysts. Hence, we explored various materials for immobilizing the active cells, in order to improve the operational stability of biocatalyst. RESULTS: It was found that Ca-alginate give the best immobilized biocatalyst, which was then coated with chitosan to further improve its mechanical strength and swelling-resistance properties. Conditions were optimized for formation of reusable immobilized beads which can be used for repeated batch asymmetric reduction of 4'-chloroacetophenone. The optimized immobilized biocatalyst was very promising, with a specific activity of 85% that of the free-cell biocatalyst (34.66 µmol/min/g dw of cells for immobilized catalyst vs 40.54 µmol/min/g for free cells in the asymmetric reduction of 4'-chloroacetophenone). The immobilized cells showed better thermal stability, pH stability, solvent tolerance and storability compared with free cells. After 25 cycles reaction, the immobilized beads still retained >50% catalytic activity, which was 3.5 times higher than degree of retention of activity by free cells reused in a similar way. The cells could be recultured in the beads to regain full activity and perform a further 25 cycles of the reduction reaction. The external mass transfer resistances were negligible as deduced from Damkohler modulus Da < <1, and internal mass transfer restriction affected the reduction action but was not the principal rate-controlling step according to effectiveness factors η < 1 and Thiele modulus 0.3<∅ <1. CONCLUSIONS: Ca-alginate coated with chitosan is a highly effective material for immobilization of Acetobacter sp. CCTCC M209061 cells for repeated use in the asymmetric reduction of ketones. Only a small cost in terms of the slightly lower catalytic activity compared to free cells could give highly practicable immobilized biocatalyst.

In vivo detoxification of furfural during lipid production by the oleaginous yeast Trichosporon fermentans.

In vivo detoxification of furfural by the oleaginous yeast, Trichosporon fermentans, under lipid-producing (i.e., nitrogen-limited) conditions was evaluated for the first time. During the initial fermentation phase, furfural was rapidly reduced to furfuryl alcohol, which is more toxic to T. fermentans than furfural. Furfuryl alcohol was subsequently oxidized to furoic acid which has low toxicity to T. fermentans and is the end product of the in vivo detoxification of furfural in this organism. These observations explain how T. fermentans can grow and accumulate lipids in medium containing furfural. They also indicate that strategies to minimize the transient production of furfuryl alcohol could further improve the capacity of the strain to produce lipids from furfural-containing lignocellulosic hydrolysates.