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d-Tagatose, a rare sugar endowed with a low-calorie property, superior taste quality, and probiotic functionality, has garnered significant research attention. However, the prevailing biological production methods relying on β-galactosidase and l-arabinose isomerase face challenges including high cost and suboptimal conversion efficiency. Consequently, it is of great research significance to find efficient alternative routes for d-tagatose synthesis. Previously, Thermotoga petrophila tagaturonate 3-epimerase was modified to function as tagatose 4-epimerase (T4E) enabling the direct conversion of d-fructose to d-tagatose. In this study, T4E was further engineered through directed evolution, specifically targeting the enhancement of its thermostability for application. This endeavor yielded promising T4E variants with superiority over those of the original enzyme. T4E I430P exhibits a half-life ( t 1/2 ) at 70 °C that is 1.83-fold that of T4E, with an increased melting temperature ( T m is 2.9 °C higher than T4E. Furthermore, whole-cell immobilization integrating these engineered T4E variants into a robust biocatalytic system was employed. This innovative approach not only underscores the practical feasibility of modifying enzymes through directed evolution but also establishes a foundation for the cost-effective, large-scale production of d-tagatose.t 1/2 was 1.69-fold that of T4E, and its T m is 2.9 °C higher than T4E. Furthermore, whole-cell immobilization integrating these engineered T4E variants into a robust biocatalytic system was employed. This innovative approach not only underscores the practical feasibility of modifying enzymes through directed evolution but also establishes a foundation for the cost-effective, large-scale production of d-tagatose.

Cladribine is a nucleoside analogue widely used in the pharmaceutical industry for the treatment of several neoplasms, including hairy‐cell leukemia among others. This compound has also shown efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this work, a green bioprocess for cladribine biosynthesis using immobilized Arthrobacter oxydans was developed. The microorganism was stabilized by entrapment immobilization in the natural matrix alginate. Different reaction parameters were optimized obtaining a biocatalyst able to achieve cladribine bioconversion values close to 85% after 1 hr, the shortest reaction times reported so far. The developed bioprocess was successfully scaled‐up reaching a productivity of 138 mg L−1 hr−1. Also, the biocatalyst was stable for 5 months in storage and in 96 hr at operational conditions. [ABSTRACT FROM AUTHOR]

Highlights • Thalassospira frigidphilosprofundus S3BA12 produced 625 U/ml of cold-active polygalacturonase. • 4-fold increase in cell mass and polygalacturonase titre from its native strain. • Polyurethane based whole cell immobilization with robustness for saline culturing. • Statistically proven correlations between pectinase subtypes. Abstract The productivity of cold-active polygalacturonase (PGase) in a newly identified marine psychrophile, Thalassospira frigidphilosprofundus S3BA12, was increased multifold by employing high-cell-density (HCD) culturing in a simulation-assisted, custom-designed single-stage-continuous immobilized reactor. The reactor was made "halophilic culturing" ready by using surface altered polyurethane foam (PUF) as a matrix, and it was uniquely packed with layers of "spacers" to increase robustness towards high salinity, prevent salt deposition and dissociation of cells. The basic limitation of low growth of a marine organism was overcome in this study, where HCD resulted in a 4-fold increase of cell mass from 0.84 dcw g/L in submerged culture to 3.22 g/L, which commensurately increased the PGase titre from 21.0 U/ml to 85.25 U/ml. The culture was sustained for 108 h, continuously producing PGase at a rate of 7.87 U/ml hr−1. This could be the highest reported cold-active PGase by any psychrophilic organism reported to date. A mechanistic modelling approach was used to develop a simulation of the process in ChemCAD 5.0, which can be used as a basis for scale-up studies. This study proved statistically that PGase titre is significantly dependent on cell mass using Anderson-Darlington normality tests and established a correlation between PGase and other pectinase subtypes using Pearson's correlation coefficient analysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol-sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml (36 h), 47.50 U ml (36 h) and 68.36 U ml (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml (18 h) on cassava, 79.17 U ml (12 h) on potato and 55.37 U ml (in 6 h and max 77.75 U ml in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells. [ABSTRACT FROM AUTHOR]

Baeyer-Villiger monooxygenases (BVMOs) are a very well-known and intensively studied class of flavin-dependent enzymes. Their substrate promiscuity, high chemo-, regio-, and enantioselectivity are prerequisites for the use in synthetic chemistry and should pave the way for successful industrial processes. Nonetheless, only a very limited number of industrial relevant transformations are known, mainly due to the lack of BVMOs stability and cofactor dependency. In this review, we focus on novel BVMO-mediated transformations, BVMOs in cascade type reactions, potential industrial applications, and how limitations have been tackled by the community. Special attention will be put on whole-cell immobilization strategies. We emphasize to bridge recent developments in fundamental research to industrial applications. [ABSTRACT FROM AUTHOR]

BACKGROUND Mycelia pellet has been exploited as a biological carrier for whole-cell immobilization due to its unique structural characteristics. An innovative two-species whole-cell immobilization system was developed initially, which was achieved simply by inoculating the marine-derived fungus Pestalotiopsis sp. J63 spores into culture medium containing another fungus Penicillium janthinellum P1 pre-grown mycelia pellets for 2 days. RESULTS The resulting co-immobilization system exhibited stronger biocatalytic activity, broader spectrum of substrates and remained more stable in media when subjected to hostile environment than free mycelia. In order to examine the potential function and prospects of this co-immobilization system, the treatment of paper mill effluent was applied as a practical example. Numerous insoluble fine fibers in the wastewater were successfully and rapidly biodegraded and removed using this novel co-immobilization system. CONCLUSION Overall, the present study offers a simple and reproducible way to architect a two-species whole-cell immobilization system, which is highly promising for biochemical and industrial processes. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: An efficient and green bioprocess is herein reported to obtain halogenated nucleosides by transglycosylation using immobilized lactic acid bacteria (LAB). Lactobacillus animalis ATCC 35046 showed a yield of 95% at 0.5h to synthesize 5-fluorouracil-2′-deoxyriboside (floxuridine). Calcium alginate was the best matrix for whole-cell immobilization by entrapment. Its productivity was 87mg/Lh in a continuous bioprocess. When adsorption techniques were evaluated, DEAE-Sepharose was the support which showed higher microbial load, its productivity being 53mg/Lh. Additionally, this microorganism was able to produce 5-bromouracil-2′-deoxyriboside, 6-chloropurine-2′-deoxyriboside and 6-bromopurine-2′-deoxyriboside. [Copyright &y& Elsevier]

Whole-cell immobilization of selenate-respiring Sulfurospirillum barnesii in polyacrylamide gels was investigated to allow the treatment of selenate contaminated (790 µg Se × L−1) synthetic wastewater with a high molar excess of nitrate (1,500 times) and sulfate (200 times). Gel-immobilized S. barnesii cells were used to inoculate a mesophilic (30°C) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (COD) × L−1 day−1. Selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39 µg Se × L−1 was obtained. Scanning electron microscopy with energy dispersive X-ray (SEM–EDX) analysis revealed spherical bioprecipitates of ≤2 µm diameter mostly on the gel surface, consisting of selenium with a minor contribution of sulfur. To validate the bioaugmentation success under microbial competition, gel cubes with immobilized S. barnesii cells were added to an Upflow Anaerobic Sludge Bed (UASB) reactor, resulting in earlier selenate (24 hydraulic retention times (HRTs)) and sulfate (44 HRTs) removal and higher nitrate/nitrite removal efficiencies compared to a non-bioaugmented control reactor. S. barnesii was efficiently immobilized inside the UASB bioreactors as the selenate-reducing activity was maintained during long-term operation (58 days), and molecular analysis showed that S. barnesii was present in both the sludge bed and the effluent. This demonstrates that gel immobilization of specialized bacterial strains can supersede wash-out and out-competition of newly introduced strains in continuous bioaugmented systems. Eventually, proliferation of a selenium-respiring specialist occurred in the non-bioaugmented control reactor, resulting in simultaneous nitrate and selenate removal during a later phase of operation. [ABSTRACT FROM AUTHOR]

Abstract: Four new whole cell biocatalysts have been selected after a high throughput screening of microorganisms from public collections: Williopsis californica CBS 2158, Williopsis saturnus NCYC 2313, Pachysolen tannophilus NCYC 1597 and Coniochaeta velutina CBS 981.68, in basis to the high yield and stereoselectivity in the oxidation of 1-tetralol. These microorganisms were immobilised in different supports by entrapment techniques, being especially remarkable the results obtained with different tailor made agar matrix from Hispanagar (Spain). The selection of the immobilization conditions for each strain was performed following a statistical design based in the response surface methodology. The immobilised biocatalysts –Williopsis californica and Williopsis saturnus – immobilised in tailor-made agar (2.5%) from Pterocladia and Gracilaria (Hispanagar S.A.) carry to 100% yield in the oxidation of cyclohexanol. The immobilised biocatalysts displayed a high S-stereoselectivity (ee>98% and 50% yield) in the oxidation of (R,S)-1-tetralol and a high yield in the oxidation of bulky substrates as 2-adamantanol (100%). The kinetic studies showed that the diffusion of reagents/products is the rate controlling step. The addition of glucose (0.5%) increases the oxidation yields. [Copyright &y& Elsevier]

Immobilization of transaminases creates promising biocatalysts for production of chiral amines in batch or continuous-flow mode reactions. E. coli cells containing overexpressed transaminases of various selectivities and hollow silica microspheres as supporting agent were immobilized by an improved sol-gel process to produce immobilized transaminase biocatalysts with suitable stability and mechanical properties for continuous-flow applications. The immobilized cell-based transaminase biocatalyst proved to be durable and easy-to-use in kinetic resolution of four racemic amines 1a–d. The batch and continuous-flow mode kinetic resolutions with transaminase biocatalyst of opposite stereopreference provided access to both enantiomers of the corresponding amines. By using the most suitable immobilized transaminase biocatalysts, this study describes the first transaminase-based approach for the production of both pure enantiomers of 1-(3,4-dimethoxyphenyl)ethan-1-amine 1d. [ABSTRACT FROM AUTHOR]

Planococcus sp. S5, a Gram-positive bacterium isolated from the activated sludge is known to degrade naproxen in the presence of an additional carbon source. Due to the possible toxicity of naproxen and intermediates of its degradation, the whole cells of S5 strain were immobilized onto loofah sponge. The immobilized cells degraded 6, 9, 12 or 15 mg/L of naproxen faster than the free cells. Planococcus sp. cells immobilized onto the loofah sponge were able to degrade naproxen efficiently for 55 days without significant damage and disintegration of the carrier. Analysis of the activity of enzymes involved in naproxen degradation showed that stabilization of S5 cells in exopolysaccharide (EPS) resulted in a significant increase of their activity. Changes in the structure of biofilm formed on the loofah sponge cubes during degradation of naproxen were observed. Developed biocatalyst system showed high resistance to naproxen and its intermediates and degraded higher concentrations of the drug in comparison to the free cells. [ABSTRACT FROM AUTHOR]

An improved sol-gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co-immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω-transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co-immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4-phenylbutan-2-amine or heptan-2-amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous-flow mode. The novel co-immobilized whole-cell system proved to be an easy-to-store and durable biocatalyst., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Methanol is a versatile compound that can be biologically synthesized from methane (CH4) by methanotrophs using a low energy-consuming and environment-friendly process. Methylocella tundrae is a type II methanotroph that can utilize CH4 as a carbon and energy source. Methanol is produced in the first step of the metabolic pathway of methanotrophs and is further oxidized into formaldehyde. Several parameters must be optimized to achieve high methanol production. In this study, we optimized the production conditions and process parameters for methanol production. The optimum incubation time, substrate, pH, agitation rate, temperature, phosphate buffer and sodium formate concentration, and cell concentration were determined to be 24 h, 50% CH4, pH 7, 150 rpm, 30°C, 100 mM and 50 mM, and 18 mg/ml, respectively. The optimization of these parameters significantly improved methanol production from 0.66 to 5.18 mM. The use of alginate-encapsulated cells resulted in enhanced methanol production stability and reusability of cells after five cycles of reuse under batch culture conditions.