Biotechnology Letters最新文献

Yeasts of the genus Pichia include osmophilic species that metabolize glycerol and produce arabitol. This research aimed to evaluate the effects of acid and salt stress on enhancing the yield of arabitol in a batch system. It also innovates in fermentation processes by proposing wild yeasts of the genus and comparing them with a yeast collection. In this study, three yeasts originally classified within the genus Pichia were evaluated: Pichia kluyveri, Pichia stipitis (Scheffersomyces stipitis), and Pichia pastoris (Komagataella phaffii). The strains were fermented individually with glycerol as a substrate. The sodium chloride concentration significantly affected the specific growth rate and the product of arabitol. The strain P. stipitis stood out for its arabitol yield (0.18 ± 0.02 g arabitol g^-1 glycerol consumed) at a pH of 6.8 and a sodium chloride concentration of 0.5 M. This study contributed to the search for environmental yeasts with potential use in arabitol production. It demonstrated that environmental isolates of this genus had superior tolerance to salt stress compared with the collection strain.

Droplet based microfluidics and automation are important technology to support bio-process and screening during the last years. Main objective of this review is to describe the current technology in monitoring secreting microbes at scale down level. During this study, questions and challenges are shown with relation to this approach specifically in droplet based microfluidics as well as in miniaturized platforms focused on screening of certain metabolites. Relevant challenges to both technologies are described in this article from a technical overview, those that are associated with the control of secondary metabolites as well as growth parameters during microbe cultivation with focus on Y. lipolidica, S. lividians and C. glutamicum. In order to achieve the objective, six studies highlighted in production of metabolites as polyphenols are discussed by using a narrative review as well as the relevancy of analytical methods that are associated with verify development of heterologous microorganisms during bioprocess.

H₂-driven biotransformations in the chemolithoautotrophic bacterium, Cupriavidus necator, are gaining attention to obtain valuable industrial molecules. However, cultivation variability and long lag phases have been reported in C. necator chemolithoautotrophic cultures. Few studies have focused on the management of these reported long lag phases. This article proposes a cultivation strategy based on serial precultures with glycerol to reduce metabolic transition. Two preculture media with and without glycerol were adapted in a seed-train to study the transition towards lithoautotrophy. Cultures were compared by monitoring chemolithoautotrophic growth in small-scale system. The presence of glycerol in fructose preculture medium completely removed the lag phase during subsequent lithoautotrophic cultivation compared to fructose only precultures (4-5 h). Glycerol can be used as carbon source to improve the transition to lithoautotrophy in C. necator, while reducing the lag phase. This preculture setup can be implemented to study the effects of any relevant biomolecule, while avoiding the random nature of culture start that makes interpretations difficult. The study provided valuable insights in the management of C. necator precultures, and lithoautotrophic cultivation systems to support the development of C. necator as a platform organism.

Chiral amines, as essential chiral building blocks in drug synthesis, present a considerable challenge in biomanufacturing due to the requirement for highly efficient stereoselective synthesis. In this study, we successfully cloned and heterologously expressed a novel (R)-amine transaminase, MagAT, from Mycolicibacterium agri. Systematic analysis showed optimal activity at pH 7.0, with the highest reaction rate occurring within 30 min at 50 ℃. However, considering overall thermal stability, 40℃ was selected as the operating temperature for subsequent experiments. Furthermore, the enzyme retained nearly 100% catalytic activity in the presence of 10% methanol, DMSO, and chloroform. Kinetic analysis demonstrated that MagAT possessed high substrate affinity, with Michaelis constants (K_m) of 5.13 ± 0.45 mM for (R)-1-phenylethan-1-amine and 3.73 ± 0.34 mM for pyruvate. Substrate specificity studies indicated that MagAT efficiently catalyzed the conversion of C3-C8 aliphatic amines (e.g., (R)-2-aminooctane) and monocyclic aromatic amines (e.g., (R)-1-phenylethan-1-amine), whereas its catalytic activity significantly declined for sterically hindered substrates such as naphthylamine. 3D structural modeling with AlphaFold3 and molecular docking showed that steric hindrance from key residues in the O-pocket critically influences substrate specificity. This study not only provides an efficient biocatalyst for the biosynthesis of chiral amines but also establishes a theoretical basis for the rational design of transaminases with broader substrate adaptability.

To enhance the applicability of Aspergillus oryzae LL1 in the food industry, the strain underwent ultraviolet irradiation through random mutagenesis to enhance the enzyme activity of extracellular leucine aminopeptidase (LAP). A. oryzae LL1 was subjected to three rounds of ultraviolet mutagenesis, and a number of 210, 280, and 290 mutant clones were selected based on LAP activity and stability analysis. The mutant clone with the highest LAP activity was selected for each mutagenesis, and then proceeded to the next round of mutagenesis. The activity of the three selected mutant strains showed significantly increased LAP activity compared to the wild-type strain. The LAP specific activity of A. oryzae LL1 was 319.9 mU/mg. The LAP specific activities of the mutant strains U01, U02, and U03 were 386.3, 449.9, and 556.5 mU/mg, respectively. After three rounds of mutagenesis, the U03 strain exhibited 1.74-fold greater specific activity of LAP than the LL1 strain. A further study evaluated the debittering effect of crude enzyme of the mutant strain U03 on isolated soy protein through hydrolysis and a decrease in the content of hydrophobic amino acids. The mutant strain U03 demonstrated a significantly higher efficiency in hydrolyzing soy protein compared to the LL1 strain, as evidenced by the degradation into small peptide fragments observed in SDS-PAGE analysis, the significant increase in peptide peaks in reverse-phase HPLC analysis, and the higher total free amino acid contents. These results suggest that the U03 strain exhibits more effective hydrolytic activity and holds potential for applications in the food industry, particularly in reducing bitterness in protein hydrolysates.

Polyphenol oxidase (PPO) enzymes perform enzymatic browning reactions by hydroxylation of monophenols to o-diphenols and oxidation of o-diphenols to o-quinones called phenolic substances in foods. Medlar fruit (Mespilus germanica L.) was used as an enzyme source which is a rich antioxidant and antiviral properties as well as its commercial value in food industry. PPO enzyme was partially purified using the homogenization step and ammonium sulfate precipitation 0-80%, respectively. Characterization studies were applied to determine the optimum substrate, buffer concentration, pH, and temperature for catechol as 0.1 M, pH: 6.8, and 15 ˚C, respectively. V_max and K_M values of medlar PPO for catechol were calculated as 12,542.46 IU and 2.5 mM, respectively. Following, PPO enzyme was purified by Sepharose 4B-L-tyrosine-p-aminobenzoic acid (S-4B-TABA) and Sepharose 6B-L-tyrosine-p-aminobenzoic acid (S-6B-TABA) affinity gels. Purification degrees were achieved as 54.0 and 4.8 for S-4B-TABA and S-6B-TABA, respectively. The medlar PPOs purified by S-4B-TABA and S-6B-TABA affinity gels exhibited a single band at a level of 40 kDa in Native PAGE and SDS-PAGE that the enzyme was concluded to have only one single subunit. Medlar PPO was firstly achieved to be purified by affinity chromatography in this study. No any study about purification of medlar PPO by affinity chromatography has been found in literature yet.

More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.

β-Nicotinamide mononucleotide (NMN) is a nucleotide that serves as a vital source of cellular energy and a direct precursor of nicotinamide adenine dinucleotide (NAD⁺), a crucial cofactor in human metabolism. Given its significant value in the fields of nutrition and healthcare, NMN biosynthesis has recently become a prominent area of research globally. In this study, we constructed the recombinant plasmid pETDuet-NPk encoding three intracellular proteins (Prs, Rk, and Nampt) and the recombinant plasmid pCDFDuet-NPC, containing the ribose ABC transporter system, the nicotinamide nucleotide transporter protein (PnuC), and nicotinic acid (NA) transporter protein (NiaP). The double plasmids were used to develop the engineered strain E. coli 2d, which facilitated a whole-cell catalytic reaction pathway for NMN production using D-ribose and nicotinamide as substrates. This approach yielded 0.139 g/L, which is 3.6 times higher than that obtained using glucose as a substrate. Additionally, the mutant strain E. coli 2d-C1, identified through UV mutagenesis and flow cytometry screening, yielded 0.91 g/L of NMN, representing a 6.5-fold increase compared to the unmutated strain E. coli 2d. This study introduces a novel approach for high-throughput NMN screening and highlights the use of cost-effective synthetic substrates to enhance NMN production.

Microbial expansins are proteins that are thought to mediate loosening of plant cell wall networks by non-catalytic disruption of non-covalent bonds between polysaccharides, particularly between cellulose chains. As a result, the expansins may facilitate the enzymatic hydrolysis of these polysaccharides. In this work, we used quartz-crystal microbalance with dissipation monitoring (QCM-D) to follow the influence of the Bacillus subtilis expansin BsEXLX1 and an inactive BsEXLX1 mutant (BsEXLX1_D81N) on the enzymatic degradation of never dried sugar beet pulp by a fungal cellobiohydrolase I (Cel7A) EC 3.2.1.176. The presence of the wild type BsEXLX1 enhanced the enzymatic degradation rate on the substrate in a dose dependent manner achieving a doubling of the rate, while the inactive BsEXLX1_D81N mutant exhibited only very limited increase in the degradation rate. This result suggests that the ability of BsEXLX1 to boost the action of Cel7A is linked to the ability of BsEXLX1 to bind to the glucan chains that in turn may increase the distance between two glucan chains enabling increased enzymatic attack.

Objectives: To expand the availability of promiscuous oleate hydratases (OAHs) for the asymmetric hydration of unactivated alkenes via sequence-based genome mining combined with targeted amino acid substitution.

Results: From 100 screened OAHs, 13 candidates were chosen, all exhibiting hydration activity toward oleic acid. These enzymes also showed significant activity with 1-decene (2 mM), with AuOAH, CkOAH, AiOAH, and CeaOAH producing (S)-2-decanol at concentrations of 822, 603, 495, and 461 μM, respectively. AuOAH, CeaOAH, and CkOAH further demonstrated notable activity with short-chain 1-heptene (2 mM), generating (S)-2-heptanol concentrations of 156, 115, and 133 μM, respectively. AuOAH, sourced from Acinetobacter ursingii for its relatively high activity and broad substrate range, was purified and characterized, showing turnover rates of 0.43-3.21 nmol min^-1 mg^-1 for 1-alkenes (C7-C13). The optimization of reaction conditions for whole-cell asymmetric hydration of 1-decene in recombinant E. coli (AuOAH) demonstrated that exogenous illumination with 561.5 nm light (9.4 µmol m^-2 s^-1) increased 1-decene conversion by approximately 1.5-fold. Similar light-induced enhancements (1.3-2.2-fold) were observed in OAHs from various sources. Under optimized conditions, recombinant E. coli (AuOAH) achieved 13.2-78.7% conversion for various unactivated alkenes (C7-C13) in an aqueous/organic two-phase system, with ee values ≥ 98%.

Conclusions: This study significantly enriches the enzymatic toolbox for asymmetric alkene hydration and illustrates the beneficial effect of light illumination on OAH-catalyzed hydration.