Editorial for the Special Issue: Environment Microorganisms and Their Enzymes with Biotechnological Application.

The ubiquitous nature of microorganisms demonstrates their ability to survive and thrive in diverse ecological settings, and their presence in extreme environments that approach the known limits of adaptable living confers importance to their role in those ecosystems [...].

Optimization of bacterioruberin production from Halorubrum ruber and assessment of its antioxidant potential.

Haloarchaea produce bacterioruberin, a major C50 carotenoid with antioxidant properties that allow for its potential application in the food, cosmetic, and pharmaceutical industries. This study aimed to optimize culture conditions for total carotenoid, predominantly comprising bacterioruberin, production using Halorubrum ruber MBLA0099. A one-factor-at-a-time and statistically-based experimental design were applied to optimize the culture conditions. Culture in the optimized medium caused an increase in total carotenoid production from 0.496 to 1.966 mg L- 1 Maximal carotenoid productivity was achieved in a 7-L laboratory-scale fermentation and represented a 6.05-fold increase (0.492 mg L-1 d-1). The carotenoid extracts from strain MBLA0099 exhibited a 1.8-10.3-fold higher antioxidant activity in vitro, and allowed for a higher survival rate of Caenorhabditis elegans under oxidative stress conditions. These results demonstrated that Hrr. ruber MBLA0099 has significant potential as a haloarchaon for the commercial production of bacterioruberin.

A Novel Carotenoid-Producing Bacterium, Paenibacillus aurantius sp. nov., Isolated from Korean Marine Environment.

The novel bacterial strain MBLB1776T was isolated from marine mud in Uljin, the Republic of Korea. Cells were Gram-positive, spore-forming, non-motile, and non-flagellated rods. Growth was observed at a temperature range of 10-45 °C, pH range of 6.0-8.0, and NaCl concentrations of 0-4% (w/v). Phylogenetic analysis of the 16S rRNA gene sequence revealed that MBLB1776T belonged to the genus Paenibacillus and was closely related to Paenibacillus cavernae C4-5T (94.83% similarity). Anteiso-C15:0, iso-C16:0, C16:0, and iso-C15:0 were the predominant fatty acids. Menaquinone 7 was identified as the major isoprenoid quinone. The major polar lipids included diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Its whole genome was 6.3 Mb in size, with a G+C content of 55.8 mol%. Average nucleotide identity and in silico DNA-DNA hybridization values were below the species delineation threshold. Gene function analysis revealed the presence of a complete C30 carotenoid biosynthetic pathway. Intriguingly, MBLB1776T harbored carotenoid pigments, imparting an orange color to whole cells. Based on this comprehensive polyphasic taxonomy, the MBLB1776T strain represents a novel species within the genus Paenibacillus, for which the name Paenibacillus aurantius sp. nov is proposed. The type strain was MBLB1776T (=KCTC 43279T = JCM 34220T). This is the first report of a carotenoid-producing Paenibacillus sp.

Whole-cell bioconversion using non-Leloir transglycosylation reactions: a review.

Microbial biocatalysts are evolving technological tools for glycosylation research in food, feed and pharmaceuticals. Advances in bioengineered Leloir and non-Leloir carbohydrate-active enzymes allow for whole-cell biocatalysts to curtail production costs of purified enzymes while enhancing glucan synthesis through continued enzyme expression. Unlike sugar nucleotide-dependent Leloir glycosyltransferases, non-Leloir enzymes require inexpensive sugar donors and can be designed to match the high value, yield and selectivity of the former. This review addresses the current state of bacterial cell-based production of glucans and glycoconjugates via transglycosylation, and describes how alterations made to microbial hosts to surpass purified enzymes as the preferred mode of catalysis are steadily being acquired through genetic engineering, rational design and process optimization. A comprehensive exploration of relevant literature has been summarized to describe whole-cell biocatalysis in non-Leloir glycosylation reactions with various donors and acceptors, and the characterization, application and latest developments in the optimization of their use.

Probiotic and antioxidant properties of C30 carotenoid-producing Lactiplantibacillus plantarum isolated from kimchi.

This study focuses on the development of functional probiotics using caroteonid-producing lactic acid bacteria (LAB) with antioxidant properties. Thirty LAB strains were evaluated for their probiotic properties. Carotenoid biosynthesis gene cluster (crtMN operon) was detected using polymer chain reaction (PCR). The carotenoid identified as 4,4'-diaponeurosporene was analyzed via UV visible absorption spectra and HPLC. Five carotenoid-producing strains showed antioxidant activities. Lactiplantibacillus plantarum MGB0112, which showed the highest carotenoid production measuring at 470 nm of absorbance per ml of culture broth (0.014 A470nm/ml), showed low pH (56.5%) and bile salt (97.8%) tolerance with high adhesion properties (55.1% for toluene). Furthermore, this strain and 4,4'-diaponeurosporene extract exhibited antioxidant activity (99.5 and 40.1%, respectively) against DPPH free radicals in vitro. Their antioxidant properties were confirmed in vivo (45.6 and 55.2% survival rates in Caenorhabditis elegans). Therefore, C30 carotenoid-producing strain MGB0112 demonstrates outstanding antioxidant effects and can be a potential functional probiotics.

4,4'-Diaponeurosporene Production as C30 Carotenoid with Antioxidant Activity in Recombinant Escherichia coli.

Carotenoids, a group of isoprenoid pigments, are naturally synthesized by various microorganisms and plants, and are industrially used as ingredients in food, cosmetic, and pharmaceutical product formulations. Although several types of carotenoids and diverse microbial carotenoid producers have been reported, studies on lactic acid bacteria (LAB)-derived carotenoids are relatively insufficient. There is a notable lack of research focusing on C30 carotenoids, the functional characterizations of their biosynthetic genes and their mass production by genetically engineered microorganisms. In this study, the biosynthesis of 4,4'-diaponeurosporene in Escherichia coli harboring the core biosynthetic genes, dehydrosqualene synthase (crtM) and dehydrosqualene desaturase (crtN), from Lactiplantibacillus plantarum subsp. plantarum KCCP11226 was constructed to evaluate and enhance 4,4'-diaponeurosporene production and antioxidant activity. The production of 4,4'-diapophytoene, a substrate of 4,4'-diaponeurosporene, was confirmed in E. coli expressing only the crtM gene. In addition, recombinant E. coli carrying both C30 carotenoid biosynthesis genes (crtM and crtN) was confirmed to biosynthesize 4,4'-diaponeurosporene and exhibited a 6.1-fold increase in carotenoid production compared to the wild type and had a significantly higher antioxidant activity compared to synthetic antioxidant, butylated hydroxytoluene. This study presents the discovery of an important novel E. coli platform in consideration of the industrial applicability of carotenoids.

Antioxidant Potential and Capacity of Microorganism-Sourced C30 Carotenoids-A Review.

Carotenoids are lipophilic tetraterpenoid pigments produced by plants, algae, arthropods, and certain bacteria and fungi. These biologically active compounds are used in the food, feed, and nutraceutical industries for their coloring and the physiological benefits imparted by their antioxidant properties. The current global carotenoid market is dominated by synthetic carotenoids; however, the rising consumer demand for natural products has led to increasing research and development in the mass production of carotenoids from alternative natural sources, including microbial synthesis and plant extraction, which holds a significant market share. To date, microbial research has focused on C40 carotenoids, but studies have shown that C30 carotenoids contain similar-and in some microbial strains, greater-antioxidant activity in both the physical and chemical quenching of reactive oxygen species. The discovery of carotenoid biosynthetic pathways in different microorganisms and advances in metabolic engineering are driving the discovery of novel C30 carotenoid compounds. This review highlights the C30 carotenoids from microbial sources, showcasing their antioxidant properties and the technologies emerging for their enhanced production. Industrial applications and tactics, as well as biotechnological strategies for their optimized synthesis, are also discussed.

Paenibacillus mellifer sp. nov., isolated from gut of the honey bee Apis mellifera.

An isolate, designated MBLB2552T, was isolated from the gut of the honey bees (Apis mellifera L.) and identified as a member of the genus Paenibacillus based on the sequences of the 16S rRNA gene sequences. The most closely related species to strain MBLB2552T were Paenibacillus timonensis 2301032 T, Paenibacillus barengoltzii NBRC 101215 T, and Paenibacillus macerans IAM 12467 T, with similarity values of 98.1, 97.21 and 97.0%, respectively, based on 16S rRNA gene sequences. The genome size and G + C content of MBLB2552T were 5.2 Mb and 52.4%, respectively. The Ortho average nucleotide identity (OrthoANI) and in silico DNA-DNA hybridization (isDDH) values between strain MBLB2552T and the type strains of the closest species were below the species delineation threshold. Comparative genomic analysis showed that most core POGs of strain MBLB2552T and other related taxa were related to translation, ribosomal structure and biogenesis (J) and carbohydrate metabolism in the COG category and KEGG pathways, respectively. Strain MBLB2552T was Gram stain-positive, spore-forming, rod-shaped, facultative anaerobic, motile, and grew at 20‒45 °C in 0‒2% (w/v) NaCl at pH 6.0‒9.0. The major polar lipids identified were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, unidentified polar lipids, and an unidentified glycolipid. We propose that strain MBLB2552T represents the type strain of the genus Paenibacillus and its name Paenibacillus mellifer sp. nov. is proposed. The type of strain was MBLB2552T (= JCM 35371 T = KCTC 43386 T).

Genomic and physiological analysis of C50 carotenoid-producing novel Halorubrum ruber sp. nov.

A novel haloarchaeal species designated as MBLA0099T was isolated from seawater near Yeongheung Island. Cells were Gram-negative, non-motile, red-pigmented, and rod-shaped. They grew at 10-45°C, within pH 5.5-9.0, and between 7.5% and 30% NaCl concentrations. Cells were able to grow without Mg2+ and were lysed in distilled water. The size of the whole-genome and G + C content of DNA was 3.02 Mb and 68.9 mol%, respectively. Phylogenetic analysis shows that the strain MBLA0099T belongs to the genus Halorubrum. The average nucleotide and amino acid identity, and in silico DNA-DNA hybridization values were below the species delineation threshold. Pan-genomic analysis revealed that 3.2% of all genes present in strain MBLA0099T were unique to the strain. The red carotenoid produced by strain MBLA0099T was subjected to spectrometric and chromatographic analyses and confirmed to be bacterioruberin as C50 carotenoid. Mevalonic acid, terpenoid backbone, and carotenoid biosynthesis pathway were annotated for strain MBLA0099T. The C50 carotenoid production by strain MBLA0099T was also enhanced under various stress conditions including relatively netural pH, high oxidative and salinity conditions. Additionally, the strain MBLA0099T-derived bacterioruberin showed the antioxidant activity with EC50 value of 12.29 µg/ml, based on the evaluation of DPPH free radical scavenging activity. The present study would be the first report on the identification of C50 carotenoid from the strain MBLA0099T representing a novel species of the genus Halorubrum, for which the name Halorubrum ruber sp. nov. is proposed. The typestrain used was MBLA0099T (= KCTC 4296T = JCM 34701T).

Fictibacillus marinisediminis sp. nov., a nitrate-reducing bacterium isolated from marine sediment in Hupo Basin, Republic of Korea.

An isolate, designated strain KIGAM418T was isolated from marine mud below 192 m depth in the Hupo Basin, Republic of Korea. Strain KIGAM418T was Gram-stain positive, spore-forming, rod-shaped, facultatively anaerobic, and grew at 10‒45 °C, in 0‒2% (w/v) NaCl at pH 4.0‒12.0. The strain tested positive for catalase, oxidase, and motility. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain KIGAM418T was related to the genus Fictibacillus. The strain showed the highest similarity to Fictibacillus rigui WPCB074T (98.0-98.1%) and Fictibacillus solisalsi YC1T (97.2-97.8%). The diagnostic diamino acid of the cell wall was meso-diaminopimelic acid. The major fatty acids were characterized as anteiso-C15:0 and iso-C15:0. Strain KIGAM418T possessed diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine as the major polar lipids and menaquinone-7 as the predominant menaquinone. The genome size and G + C content were 4.56 Mb and 43.2 mol%, respectively. According to predicted functional genes of the genome, the category of amino acid transport and metabolism was mainly distributed. Based on the polyphasic taxonomic data, strain KIGAM418T represents a novel species of the genus Fictibacillus, for which the name Fictibacillus marinisediminis sp. nov. is proposed. The type strain is KIGAM418T (= KCTC 43291 T = JCM 34437 T).

Genomic and Physiological Characterization of Metabacillus flavus sp. nov., a Novel Carotenoid-Producing Bacilli Isolated from Korean Marine Mud.

The newly isolated strain KIGAM252T was found to be facultatively anaerobic, Gram-stain-positive, spore-forming, and rod-shaped. They grew at 10-45 °C, pH 6.0-10.0, and were able to tolerate up to 6% NaCl in the growth medium. Phylogenetic analysis indicated that the KIGAM252T strain was related to the genus Metabacillus. The cell membrane fatty acid composition of strain KIGAM252T included C15:0 anteiso and C15:0 iso (25.6%) as the major fatty acids, and menaquinone 7 was the predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The size of the whole genome was 4.30 Mbp, and the G + C content of the DNA was 43.8%. Average nucleotide and amino acid identity and in silico DNA-DNA hybridization values were below the species delineation threshold. Pan-genomic analysis revealed that 15.8% of all genes present in strain KIGAM252T was unique to the strain. The analysis of the secondary biosynthetic pathway predicted the carotenoid synthetic gene cluster in the strain KIGAM252T. Based on these current polyphasic taxonomic data, strain KIGAM252T represents a novel species of the genus Metabacillus that produces carotenoids, for which we propose the name Metabacillus flavus sp. nov. The type of strain was KIGAM252T (=KCTC 43261T = JCM 34406T).

Enhanced Production of C30 Carotenoid 4,4'-Diaponeurosporene by Optimizing Culture Conditions of Lactiplantibacillus plantarum subsp. plantarum KCCP11226T.

The rising demand for carotenoids can be met by microbial biosynthesis as a promising alternative to chemical synthesis and plant extraction. Several species of lactic acid bacteria (LAB) specifically produce C30 carotenoids and offer the added probiotic benefit of improved gut health and protection against chronic conditions. In this study, the recently characterized Lactiplantibacillus plantarum subsp. plantarum KCCP11226T produced the rare C30 carotenoid, 4,4'-diaponeurosporene, and its yield was optimized for industrial production. The one-factor-at-a-time (OFAT) method was used to screen carbon and nitrogen sources, while the abiotic stresses of temperature, pH, and salinity, were evaluated for their effects on 4,4'-diaponeurosporene production. Lactose and beef extract were ideal for optimal carotenoid production at 25°C incubation in pH 7.0 medium with no salt. The main factors influencing 4,4'-diaponeurosporene yields, namely lactose level, beef extract concentration and initial pH, were enhanced using the Box-Behnken design under response surface methodology (RSM). Compared to commercial MRS medium, there was a 3.3-fold increase in carotenoid production in the optimized conditions of 15% lactose, 8.3% beef extract and initial pH of 6.9, producing a 4,4'-diaponeurosporene concentration of 0.033 A470/ml. To substantiate upscaling for industrial application, the optimal aeration rate in a 5 L fermentor was 0.3 vvm. This resulted in a further 3.8-fold increase in 4,4'-diaponeurosporene production, with a concentration of 0.042 A470/ml, compared to the flask-scale cultivation in commercial MRS medium. The present work confirms the optimization and scale-up feasibility of enhanced 4,4'-diaponeurosporene production by L. plantarum subsp. plantarum KCCP11226T.

Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9 kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35 °C and pH 5. rLgGad showed a melting temperature of 43.84 °C. At 30 °C, more than 80% of the activity was maintained even after 7 h; however, it rapidly decreased at 50 °C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3 min- 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., low salt-tolerant haloarchaea isolated from seawater and sediment.

Three novel halophilic archaea were isolated from seawater and sediment near Yeoungheungdo Island, Republic of Korea. The genome size and G + C content of the isolates MBLA0076T, MBLA0077T, and MBLA0078T were 3.56, 3.48, and 3.48 Mb and 61.7, 60.8, and 61.1 mol%, respectively. The three strains shared 98.5-99.5 % sequence similarity of the 16 S rRNA gene, whereas their sequence similarity to the 16 S rRNA gene of type strains was below 98.5 %. Phylogenetic analysis based on sequences of the 16 S rRNA and RNA polymerase subunit beta genes indicated that the isolates belonged to the genus Haloferax. The orthologous average nucleotide identity, average amino-acid identity, and in silico DNA-DNA hybridization values were below species delineation thresholds. Pan-genomic analysis indicated that the three novel strains and 11 reference strains had 8981 pan-orthologous groups in total. Fourteen Haloferax strains shared 1766 core pan-genome orthologous groups, which were mainly related to amino acid transport and metabolism. Cells of the three isolates were gram-negative, motile, red-pink pigmented, and pleomorphic. The strains grew optimally at 30 °C (MBLA0076T) and 40 °C (MBLA0077T, MBLA0078T) in the presence of 1.28 M (MBLA0077T) and 1.7 M (MBLA0076T, MBLA0078T) NaCl and 0.1 M (MBLA0077T), 0.2 M (MBLA0076T), and 0.3 M (MBLA0078T) MgCl2·6H2O at pH 7.0-8.0. Cells of all isolates lysed in distilled water; the minimum NaCl concentration necessary to prevent lysis was 0.43 M. The major polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and sulphated diglycosyl archaeol-1. Based on their phenotypic and genotypic properties, MBLA0076T, MBLA0077T, and MBLA0078T were described as novel species of Haloferax, for which we propose the names Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., respectively. The respective type strains of these species are MBLA0076T (= KCTC 4288T = JCM 34,169T), MBLA0077T (= KCTC 4289T = JCM 34,170T), and MBLA0078T (= KCTC 4290T = JCM 34,171T).

Nocardioides luti sp. nov., belonging to the family Nocardioidaceae isolated from kaolinite, exhibiting the biosynthesis potential of alkylresorcinol.

A novel Gram-staining-positive, short rod-shaped, non-motile, and non-pigmented actinobacterial strain (KIGAM211T) was isolated from kaolinite, a soft white clay mineral, collected from Sancheong in the Republic of Korea. On the basis of 16S rRNA gene sequence analysis, strain KIGAM211T was determined to belong to the genus Nocardioides and was most closely related to N. ungokensis UKS-03T (97.5% similarity). Cells could grow between 4 and 35 °C (optimum 30 °C), 0-3% (w/v) NaCl concentration (optimum 0%) and pH 5.5-8.5 (optimum 7.0) on R2A agar. Morphological appearance of colonies was cream-white, arranged singly or in groups. Biochemical characterization of strain KAGAM211T indicated that it could hydrolyze casein, gelatin, Tweens 40 and tyrosine. Furthermore, the strain was positive for both oxidase and catalase activity. Strain KIGAM211T was characterized chemotaxonomically by MK-8 (H4) as the predominant menaquinone and phosphatidylglycerol (PG) and phosphatidylinositol (PI) as the major polar lipids. Major fatty acids were iso-C16:0 and C18:1 ω9c. The Ortholog average nucleotide identity (OrthoANI) and in silico DNA-DNA hybridization (isDDH) values between strain KIGAM211T and its most closely related strains of the Nocardioides genus were < 82% and < 24%, respectively, suggesting that strain KIGAM211T represent a novel species. The whole genome size of KIGAM211T was 4.52 Mb, comprising a total of 4,294 genes with DNA G + C content of 72.3 mol%. The genome of strain KIGAM211T also comprises the biosynthetic gene cluster for alkylresorcinol as secondary metabolite. The results of physiological, taxonomical, phylogenetic, and whole genome analyses allowed for differentiation of strain KIGAM211T from the recognized Nocardioides species. Therefore, strain KIGAM211T is considered to represent a novel species, for which the name Nocardioides luti sp. nov. (type strain KIGAM211T = KCTC 49364T = JCM 33859T) is proposed.

Halobellus ruber sp. nov., a deep red-pigmented extremely halophilic archaeon isolated from a Korean solar saltern.

A novel halophilic archaeon, strain MBLA0160T, was isolated from a solar saltern in Sorae, Republic of Korea. The cells are deep-red pigmented, Gram-negative, rod shaped, motile, and lysed in distilled water. The strain MBLA0160T grew at 25-45 °C (optimum 37 °C), in 15-30% (w/v) NaCl (optimum 20%) and 0.1-1.0 M MgCl2 (optimum 0.3-0.5 M) at pH 5.0-9.0 (optimum 7.0). Phylogenetic analysis based on the 16S rRNA sequence showed that this strain was related to two species within the genus Halobellus (Hbs.), with 98.4% and 95.8% similarity to Hbs. salinus CSW2.24.4 T and Hbs. clavatus TNN18T, respectively. The major polar lipids of the strain MBLA160T were phosphatidylglycerol, phosphatidylglycerol sulfate, and phosphatidylglycerol phosphate methyl ester. The genome size, G + C content, and N50 value of MBLA0160T were 3.49 Mb, 66.5 mol%, and 620,127 bp, respectively. According to predicted functional proteins of strain MBLA0160T, the highest category was amino acid transport and metabolism. Genome rapid annotation showed that amino acid and derivatives was the most subsystem feature counts. Pan-genomic analysis showed that strain MBLA0160T had 97 annotated unique KEGG, which were mainly included metabolism and environmental information processing. Ortholog average nucleotide identities (OrthoANI) and in silico DNA-DNA hybridization (isDDH) values between the strain MBLA0160T and other strains of the genus Halobellus were under 84,4% and 28.1%, respectively. The genome of strain MBLA0160T also contain the biosynthetic gene cluster for C50 carotenoid as secondary metabolite. Based on the phylogenetic, phenotypic, chemotaxonomic properties, and comparative genomic analyses, strain MBLA0160T is considered to represent a novel species of the genus Halobellus, for which the name Halobellus ruber sp. nov. is proposed. The type strain is MBLA0160T (= KCTC 4291 T = JCM 34172 T).

Biosynthesis of glyceride glycoside (nonionic surfactant) by amylosucrase, a powerful glycosyltransferase.

Amylosucrase (ASase, E.C. 2.4.1.4) is a powerful transglycosylation enzyme that can transfer glucose from sucrose to the hydroxyl (-OH) group of various compounds. In this study, recombinant ASases from Deinococcus geothermalis (DgAS) and Bifidobacterium thermophilum (BtAS) were used to synthesize biosurfactants based on the computational analysis of predicted docking simulations. Successful predictions of the binding affinities, conformations, and three-dimensional structures of three surfactants were computed from receptor-ligand binding modes. DgAS and BtAS were effective in the synthesis of biosurfactants from glyceryl caprylate, glyceryl caprate, and polyglyceryl-2 caprate. The results of the transglycosylation reaction were consistent for both ASases, with glyceryl caprylate acceptor showing the highest concentration, as confirmed by thin layer chromatography. Furthermore, the transglycosylation reactions of DgAS were more effective than those of BtAS. Among the three substrates, glyceryl caprylate glycoside and glyceryl caprate glycoside were successfully purified by liquid chromatography-mass spectrometry (LC-MS) with the corresponding molecular weights.

Genome analysis of 1-deoxynojirimycin (1-DNJ)-producing Bacillus velezensis K26 and distribution of Bacillus sp. harboring a 1-DNJ biosynthetic gene cluster.

1-Deoxynojirumycin (1-DNJ) is a representative iminosugar with α-glucosidase inhibition (AGI) activity. In this study, the full genome sequencing of 1-DNJ-producing Bacillus velezensis K26 was performed. The genome consists of a circular chromosome (4,047,350 bps) with two types of putative virulence factors, five antibiotic resistance genes, and seven secondary metabolite biosynthetic gene clusters. Genomic analysis of a wide range of Bacillus species revealed that a 1-DNJ biosynthetic gene cluster was commonly present in four Bacillus species (B. velezensis, B. pseudomycoides, B. amyloliquefaciens, and B. atrophaeus). In vitro experiments revealed that the increased mRNA expression levels of the three 1-DNJ biosynthetic genes were closely related to increased AGI activity. Genomic comparison and alignment of multiple gene sequences indicated the conservation of the 1-DNJ biosynthetic gene cluster in each Bacillus species. This genomic analysis of Bacillus species having a 1-DNJ biosynthetic gene cluster could provide a basis for further research on 1-DNJ-producing bacteria.

4,4'-Diaponeurosporene from Lactobacillus plantarum subsp. plantarum KCCP11226: Low Temperature Stress-Induced Production Enhancement and In Vitro Antioxidant Activity.

Carotenoids, which have biologically beneficial effects and occur naturally in microorganisms and plants, are pigments widely applied in the food, cosmetics and pharmaceutical industries. The compound 4,4'-diaponeurosporene is a C30 carotenoid produced by some Lactobacillus species, and Lactobacillus plantarum is the main species producing it. In this study, the antioxidant activity of 4,4'-diaponeurosporene extracted from L. plantarum subsp. plantarum KCCP11226 was examined. Maximum carotenoid content (0.74 ± 0.2 at A470) was obtained at a relatively low temperature (20°C). The DPPH radical scavenging ability of 4,4'-diaponeurosporene (1 mM) was approximately 1.7-fold higher than that of butylated hydroxytoluene (BHT), a well-known antioxidant food additive. In addition, the ABTS radical scavenging ability was shown to be 2.3- to 7.5-fold higher than that of BHT at the range of concentration from 0.25 mM to 1 mM. The FRAP analysis confirmed that 4,4'- diaponeurosporene (0.25 mM) was able to reduce Fe3+ by 8.0-fold higher than that of BHT. Meanwhile, 4,4'-diaponeurosporene has been confirmed to be highly resistant to various external stresses (acid/bile, high temperature, and lysozyme conditions). In conclusion, L. plantarum subsp. plantarum KCCP11226, which produces 4,4'-diaponeurosporene as a functional antioxidant, may be a potentially useful strain for the development of functional probiotic industries.

Molecular Docking and Kinetic Studies of the A226N Mutant of Deinococcus geothermalis Amylosucrase with Enhanced Transglucosylation Activity.

Amylosucrase (ASase, E.C. 2.4.1.4) is capable of efficient glucose transfer from sucrose, acting as the sole donor molecule, to various functional acceptor compounds, such as polyphenols and flavonoids. An ASase variant from Deinococcus geothermalis, in which the 226th alanine is replaced with asparagine (DgAS-A226N), shows increased polymerization activity due to changes in the flexibility of the loop near the active site. In this study, we further investigated how the mutation modulates the enzymatic activity of DgAS using molecular dynamics and docking simulations to evaluate interactions between the enzyme and phenolic compounds. The computational analysis revealed that the A226N mutation could induce and stabilize structural changes near the substratebinding site to increase glucose transfer efficiency to phenolic compounds. Kinetic parameters of DgAS-A226N and WT DgAS were determined with sucrose and 4-methylumbelliferone (MU) as donor and acceptor molecules, respectively. The kcat/Km value of DgAS-A226N with MU (6.352 mM-1min-1) was significantly higher than that of DgAS (5.296 mM-1min-1). The enzymatic activity was tested with a small phenolic compound, hydroquinone, and there was a 1.4-fold increase in α-arbutin production. From the results of the study, it was concluded that DgAS-A226N has improved acceptor specificity toward small phenolic compounds by way of stabilizing the active conformation of these compounds.