Next-Generation Sequencing for Whole-Genome Characterization of Weissella cibaria UTNGt21O Strain Originated From Wild Solanum quitoense Lam. Fruits: An Atlas of Metabolites With Biotechnological Significance

The whole genome of Weissella cibaria strain UTNGt21O isolated from wild fruits of Solanum quitoense (naranjilla) shrub was sequenced and annotated. The similarity proportions based on the genus level, as a result of the best hits for the entire contig, were 54.84% with Weissella, 6.45% with Leuconostoc, 3.23% with Lactococcus, and 35.48% no match. The closest genome was W. cibaria SP7 (GCF_004521965.1) with 86.21% average nucleotide identity (ANI) and 3.2% alignment coverage. The genome contains 1,867 protein-coding genes, among which 1,620 were assigned with the EggNOG database. On the basis of the results, 438 proteins were classified with unknown function from which 247 new hypothetical proteins have no match in the nucleotide Basic Local Alignment Search Tool (BLASTN) database. It also contains 78 tRNAs, six copies of 5S rRNA, one copy of 16S rRNA, one copy of 23S rRNA, and one copy of tmRNA. The W. cibaria UTNGt21O strain harbors several genes responsible for carbohydrate metabolism, cellular process, general stress responses, cofactors, and vitamins, conferring probiotic features. A pangenome analysis indicated the presence of various strain-specific genes encoded for proteins responsible for the defense mechanisms as well as gene encoded for enzymes with biotechnological value, such as penicillin acylase and folates; thus, W. cibaria exhibited high genetic diversity. The genome characterization indicated the presence of a putative CRISPR-Cas array and five prophage regions and the absence of acquired antibiotic resistance genes, virulence, and pathogenic factors; thus, UTNGt21O might be considered a safe strain. Besides, the interaction between the peptide extracts from UTNGt21O and Staphylococcus aureus results in cell death caused by the target cell integrity loss and the release of aromatic molecules from the cytoplasm. The results indicated that W. cibaria UTNGt21O can be considered a beneficial strain to be further exploited for developing novel antimicrobials and probiotic products with improved technological characteristics.

Bacterial Cellulose Containing Combinations of Antimicrobial Peptides with Various QQ Enzymes as a Prototype of an “Enhanced Antibacterial” Dressing: In Silico and In Vitro Data

To improve the action of already in use antibiotics or new antimicrobial agents against different bacteria, the development of effective combinations of antimicrobial peptides (AMPs) with enzymes that can quench the quorum (QQ) sensing of bacterial cells was undertaken. Enzymes hydrolyzing N-acyl homoserine lactones (AHLs) and peptides that are signal molecules of Gram-negative and Gram-positive bacterial cells, respectively, were estimated as “partners” for antibiotics and antimicrobial peptides in newly designed antimicrobial–enzymatic combinations. The molecular docking of six antimicrobial agents to the surface of 10 different QQ enzyme molecules was simulated in silico. This made it possible to choose the best variants among the target combinations. Further, bacterial cellulose (BC) was applied as a carrier for uploading such combinations to generally compose prototypes of effective dressing materials with morphology, providing good absorbance. The in vitro analysis of antibacterial activity of prepared BC samples confirmed the significantly enhanced efficiency of the action of AMPs (including polymyxin B and colistin, which are antibiotics of last resort) in combination with AHL-hydrolyzing enzymes (penicillin acylase and His6-tagged organophosphorus hydrolase) against both Gram-negative and Gram-positive cells.

Penicillin Acylase from Streptomyces lavendulae and Aculeacin A Acylase from Actinoplanes utahensis: Two Versatile Enzymes as Useful Tools for Quorum Quenching Processes

Many Gram-negative bacteria produce N-acyl-homoserine lactones (AHLs), quorum sensing (QS) molecules that can be enzymatically inactivated by quorum quenching (QQ) processes; this approach is considered an emerging antimicrobial alternative. In this study, kinetic parameters of several AHLs hydrolyzed by penicillin acylase from Streptomyces lavendulae (SlPA) and aculeacin A acylase from Actinoplanes utahensis (AuAAC) have been determined. Both enzymes catalyze efficiently the amide bond hydrolysis in AHLs with different acyl chain moieties (with or without 3-oxo modification) and exhibit a clear preference for AHLs with long acyl chains (C12-HSL > C14-HSL > C10-HSL > C8-HSL for SlPA, whereas C14-HSL > C12-HSL > C10-HSL > C8-HSL for AuAAC). Involvement of SlPA and AuAAC in QQ processes was demonstrated by Chromobacterium violaceum CV026-based bioassays and inhibition of biofilm formation by Pseudomonas aeruginosa, a process controlled by QS molecules, suggesting the application of these multifunctional enzymes as quorum quenching agents, this being the first time that quorum quenching activity was shown by an aculeacin A acylase. In addition, a phylogenetic study suggests that SlPA and AuAAC could be part of a new family of actinomycete acylases, with a preference for substrates with long aliphatic acyl chains, and likely involved in QQ processes.

Pengaruh pH dan Jenis Pelarut terhadap Ekstraksi Batch Asam 6-Aminopenisilinat

Abstrak. Asam 6-aminopenisilinat (6-APA) merupakan bahan dasar pembuatan penisilin semi-sintetis. Dalam skala komersial, 6-APA dapat diproduksi dengan cara enzimatis atau kimiawi. Pada umumnya, produksi 6-APA dilakukan secara enzimatis, yaitu dengan mengkonversi penisilin G menjadi 6-APA dengan bantuan penisilin asilase. Karena konversi merupakan reaksi kesetimbangan, maka produk yang didapat adalah campuran penisilin G, 6-APA, dan asam fenil asetat (PAA) sehingga untuk memperoleh 6-APA murni dilakukan proses ekstraksi, pemekatan, dan kristalisasi. Proses ini dipengaruhi oleh beberapa variabel operasi, yaitu temperatur, pH, dan jenis pelarut. Dalam penelitian ini akan dipelajari pengaruh pH dan jenis pelarut terhadap proses pemisahan 6-APA. Temperatur operasi adalah kondisi ruang dan perbandingan volume pelarut dengan volume larutan yang akan diekstraksi adalah 1:1. Variasi pH ekstraksi dilakukan antara rentang 2,0-5,0, sedangkan jenis pelarut yang digunakan adalah n-butil asetat, iso butil asetat, metil isobutil keton, dan iso amil asetat. Rentang pH terbaik untuk pemisahan 6-APA adalah 2,0-3,0 dengan pelarut metil isobutil keton. Pada kondisi ini, perolehan penisilin G adalah 98%, 6-APA 5%, dan PAA 99%. Sebagian besar 6-APA pada fase aquatik dapat diproses untuk pemurnian selanjutnya. Kata kunci: Penisilin G, 6-APA, PAA, ekstraksi, pelarut, pH. Abstract. Influence of pH and Solvent Types on 6-Aminopenicillinic Acid Batch Extraction. 6-aminopenicillinic acid (6-APA) is the raw material for producing semi-synthetic penicillin. In commercial scale, penicillin G is converted into 6-APA enzymatically by penicillin acylase. Due to the nature of equilibrium reaction, the products are in mixture solution of penicillin G, 6-APA, and phenyl acetic acid (PAA). In order to purify the targeted 6-APA, steps of extraction, concentration, and crystallisation are thereby compulsory. Extraction process is influenced by operation variables, among other things, temperature, pH, and solvent types. In this experiment, we observed the aspects of pH and solvent type, while temperature was set in room condition. Volume ratio of solvent to extracted solution was 1:1 and pH was varied between 2.0 and 5.0. There were four solvents tested: n-butyl acetate, isobutyl acetate, methyl isobutyl ketone, and isoamyl acetate. The results suggested that optimum process was attained from pH 2.0 to 3.0, using methyl isobutyl ketone as solvent. In this regard, the yield of penicillin G (98%), 6-APA 95%) and PAA (99%); hence, most of the 6-APA was concentrated within the aquatic phase, representing the ease of further process. Keywords: Penicillin G, 6-APA, PAA, extraction, solvent, pH. Graphical Abstract

Studying the Possibilities of Using 2-Halogen-Substituted Acetamides As Acyl Donors in Penicillin Acylase-Catalyzed Reactions

The possibility of using amides of halogen-substituted acetic acids as acyl donors in penicillin acylase-catalyzed reactions has been investigated, and the ability of this group of compounds to inactivate enzymes in the course of the catalytic conversion has been established. The strongest inactivating effect was demonstrated by iodoacetamide and bromoacetamide. However, the negative contribution of this side activity can be minimized by decreasing the temperature, when the rate of acyl donor conversion by penicillin acylases is still high enough, but the impact of enzyme inactivation becomes less significant. The catalytic activity of penicillin acylase from Alcaligenes faecalis in the conversion of 2-haloacetamides was significantly (5-8 times) higher than that of penicillin acylase from Escherichia coli.