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The rising utilization of PLA/PBAT-ST20 presents potential ecological risks stemming from its casual disposal and incomplete degradation. To solve this problem, this study investigated the degradation capabilities of PLA/PBAT-ST20 by a co-culture system comprising two thermophilic bacteria, Pseudomonas G1 and Kocuria G2, selected and identified from the thermophilic phase of compost. Structural characterization results revealed that the strains colonized the PLA/PBAT-ST20's surface, causing holes and cracks, with an increase in the carbonyl index (CI) and polydispersity index (PDI), indicating oxidative degradation. Enzyme activity results demonstrated that the co-culture system significantly enhanced the secretion and activity of proteases and lipases, promoting the breakdown of ester bonds. LC-QTOF-MS results showed that various intermediate products were obtained after degradation, ultimately participating in the TCA cycle (ko00020), further completely mineralized. Additionally, after 15-day compost, the co-culture system achieved a degradation rate of 72.14 ± 2.1 wt% for PBAT/PLA-ST20 films, with a decrease in the abundance of plastic fragments of all sizes, demonstrating efficient degradation of PLA/PBAT-ST20 films. This study highlights the potential of thermophilic bacteria to address plastic pollution through biodegradation and emphasizes that the co-culture system could serve as an ideal solution for the remediation of PLA/PBAT plastics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The protective mode of PostbioYDFF-3 (referred to as postbiotics) on the quality stability of refrigerated fillets was explored from the aspects of endogenous enzyme activity and the abundance of spoilage microorganisms. Compared to the control group, the samples soaked in postbiotics showed significant reductions in TVC, TVB-N and TBARS values by 39.6%, 58.6% and 25.5% on day 5, respectively. In addition, the color changes, biogenic amine accumulation and texture softening of the fish fillets soaked in postbiotics were effectively suppressed. Furthermore, the activity of endogenous enzyme activities was detected. The calpain activities were significantly inhibited (p < 0.05) after soaking in postbiotics, which declined by 23%. Meanwhile, high throughput sequencing analysis further indicated that the growth of spoilage microorganism such as Acinetobacter and Pseudomonas were suppressed. Overall, the PostbioYDFF-3 was suitable for preserving fish meat., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The following are the supplementary data related to this article. Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2024.139345., (Copyright © 2024. Published by Elsevier Ltd.)

1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in the isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a five gene-cluster encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. A glutamine synthetase-like (GS-like) enzyme (NpaA1) initiates 1NA glutamylation, and this enzyme exhibits a broad substrate selectivity toward a variety of anilines and naphthylamine derivatives. Structural analysis revealed that the aromatic residues in the 1NA entry tunnel and the V201 site in the large substrate-binding pocket significantly influence NpaA1's substrate preferences. The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites., Competing Interests: SZ, SD, TL, MM, DL, JS, NZ No competing interests declared, (© 2024, Zhang, Deng et al.)

In seeding plants, biosynthesis of the phytohormone ethylene, which regulates processes including fruit ripening and senescence, is catalyzed by 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The plant pathogen Pseudomonas savastanoi (previously classified as: Pseudomonas syringae) employs a different type of ethylene-forming enzyme (psEFE), though from the same structural superfamily as ACC oxidase, to catalyze ethylene formation from 2-oxoglutarate (2OG) in an arginine dependent manner. psEFE also catalyzes the more typical oxidation of arginine to give L-Δ 1 -pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO 2 . We report on the effects of C3 and/or C4 substituted 2OG derivatives on the reaction modes of psEFE. 1 H NMR assays, including using the pure shift method, reveal that, within our limits of detection, none of the tested 2OG derivatives is converted to an alkene; some are converted to the corresponding β-hydroxypropionate or succinate derivatives, with only the latter being coupled to arginine oxidation. The NMR results reveal that the nature of 2OG derivatization can affect the outcome of the bifurcating reaction, with some 2OG derivatives exclusively favoring the arginine oxidation pathway. Given that some of the tested 2OG derivatives are natural products, the results are of potential biological relevance. There are also opportunities for therapeutic or biocatalytic regulation of the outcomes of reactions catalyzed by 2OG-dependent oxygenases by the use of 2OG derivatives., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C 4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an S N 2-like attack at C 4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology., (© 2024 Wiley-VCH GmbH.)

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETase Y239R/F244G/Y250G and ScPETase A212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Yunpeng Bai reports financial support was provided by Ministry of Science and Technology of the People’s Republic of China. Yunpeng Bai has patent pending to Yunpeng Bai. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background/purposes: The continuously increasing carbapenem resistance within Enterobacterales and Pseudomonas poses a threat to public health, nevertheless, the molecular characteristics of which in southern China still remain limited. And carbapenemase identification is a key factor in effective early therapy of carbapenem-resistant bacteria infections. We aimed to determine the molecular characteristics of these pathogens and compare commercial combined disc tests (CDTs) with the modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) in detecting and distinguishing carbapenemases using whole genome sequencing (WGS)., Methods: A total of 78 Enterobacterales , 30 Pseudomonas were obtained from two tertiary hospitals in southern China. Susceptibility tests were conducted using an automated VITEK2 compact system with confirmation via the Kirby-Bauer method. The WGS was conducted on all clinical isolates and the molecular characteristics were analyzed by screening the whole genome sequences. CDTs with or without cloxacillin, mCIM, and eCIM, were performed and compared by taking WGS results as the benchmark., Results: A total of 103 carbapenem non-susceptible and 5 carbapenem susceptible bacteria were determined, with Klebsiella pneumoniae (42.7%), Pseudomonas aeruginosa (23.3%) and Escherichia coli (18.4%) being most prevalent. Carbapenemase genes were detected in 58 (56.3%) of the 103 carbapenem-non-susceptible clinical isolates, including 46 NDM, 6 KPC, 3 IMP, 1 IPM+VIM,1NDM+KPC, and 1 OXA-181. Carbapenemase-producing isolates were detected more frequently in Enterobacterales (76.3%). Among K. pneumoniae , the major sequence types were st307 and st11, while among E. coli and P. aeruginosa , the most prevalent ones were st410 and st242 respectively. For carbapenemase detection in Enterobacterales , the mCIM method achieved 100.00% (95% CI, 92.13-100.00%) sensitivity and 94.44% (70.63-99.71%) specificity (kappa, 0.96); for Pseudomonas , detection sensitivity was 100% (5.46-100.00%), and 100% (84.50-100.00%) specificity (kappa, 0.65). Commercial CDT carbapenemase detection sensitivity for Enterobacterales was 96.49% (86.84-99.39%), and 95.24% (74.13-99.75%) specificity (kappa, 0.90); for Pseudomonas , carbapenemase detection sensitivity was 100.00% (5.46-100.00%) and 37.93% (21.30-57.64%) specificity (kappa, 0.04). When cloxacillin testing was added, CDT specificity reached 84.61% (64.27-94.95%)., Conclusion: The molecular epidemiology of carbapenem-non-susceptible isolates from pediatric patients in Southern China exhibited distinctive characteristics. Both the mCIM-eCIM combination and CDT methods effectively detected and differentiated carbapenemases among Enterobacterales isolates, and the former performed better than CDT among Pseudomonas ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liang, Chen, Liang, Li, Cai, Lai, Zhong, Xie, Huang, Gao and Long.)

The acyltransferase from Pseudomonas protegens (PpATase) catalyzes in nature the reversible transformation of monoacetylphloroglucinol to diacetylphloroglucinol and phloroglucinol. Interestingly, this enzyme has been shown to catalyze the promiscuous transformation of 3-hydroxyphenyl acetate to 2',4'-dihydroxyacetophenone, representing a biological version of the Fries rearrangement. In the present study, we report a mechanistic investigation of this activity of PpATase using quantum chemical calculations. A detailed mechanism is proposed, and the energy profile for the reaction is presented. The calculations show that the acylation of the enzyme is highly exothermic, while the acetyl transfer back to the substrate is only slightly exothermic. The deprotonation of the C6-H of the substrate is rate-limiting, and a remote aspartate residue (Asp137) is proposed to be the general base group in this step. Analysis of the binding energies of various acetyl acceptors shows that PpATase can promote both intramolecular and intermolecular Fries rearrangement towards diverse compounds., (© 2023 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Marine environments harbor a plethora of microorganisms that represent a valuable source of new biomolecules of biotechnological interest. In particular, enzymes from marine bacteria exhibit unique properties due to their high catalytic activity under various stressful and fluctuating conditions, such as temperature, pH, and salinity, fluctuations which are common during several industrial processes. In this study, we report a new esterase (EstGoM) from a marine Pseudomonas sp. isolated at a depth of 1000 m in the Gulf of Mexico. Bioinformatic analyses revealed that EstGoM is an autotransporter esterase (type Va) and belongs to the lipolytic family II, forming a new subgroup. The purified recombinant EstGoM, with a molecular mass of 67.4 kDa, showed the highest hydrolytic activity with p-nitrophenyl octanoate (p-NP C8), although it was also active against p-NP C4, C5, C10, and C12. The optimum pH and temperature for EstGoM were 9 and 60 °C, respectively, but it retained more than 50% of its activity over the pH range of 7-11 and temperature range of 10-75 °C. In addition, EstGoM was tolerant of up to 1 M NaCl and resistant to the presence of several metal ions, detergents, and chemical reagents, such as EDTA and β-mercaptoethanol. The enzymatic properties of EstGoM make it a potential candidate for several industrial applications., (© 2024. The Author(s).)

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, Chd H114A exhibited catalytic activity with a k cat value of 1.07 s -1 , ~ 5% of wild-type (WT) Chd, and a K M of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and Chd H114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on Chd H114A , Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented., (© 2024. The Author(s), under exclusive licence to Society for Biological Inorganic Chemistry (SBIC).)

Objectives: To characterize VIM-type metallo-β-lactamase (MBL)-encoding genomic islands (GIs) in Pseudomonas aeruginosa and P. putida group isolates from Polish hospitals from 2001-2015/16., Methods: Twelve P. aeruginosa and 20 P. putida group isolates producing VIM-like MBLs were selected from a large collection of these based on epidemiological and typing data. The organisms represented all major epidemic genotypes of these species spread in Poland with chromosomally located blaVIM gene-carrying integrons. The previously determined short-read sequences were complemented by long-read sequencing in this study. The comparative structural analysis of the GIs used a variety of bioinformatic tools., Results: Thirty different GIs with blaVIM integrons were identified in the 32 isolates, of which 24 GIs from 26 isolates were integrative and conjugative elements (ICEs) of the clc family. These in turn were dominated by 21 variants of the GI2/ICE6441 subfamily with a total of 19 VIM integrons, each inserted in the same position within the ICE's Tn21-like transposon Tn4380. The three other ICEs formed a novel ICE6705 subfamily, lacking Tn4380 and having different VIM integrons located in another site of the elements. The remaining six non-ICE GIs represented miscellaneous structures. The presence of various integrons in the same ICE sublineage, and of the same integron in different GIs, indicated circulation and recombination of the integron-carrying genetic platforms across Pseudomonas species/genotypes., Conclusions: Despite the general diversity of the blaVIM-carrying GIs in Pseudomonas spp. in Poland, a clear predominance of broadly spread and rapidly evolving clc-type ICEs was documented, confirming their significant role in antimicrobial resistance epidemiology., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the δ-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase ( ppk ) and exopolyphosphatase ( ppx ) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ ppk ] and exopolyphosphatase [ ppx ]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana .

Our previous studies on FabG have identified two compounds 5-bromo-2-(thiophene-2-carboxamido) benzoic acid (A) and ethyl 6-bromo-2-((dimethylamino)methyl)-5-hydroxy-1-phenyl-1H-indole-3-carboxylate(B) as best hits with allosteric mode of inhibition. FabG is an integral part of bacterial fatty acid biosynthetic system FAS II shown to be an essential gene in most ESKAPE Pathogens. The current work is focussed on lead expansion of these two hit molecules which ended up with forty-three analogues (twenty-nine analogues from lead compound A and fourteen compounds from lead compound B). The enzyme inhibition studies revealed that compound 15 (effective against EcFabG, AbFabG, StFabG, MtFabG1) and 19 (inhibiting EcFabG and StFabG) had potency of broad-spectrum inhibition on FabG panel., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Masson SAS.)

Alginate lyases are epitomized as prospective therapeutic mediators for treating Pseudomonas aeruginosa infections, particularly in the cystic fibrosis airway through alginate degradation thereby improving the efficacy of anti-pseudomonal antibiotics. Investigation of metal-binding residues is significant for expounding the ion specificity of an enzyme and will provide a broad understanding of the potential roles of metal ions in enzyme function and stability. However, experimental analysis of metal ion-binding sites in proteins is time consuming and expensive. Concerning the clinical importance of this therapeutic enzyme, the present study was focused on the prediction and characterization of metal ion-binding sites of different alginate lyases reported in the literature through a computational approach using a Metal Ion-Binding Site Prediction and Docking Server. 3D structures of different alginate lyase from different organisms were retrieved, and these retrieved proteins were docked with twelve different metal ions such as Ca 2+ , Cu 2+ , Fe 3+ , Mg 2+ , Mn 2+ , Zn 2+ , Cd 2+ , Fe 2+ , Ni 2+ , Hg 2+ , Co 2+ , and Cu + . The binding affinity and interacting amino acids for alginate lyases produced by different microorganisms were compared and analysed. Further analysis on active site residues of reported alginate lyase and subsequent experiments will reveal the function of different metal ions in enhancing or inhibiting the catalysis of alginate lyase and will help in exploiting the enzyme as an efficient therapeutic agent as well as for industrial applications., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nonribosomal peptide synthetases (NRPSs) are typically multimodular enzymes that assemble amino acids or carboxylic acids into complex natural products. Here, we characterize a monomodular NRPS, PvfC, encoded by the Pseudomonas virulence factor ( pvf ) gene cluster that is essential for virulence and signaling in different bacterial species. PvfC exhibits a unique adenylation-thiolation-reductase (ATR) domain architecture that is understudied in bacteria. We show that the activity of PvfC is essential in the production of seven leucine-derived heterocyclic natural products, including two pyrazines, a pyrazinone, and a rare disubstituted imidazole, as well as three pyrazine N -oxides that require an additional N- oxygenation step. Mechanistic studies reveal that PvfC, without a canonical peptide-forming domain, makes a dipeptide aldehyde intermediate en route to both the pyrazinone and imidazole. Our work identifies a novel biosynthetic route for the production of pyrazinones, an emerging class of signaling molecules and virulence factors. Our discovery also showcases the ability of monomodular NRPSs to generate amino acid- and dipeptide-aldehydes that lead to diverse natural products. The diversity-prone biosynthesis by the pvf -encoded enzymes sets the stage for further understanding the functions of pvf in bacterial cell-to-cell signaling.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5-10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20-70 °C compared to PsCPG2. The K m value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The V max value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90  µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.

Tryptophan metabolism plays a role in the occurrence and development of hepatocellular carcinoma cells. By degrading certain amino acids, tumor growth can be limited while maintaining the body's normal nutritional requirements. Tryptophan side-chain oxidase (TSO) enzyme can degrade tryptophan, and its inhibitory effect on hepatocellular carcinoma cells is worthy of further study. To investigate the degradation effect on tryptophan, TSO was isolated and purified from qq Pseudomonas. The reaction products were identified with high performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). De novo sequencing provided the complete amino acid sequence of TSO. The results of CCK-8, colony formation, transwell, and qPCR confirmed that TSO had inhibitory effects on the proliferation and migration of HCCLM3 (human hepatocarcinoma cell line) and HepG2 cells. The results of flow cytometry confirmed its apoptotic activity. In animal experiments, we found that the tumor-suppressive effect was better in the oncotherapy group than the intraperitoneal injection group. The results of immunohistochemistry also suggested that TSO could inhibit proliferation and promote apoptosis. In conclusion, a specific enzyme that can degrade tryptophan and inhibit the growth of hepatoma cells was authenticated, and its basic information was obtained by extraction/purification and amino acid sequencing.

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs., (© 2021. The Author(s).)

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli . Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40°C, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active serine (S) located within a highly conserved catalytic triad of Ser 155 , Asp 253 , and His 282 residues. The catalytic efficiency ( k cat / K m ) of Est19 for the p NPC6 substrate is 148.68 s -1 mM -1 at 40 °C. Replacing Glu 154 juxtaposed to the critical catalytic serine with Asp (E154→D substitution) reduced the activity and catalytic efficiency of the enzyme two-fold, with little change in the substrate affinity. The wild-type enzyme retained near complete activity over a temperature range of 10-60 °C, while ~50% of its activity was retained at 0 °C. A phylogenetic analysis suggested that Est19 and its homologs may represent a new subfamily of HSL. The thermal stability and stereo-specificity suggest that the Est19 esterase may be useful for cold and chiral catalyses.

In the present study, a magnetic mimic multi-enzyme system was developed by encapsulating the aryloxyphenoxypropionate (AOPP) herbicide hydrolase QpeH and alcohol oxidase (AOx) in zeolitic imidazolate framework (ZIF-8) nanocrystals with magnetic Fe 3 O 4 nanoparticles (MNPs) to detect AOPP herbicides. The structural, protein loading capacity and loading ratio, porosity, and magnetic properties of QpeH/AOx@mZIF-8 were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, nitrogen sorption, and vibrating sample magnetometry. An AOPP herbicide colorimetric biosensor made with QpeH/AOx@mZIF-8 had the highest sensitivity toward quizalofop-P-ethyl (QpE) with a limit of detection of 8.2 μM. This system was suitable to detect two other AOPP herbicides, including fenoxaprop-P-ethyl (FpE) and haloxyfop-P-methyl (HpE). The practical application of the biosensor was verified through quantitative analysis of QpE residues in industrial wastewater and field soils. Furthermore, QpeH/AOx@mZIF-8 exhibited excellent long-term storage stability (at least 50 days), easy separation by magnet, and reusability (at least 10 cycles), supporting its promising role in simple and low-cost detection of AOPP herbicides in real environmental samples.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG 0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ) 2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis., (© 2021 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70-90oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117-125. https://doi.org/10.18388/abp.2015&#95;1074).

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, that is, isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for K S (9.3-fold higher) and k cat (4.8-fold lower) compared with corresponding K M and k cat values of isolated CHMO, but a favorable K I for cyclohexanone (5.3-fold higher). The unfavorable K S and k cat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8-fold higher K S , but 1.3- and 4.2-fold higher k cat and K I values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

4-Nitrophenol, a priority pollutant, is degraded by Gram-positive and Gram-negative bacteria via 1,2,4-benzenetriol (BT) and hydroquinone (HQ), respectively. All enzymes involved in the two pathways have been functionally identified. So far, all Gram-negative 4-nitrophenol utilizers are from the genera Pseudomonas and Burkholderia. But it remains a mystery why pnpG , an apparently superfluous BT 1,2-dioxygenase-encoding gene, always coexists in the catabolic cluster ( pnpABCDEF ) encoding 4-nitrophenol degradation via HQ. Here, the physiological role of pnpG in Burkholderia sp. strain SJ98 was investigated. Deletion and complementation experiments established that pnpG is essential for strain SJ98 growing on 4-nitrocatechol rather than 4-nitrophenol. During 4-nitrophenol degradation by strain SJ98 and its two variants ( pnpG deletion and complementation strains), 1,4-benzoquinone and HQ were detected, but neither 4-nitrocatechol nor BT was observed. When the above-mentioned three strains (the wild type and complementation strains with 2,2'-dipyridyl) were incubated with 4-nitrocatechol, BT was the only intermediate detected. The results established the physiological role of pnpG that encodes BT degradation in vivo . Biotransformation analyses showed that the pnpA- deleted strain was unable to degrade both 4-nitrophenol and 4-nitrocatechol. Thus, the previously characterized 4-nitrophenol monooxygenase PnpA SJ98 is also essential for the conversion of 4-nitrocatechol to BT. Among 775 available complete genomes for Pseudomonas and Burkholderia , as many as 89 genomes were found to contain the putative pnpBCDEFG genes. The paucity of pnpA (3 in 775 genomes) implies that the extension of BT and HQ pathways enabling the degradation of 4-nitrophenol and 4-nitrocatechol is rarer, more recent, and likely due to the release of xenobiotic nitroaromatic compounds. IMPORTANCE An apparently superfluous gene ( pnpG ) encoding BT 1,2-dioxygenase is always found in the catabolic clusters involved in 4-nitrophenol degradation via HQ by Gram-negative bacteria. Our experiments reveal that pnpG is not essential for 4-nitrophenol degradation in Burkholderia sp. strain SJ98 but instead enables its degradation of 4-nitrocatechol via BT. The presence of pnpG genes broadens the range of growth substrates to include 4-nitrocatechol or BT, intermediates from the microbial degradation of many aromatic compounds in natural ecosystems. In addition, the existence of pnpCDEFG in 11.6% of the above-mentioned two genera suggests that the ability to degrade BT and HQ simultaneously is ancient. The extension of BT and HQ pathways including 4-nitrophenol degradation seems to be an adaptive evolution for responding to synthetic nitroaromatic compounds entering the environment since the industrial revolution.

Background: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn 2+ . So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn 2+ . We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment., Results: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn 2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn 2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O 2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity., Conclusions: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 2 3  + 4 were performed to evaluate the following parameters: molar mass of PEG (M PEG ), concentration of PEG (C PEG ), concentration of citrate buffer (C Cit ), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of M PEG  = 10,000 g/mol, C PEG  = 22 wt%, C Cit  = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 μmol Trolox equivalent/mg protein, for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 μmol Trolox/mg protein). Nevertheless, the IC 50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates., (© 2020 American Institute of Chemical Engineers.)

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine., (© 2021 The Protein Society.)

Several thousand different terpenoid structures are known so far, and many of them are interesting for applications as pharmaceuticals, flavors, fragrances, biofuels, insecticides, or fine chemical intermediates. One prominent example is camphor, which has been utilized since ancient times in medical applications. Especially (-)-camphor is gaining more and more interest for pharmaceutical applications. Hence, a commercial reliable source is needed. The natural sources for (-)-camphor are limited, and the oxidation of precious (-)-borneol would be too costly. Hence, synthesis of (-)-camphor from renewable alpha-pinene would be an inexpensive alternative. As the currently used route for the conversion of alpha-pinene to camphor produces a mixture of both enantiomers, preferably catalytic methods for the separation of this racemate are demanded to yield enantiopure camphor. Enzymatic kinetic resolution is a sustainable way to solve this challenge but requires suitable enzymes. In this study, the first borneol dehydrogenase from Pseudomonas sp. ATCC 17453, capable of catalyzing the stereoselective reduction of camphor, was examined. By using a targeted enzyme engineering approach, enantioselective enzyme variants were created with E-values > 100. The best variant was used for the enzymatic kinetic resolution of camphor racemate, yielding 79% of (-)-camphor with an ee of > 99%. KEY POINTS: • Characterization of a novel borneol dehydrogenase (BDH) from P. putida. • Development of enantioselective BDH variants for the reduction of camphor. • Enzymatic kinetic resolution of camphor with borneol dehydrogenase.

Currently, the biotechnological preparation of fragrances using natural materials attracted growing attention. Enzymatic synthesis of vanillin from isoeugenol by recombinant isoeugenol monooxygenase from Pseudomonas nitroreducens Jin1 was systematically investigated herein. With series of work on the construction of recombinant E. coli over-expressing isoeugenol monooxygenase, optimization of the culture conditions for enzyme production and reaction process for converting isoeugenol into vanillin, an increase of 22-fold in the enzyme activity (2050 U/L) was obtained, and the conversion was significantly increased at high substrate concentration with the aid of magnetic chitosan membrane for product isolation in situ. Under optimal conditions, the product concentration and space-time yield reached 252 mM and 115 g/L/d, respectively, and vanillin was obtained in 82.3% yield and > 99% purity in the gram preparative scale. The developed bioprocess showed application potential for efficient preparation of vanillin from inexpensive natural resources.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 μM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 μM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 μM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

Background: L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds, such as l-theanine. Pseudomonas nitroreducens SP.001 with intracellular l-glutaminase activity has been screened previously. In the present study, three physical permeabilization methods were used to improve l-glutaminase activity. Then, the whole-cell immobilization conditions of permeabilized cells using sodium alginate as an embedding agent were optimized to enhance the enzyme's stability and reusability. The characteristics of the immobilized cells were investigated in comparison with those of permeabilized cells., Results: The results obtained showed that cell permeabilization using osmotic shock with 155 g L -1 sucrose markedly improved enzyme activity. Then, an effective procedure for immobilization of permeabilized P. nitroreducens cells was established. The optimum conditions for cell immobilization were: sodium alginate 40 g L -1 , calcium chloride 30 g L -1 , cell mass 100 g L -1 and a curing time of 3 h. After successful immobilization, characterization studies revealed that the thermostability and pH resistance of l-glutaminase from immobilized cells were enhanced compared to those from permeabilized cells. Moreover, the immobilized biocatalyst could be reused up to 10 times and retained 80% of its activity., Conclusion: The stability and reusability of the permeabilized cells were improved through the immobilization. These findings indicated that immobilized whole-cell l-glutaminase from P. nitroreducens SP.001 possesses more potential for various industrial biotechnological applications than free cells. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

The NitroSpeed-Carba NP test was used to rapidly detect and discriminate between the different types of carbapenemases (classes A, B, and D) within 30 minutes among a collection of 202 Pseudomonas sp. strains (mostly Pseudomonas aeruginosa). A total of 99 carbapenemase-(including enzymes exhibiting weak carbapenemase activity such as several Guyana Extended-Spectrum (GES)-ß-lactamases) and 103 non-carbapenemase producers were tested, and the overall specificity and sensitivity were 100% and 99%, respectively. The NitroSpeed-Carba NP test is a rapid, specific, sensitive, and easy-to-implement technique for identification of carbapenemase-producing Pseudomonas spp., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nicotine and nicotinic acid (NA) are both considered to be representatives of N -heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the cooccurrence of these two pathways has only been observed in Pseudomonas sp. strain JY-Q. The nicotine pyrrolidine catabolism pathway of strain JY-Q consists of the functional modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenase (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm enzymes (namely, Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 were both in-frame deleted, the mutant still grew well in basic salt medium (BSM) supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains reorganized domains similar to those of Spm. When the JY-Q&#95; nicAB gene ( nicAB in strain JY-Q) was introduced into another Pseudomonas strain, one that is unable to degrade NA, the resultant recombinant strain exhibited the ability to transform SP to HSP, but without the ability to metabolize NA. Here, we conclude that NicAB in strain JY-Q exhibits an additional role in SP transformation. The other genes in the NA cluster, NicXDFE (Nic2 homolog), then also exhibit a role in subsequent HSP metabolism for energy yield. This finding also suggests that the cooccurrence of nicotine and NA degradation genes in strain JY-Q represents an advantage for JY-Q, making it more effective and flexible for the degradation of nicotine. IMPORTANCE 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) are both valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway, in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. strain JY-Q. However, when two homologous Spm genes ( spm1 and spm2 ) were knocked out, the mutant retained the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have an alternative pathway for SP conversion. In this research, we showed that JY-Q&#95;NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q&#95;NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates., (Copyright © 2021 American Society for Microbiology.)

Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized d-sorbitol as its carbon source, among the four polyols examined (d-galactitol, d-mannitol, d-sorbitol and d-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153 and Lys157 as catalytic active site residues and existed as an ∼67-kDa dimer in size-exclusion chromatography. PmSDH converted d-sorbitol to d-fructose using nicotinamide adenine dinucleotide (NAD+) as a cofactor and, vice versa, d-fructose to d-sorbitol using nicotinamide adenine dinucleotide reduced (NADH) as a cofactor. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4-25°C. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD , hcdE , hcdF , and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The K m and k cat values of 6.5 µM and 4.57 s -1 , respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.

Pseudomonas aeruginosa infects patients with cystic fibrosis, burns, wounds and implants. Previously, our group showed that elevated Ca 2+ positively regulates the production of several virulence factors in P. aeruginosa , such as biofilm formation, production of pyocyanin and secreted proteases. We have identified a Ca 2+ -regulated β-propeller putative phytase, CarP, which is required for Ca 2+ tolerance, regulation of the intracellular Ca 2+ levels, and plays a role in Ca 2+ regulation of P. aeruginosa virulence. Here, we studied the conservation of carP sequence and its occurrence in diverse phylogenetic groups of bacteria. In silico analysis revealed that carP and its two paralogues PA2017 and PA0319 are primarily present in P. aeruginosa and belong to the core genome of the species. We identified 155 single nucleotide alterations within carP , 42 of which lead to missense mutations with only three that affected the predicted 3D structure of the protein. PCR analyses with carP -specific primers detected P. aeruginosa specifically in 70 clinical and environmental samples. Sequence comparison demonstrated that carP is overall highly conserved in P. aeruginosa isolated from diverse environments. Such evolutionary preservation of carP illustrates its importance for P. aeruginosa adaptations to diverse environments and demonstrates its potential as a biomarker.

Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD + , suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Given the excellent characteristics of alginate, it is an industrially important polysaccharide. Mannuronan C5-epimerase (MC5E) is an alginate-modifying enzyme that catalyzes the conversion of β-D-mannuronate (M) to its C5 epimer α-L-guluronate (G) in alginate. Both the biological activities and physical properties of alginate are determined by M/G ratios and distribution patterns. Therefore, MC5E is regarded as a biotechnological tool for modifying and processing alginate. Various MC5Es derived from brown algae, Pseudomonas and Azotobacter have been isolated and characterized. With the rapid development of structural biology, the crystal structures and catalytic mechanisms of several MC5Es have been elucidated. It is necessary to comprehensively understand the research status of this alginate-modifying enzyme. In this review, the properties and potential applications of MC5Es isolated from different kinds of organisms are summarized and reviewed. Moreover, future research directions of MC5Es as well as strategies to enhance their properties are elucidated, highlighted, and prospected., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although bacteria with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity have been used to mitigate biotic and abiotic stresses in crops, it is not well known whether the ACC deaminase gene ( acdS ) in Pseudomonas azotoformans is related to the alleviation of salt stress by the bacterium. This study aimed to evaluate the effects of acdS in P. azotoformans strain CHB 1107 on the nutrient uptake and growth of tomato plants under salt stress. The acdS mutant (CHB 1107 M) of P. azotoformans CHB 1107 was obtained through bacterial conjugation. Wild-type (CHB 1107 WT) and CHB 1107 M were used to inoculate tomato plants grown in a soil or solution with an electrical conductivity of 6 dS/m adjusted by NaCl. CHB 1107 M completely lost the ability to produce ACC deaminase, whereas the complementation of acdS in CHB 1107 M preserved its ACC deaminase activity. CHB 1107 WT significantly reduced the production of ethylene and proline by tomato plants under salt stress, increasing the shoot and root dry weights of tomato plants compared with the noninoculated control and CHB 1107 M. In addition, tomato plants inoculated with CHB 1107 M showed a significant reduction in K (27.5%), Ca (23.0%), and Mn uptake (17.5%) compared with those inoculated with CHB 1107 WT. In contrast, CHB 1107 WT significantly reduced Na uptake by tomato plants in comparison to CHB 1107 M in saline soil conditions. In addition, the inoculation of tomato plants with CHB 1107 WT resulted in a higher K/Na ratio than in those inoculated with CHB 1107 M and the noninoculated control. These findings suggest that acdS in P. azotoformans is associated with the amelioration of salinity stress in tomato. Plant transformation with acdS and the field application of P. azotoformans may be used as potential management tools for crops under salt stress.

Polyurethanes (PU) are multifunctional polymers, used in automotive industry, in coatings, rigid and flexible foams, and also in biomimetic materials. In the same way as all plastic waste, the incorrect disposal of these materials leads to the accumulation of polyurethanes in the environment. To reduce the amount of waste as well as add value to degradation products, bioremediation methods have been studied for waste management of PU. Enzymes of the hydrolases class have been experimentally tested for enzymatic degradation of PU, with very promising results. In this work, two enzymes that can degrade polyurethanes were studied by molecular dynamics simulations: a protease and an esterase, both from Pseudomonas. From molecular dynamics simulations analysis, it was observed the stability of the structures, both in the simulations of the free enzymes and in the simulations of the complexes with a PU monomer. Hydrogen bonds were formed with the monomer and the enzymes throughout the simulation time, and the interaction free energy was found to be strongly negative, pointing to strong interactions in both cases.

Here, we have analyzed the enzyme ornithine carbamoyltransferase (OCTase) in different classes of microorganisms belonging to psychrophiles, mesophiles and thermophiles. This OCTase catalyzes the formation of citrulline from carbamoyl phosphate (CP) and ornithine (ORN) in arginine biosynthesis pathway and has certain unique adaptations to regulate metabolic pathways in extreme conditions. The tertiary structure of OCTase showed two binding domains, the CP domain and ORN-binding domain at N and C terminals, respectively. We propose general acid-base catalysis in Pseudomonas gessardii between His259 and Asp220 in which later may act as a recipient of proton in the process. The comparative docking analysis showed that substrate-binding loops have been evolved to accommodate their lifestyles across the physiological temperature range where two substrates bind on two distinct loops in psychrophiles and mesophiles, whereas both the substrates bind on a single-substrate-binding loop in thermophiles and bring down the flexibility of the active site pocket to improve its evolutionary fitness.

Pseudomonas asiatica and Pseudomonas monteilii , belonging to the Pseudomonas putida phylogenetic group, are occasionally isolated from clinical samples, partly because they are often misidentified as P. putida in clinical laboratories. There are five reports describing carbapenem-resistant clinical isolates of these species. Carbapenem-resistant strains of P. asiatica and P. monteilii were isolated from stool samples. These isolates were sequenced using Illumina MiSeq and reidentified using average nucleotide identity (ANI) based on comparisons of their whole-genome sequences using the OrthoANI algorithm. The clonal relatedness of the isolates was assessed by pulse-field gel electrophoresis (PFGE). The size of plasmids conveying bla VIM-2 was examined by Southern blotting. A total of six carbapenem-resistant clinical isolates of P. asiatica (two isolates) and P. monteilii (four isolates) were obtained from stool samples from five patients in a Japanese hospital. All isolates harboured blaVIM-2 . The two isolates of P. asiatica had a different pattern in the PFGE analysis, with both having a 23 kb plasmid. Of the four isolates of P. monteilii with similar patterns in the PFGE analysis, three had 320 kb plasmids and one had a 240 kb plasmid. The genetic environments of the 320/240 kb and 23 kb plasmids differed. The results strongly indicated that carbapenem-resistant P. asiatica and P. monteilii producing metallo-β-lactamase are emerging in Japan. This is the first report of carbapenem-resistant P. asiatica and P. monteilii in Japan.

γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The present study demonstrated atrazine detoxification by intracellular crude enzyme extracts of Pseudomonas spp. strains ACB and TLB. Indigenous bacterial protein-based remediation techniques could be an alternative to bioaugmentation which pose multiple challenges when applied to the field. Intracellular enzymes were extracted from strains ACB and TLB and their degradation potential of 10 mg L -1 was determined using Gas Chromatography; further, enzyme extracts were subjected to protein profiling studies. In span of 6 h, enzyme extracts of strain ACB showed maximum degradation at 30 °C and 40 °C (71%) and enzyme extracts of strain TLB showed maximum degradation at 40 °C (48%). Atrazine degradation by enzyme extracts of strain ACB showed maximum degradation at pH 7 (71%) and pH 6 (69%) in 6 h. Similarly, enzyme extracts of strain TLB showed maximal degradation at pH 6 (46%) in 6 h. The present study demonstrated, for the first time, efficient atrazine remediation by intracellular crude enzyme extracts from epiphytic root bacteria at a range of temperature and pH conditions. Protein profiling studies indicated that atrazine induced expression of CoA ester lyase and alkyl hydroperoxide reductase in the strains ACB and TLB respectively. Expressions of these proteins have never been associated with atrazine exposure.

Background: L-Methioninase (EC 4.4.1.11; MGL) is a pyridoxal phosphate (PLP)-dependent enzyme that is produced by a variety of bacteria, fungi, and plants. L-methioninase, especially from Pseudomonas and Citrobacter sp., is considered as the efficient therapeutic enzyme, particularly in cancers such as glioblastomas, medulloblastoma, and neuroblastoma that are more sensitive to methionine starvation., Objective: The low stability is one of the main drawbacks of the enzyme; in this regard, in the current study, different features of the enzyme, including phylogenetic, functional, and structural from Pseudomonas, Escherichia, Clostridium, and Citrobacter strains were evaluated to find the best bacterial L-Methioninase., Methods: After the initial screening of L-Methioninase sequences from the above-mentioned bacterial strains, the three-dimensional structures of enzymes from Escherichia fergusonii, Pseudomonas fluorescens, and Clostridium homopropionicum were determined through homology modeling via GalaxyTBM server and refined by GalaxyRefine server., Results and Conclusion: Afterwards, PROCHECK, verify 3D, and ERRAT servers were used for verification of the obtained models. Moreover, antigenicity, allergenicity, and physico-chemical analysis of enzymes were also carried out. In order to get insight into the interaction of the enzyme with other proteins, the STRING server was used. The secondary structure of the enzyme is mainly composed of random coils and alpha-helices. However, these outcomes should further be validated by wet-lab investigations., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)