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Modification of kraft lignin (KL), traditionally uses harsh and energy-demanding physical and chemical processes. In this study, the potential of the bacterial laccase CotA (spore coating protein A) for oxidation of KL under mild conditions was assessed. Thereby, the efficiency of CotA to oxidize both softwood and hardwood KL of varying purity at alkaline conditions was examined. For the respective type of wood, the highest oxidation activity by CotA was determined for the medium ash content softwood KL (MA_S) and the medium ash content hardwood KL (MA_H), respectively. By an up to 95% decrease in fluorescence and up to 65% in phenol content coupling of the structural lignin units was indicated. These results correlated with an increase in viscosity and molecular weight, which increased nearly 2 and 20-fold for MA_H and about 1.3 and 6.0-fold for MA_S, respectively. Thus, this study confirms that the CotA laccase can oxidize a variety of KL at alkaline conditions, while the origin and purity of KL were found to have a major impact on the efficiency of oxidation. Under the herein tested conditions, it was observed that the MA_H KL showed the highest susceptibility to CotA oxidation when compared to the other hardwood KLs and the softwood KLs. Therefore, this could be a viable method to produce sustainable resins and adhesives.

Herein, we report a novel two-step method for the covalent, site-directed, and efficient immobilization of proteins on lab-made paper sheets. First, paper fibers were modified with a peptidic anchor comprising enzyme recognition motifs. Four different conjugation strategies for peptide immobilization were evaluated with respect to reproducibility and fiber loading efficiency. After manufacturing of the peptide-preconditioned paper, oriented conjugation of the model protein tGFP containing a C-terminal recognition sequence for either sortase A or microbial transglutaminase was assessed semiquantitatively by fluorescence measurement and inspected by confocal laser scanning microscopy (CLSM). The two enzymes utilized for protein conjugation used the same oligoglycine peptide anchor, and both proved to be suitable for controlled oriented linkage of substrate proteins at physiological conditions.

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_ 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_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_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.)

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.)

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. strain JY-Q still has difficulties degrading high concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators, namely, NicR2A and NicR2Bs (NicR2B1 plus NicR2B2), can repress nicotine degradation gene expression. When both nicR2A and nicR2Bs were deleted, the resulting mutant JY-Q Δ nicR2A Δ nicR2B1 Δ nicR2B2 (QΔABs) exhibits a 17% higher nicotine degradation efficiency than wild-type JY-Q. Transcriptome sequencing (RNA-seq) analysis showed that the transcription levels (fragments per kilobase per million [FPKM] value) of six genes were higher than those of the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, P RS28250 , P RS09985 , and P RS24685 , were identified. Their promoter activities were evaluated by comparing their expression levels using reverse transcriptase quantitative PCR (RT-qPCR). We found that the transcription levels of RS28250 , RS09985 , and RS24685 were respectively 16.8, 2.6, and 1.6 times higher than that of hspB2 , encoding 6-hydroxy-3-succinylpyridine hydroxylase, which is involved in nicotine degradation. Thus, two strong endogenous promoters, namely, P RS28250 and P RS09985 , were selected to replace the original promoters of nic2 gene clusters. The effect of the endogenous ectopic promoter was also related to the position of target gene clusters. When the promoter P RS28250 replaced the promoter of hspB2 , the resultant mutant QΔABs-Δ P hspB2 :: P RS28250 exhibited nicotine-degrading efficiency 69% higher than that of JY-Q. This research suggests a feasible strategy to enhance strains' capacity for nicotine degradation by removal of repressing regulatory proteins and replacing the target promoter with strong endogenous ectopic promoters. IMPORTANCE This study evaluated the differential effects of homologous NicR2A and NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. strain JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild-type (WT) strain JY-Q by removing repressing regulatory proteins NicR2A and NicR2Bs and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving the pollutant-degrading capacity of natural strains through genomic modification., (Copyright © 2021 American Society for Microbiology.)

α-Glucosidase, Agl2, from Xanthomonas campestris was successfully overexpressed in Escherichia coli BL21(DE3) cells and purified with Ni columns. The enzyme exhibits glycosylation abilities towards a wide range of phenolic substrates, including phenol, vanillin, and ethyl vanillin, with maltose as the glycosyl donor. The catalytic properties of the purified enzyme were further investigated. It was observed that the synthesized glycosides started to degrade with prolonged catalytic time, giving an "n"-shaped kinetic profile. To understand such catalytic behavior, the Agl2-catalyzed glycosylation process was investigated kinetically. Based on the obtained parameters, it was concluded that although the substrate conversions are thermodynamically restricted in a batch system, the glycosylation efficiency can be kinetically controlled by the glycosylation/hydrolysis selectivity. Glucose was produced by both glycosylation and hydrolysis, significantly impacting the glycosylation efficiency. This study provides a mechanistic understanding of the α-glucosidase-catalyzed glycosylation process in a water-based system. The developed kinetic model was successful in explaining and analyzing the catalytic process. It is suggested that when α-glucosidase is employed for glycosylation in a water-enriched environment, the catalytic efficiency is mainly impacted by the enzyme's glycosylation/hydrolysis selectivity and glucose content in the catalytic environment.

Oil palm trunk (OPT) is one of the most promising lignocellulosic bioresources. To develop effective biodegradation, thermophilic, anaerobic microorganisms were screened from bovine manure compost using fibrillated OPT (f-OPT) pretreated by wet disk milling as the substrate. One thermophilic, anaerobic bacterium, strain CL-2, whose 16S rDNA gene has 98.6% sequence identity with that of Caldicoprobacter faecale DSM 20678 T , exhibited high degradation activity (32.7% reduction in total dry solids of f-OPT). Strain CL-2 did not use cellulose as a carbon source, but used hemicelluloses such as xylan, arabinoxylan, starch and pectin at 70 °C. Phylogenetic and morphologic analyses and the polysaccharide use suggest that CL-2 may be classified as a novel species of Caldicoprobacter, named Caldicoprobacter sp. CL-2. To characterize enzymatic activities of CL-2, extracellular enzymes were prepared from culture broth using beechwood xylan as the carbon source. The extracellular enzymes showed high xylanase activity, but low cellulase activity, suggesting that f-OPT degradation may depend on xylanase activity. To understand the xylanase system of CL-2, a major xylanase was cloned and characterized. The xylanase (CalXyn11A) had a modular structure consisting of a glycoside hydrolase (GH) family-11 domain and a family 36 carbohydrate-binding module. CalXyn11A did not show f-OPT degradation activity, but a strong synergistic effect was observed when CalXyn11A was added to the extracellular enzyme preparation. These results indicate that, rather than working alone, CalXyn11A has an important role in enhancing total lignocellulose degradation activity by cooperation with other GHs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aminoglycoside modifying enzyme (AME) ANT(2″)-Ia is a significant target for next generation antibiotic development. Structural studies of a related aminoglycoside-modifying enzyme, ANT(3″)(9), revealed this enzyme contains dynamic, disordered, and well-defined segments that modulate thermodynamically before and after antibiotic binding. Characterizing these structural dynamics is critical for in situ screening, design, and development of contemporary antibiotics that can be implemented in a clinical setting to treat potentially lethal, antibiotic resistant, human infections. Here, the first NMR structural ensembles of ANT(2″)-Ia are presented, and suggest that ATP-aminoglycoside binding repositions the nucleotidyltransferase (NT) and C-terminal domains for catalysis to efficiently occur. Residues involved in ligand recognition were assessed by site-directed mutagenesis. In vitro activity assays indicate a critical role for I129 toward aminoglycoside modification in addition to known catalytic D44, D46, and D48 residues. These observations support previous claims that ANT aminoglycoside sub-class promiscuity is not solely due to binding cleft size, or inherent partial disorder, but can be controlled by ligand modulation on distinct dynamic and thermodynamic properties of ANTs under cellular conditions. Hydrophobic interactions in the substrate binding cleft, as well as solution dynamics in the C-terminal tail of ANT(2″)-Ia, advocate toward design of kanamycin-derived cationic lipid aminoglycoside analogs, some of which have already shown antimicrobial activity in vivo against kanamycin and gentamicin-resistant P. aeruginosa. This data will drive additional in silico, next generation antibiotic development for future human use to combat increasingly prevalent antimicrobial resistance., (© 2017 The Protein Society.)

The response of Clostridium tyrobutyricum to butyric acid stress involves various stress-related genes, and therefore overexpression of stress-related genes can improve butyric acid tolerance and yield. Class I heat shock proteins (HSPs) play an important role in the process of protecting bacteria from sudden changes of extracellular stress by assisting protein folding correctly. The results of quantitative real-time PCR indicated that the Class I HSGs grpE, dnaK, dnaJ, groEL, groES, and htpG were significantly upregulated under butyric acid stress, especially the dnaK and groE operons. Overexpression of groESL and htpG could significantly improve the tolerance of C. tyrobutyricum to butyric acid, while overexpression of dnaK and dnaJ showed negative effects on butyric acid tolerance. Acid production was also significantly promoted by increased GroESL expression levels; the final butyric acid and acetic acid concentrations were 28.2 and 38% higher for C. tyrobutyricum ATCC 25755/groESL than for the wild-type strain. In addition, when fed-batch fermentation was carried out using cell immobilization in a fibrous-bed bioreactor, the butyric acid yield produced by C. tyrobutyricum ATCC 25755/groESL reached 52.2 g/L, much higher than that for the control. The improved butyric acid yield is probably attributable to the high GroES and GroEL levels, which can stabilize the biosynthetic machinery of C. tyrobutyricum under extracellular butyric acid stress.

Adsorption of proteins and adhesion of bacteria to a surface is affected by chemical and physical interactions. In this study, polymer coatings and their ability to adsorb avidin and Staphylococcus aureus were investigated. The surface chemistry and topography of the polymer coatings was modified by changing the weight ratio of the hydrophobic polystyrene (PS) and the hydrophilic acrylonitrile butadiene styrene (ABS) components in the polymer blend. Avidin adsorbed less to the ABS phase compared with the PS phase. The side-on orientation of avidin on the ABS surface, however, resulted in a higher specific binding of biotinylated bovine serum albumin. Steric effects and hydrophobic protein-surface interactions decreased the activity of avidin on the PS phase. The increased hydrophobicity and roughness of the polymer coatings enhanced the adhesion of S. aureus. The avidin-coated latex surface with 55% relative surface coverage of the PS phase showed anti-microbial behavior., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study evaluates the hydrolysis specificity of newly developed protease from Exiguobacterium sp. SWJS2 (EP) based on the released free amino acid (FAA) during enzymolysis and through comparing with commercially available papain and Alcalase 2.4L. Results showed that EP had great potential in producing hydrolysates with better nutrition and less bitterness. The percentages of essential amino acids in the EP-treated Coilia mystus and soybean protein were 72 and 70 %, respectively. And, corresponding hydrophobic amino acids were 74 and 72 %, respectively (i.e. <60 % for papain- or Alcalase 2.4L-treated samples). The differences in FAA releasing rates between EP and the two commercial proteases suggested that EP could become a new commercial protease that offers different reaction rates and extends protease application scope. Track changes in FAA profiles throughout EP hydrolysis revealed that the release of each amino acid exhibited its distinct regularity as the hydrolysis proceeded, which also varied with the substrate proteins. EP hydrolysis of C. mystus and soybean protein led to greater production rates of Phe, Leu, Val, Ile and Ala, suggesting that EP might have higher preference to these amino acids.