Lysine Decarboxylase Activity Research Articles

Assessment of the Safety and Probiotic Properties of Enterococcus faecium B13 Isolated from Fermented Chili

Enterococcus faecium B13, selected from fermentation chili, has been proven to promote animal growth by previous studies, but it belongs to opportunistic pathogens, so a comprehensive evaluation of its probiotic properties and safety is necessary. In this study, the probiotic properties and safety of B13 were evaluated at the genetic and phenotype levels in vitro and then confirmed in vivo. The genome of B13 contains one chromosome and two plasmids. The average nucleotide identity indicated that B13 was most closely related to the fermentation-plant-derived strain. The strain does not carry the major virulence genes of the clinical E. faecium strains but contains aac(6′)-Ii, ant (6)-Ia, msrC genes. The strain had a higher tolerance to acid at pH 3.0, 4.0, and 0.3% bile salt and a 32.83% free radical DPPH clearance rate. It can adhere to Caco-2 cells and reduce the adhesion of E. coli to Caco-2 cells. The safety assessment revealed that the strain showed no hemolysis and did not exhibit gelatinase, ornithine decarboxylase, lysine decarboxylase, or tryptophanase activity. It was sensitive to twelve antibiotics but was resistant to erythromycin, rifampicin, tetracycline, doxycycline, and minocycline. Experiments in vivo have shown that B13 can be located in the ileum and colon and has no adverse effects on experiment animals. After 28 days of feeding, B13 did not remarkable change the α-diversity of the gut flora or increase the virulence genes. Our study demonstrated that E. faecium B13 may be used as a probiotic candidate.

Investigation of enzymatic quality and quantity using pyridoxal 5′-phosphate (PLP) regeneration system as a decoy in Escherichia coli

Pyridoxal 5′-phosphate (PLP), an essential cofactor for multiple enzymes, was used as a protein decoy to prompt enzyme expression and activity for the first time. The best chassis, denoted as WJK, was developed using a pyridoxal kinase (PdxK) and integrated at the HK022 phage attack site of Escherichia coli W3110. When compared with the original strain, the amount and activity of lysine decarboxylase (CadA) in WJK were significantly increased by 100 % and 120 %, respectively. When supplementary nineteen amino acids as second carbon source, cell growth and protein trade-off were observed. The transcriptional levels of genes from glycolysis to TCA cycle, adhE, argH and gdhA were dominating and redirected more flux into α-ketoglutarate, thus facilitated cell growth. Stepwise improvement was conducted with pyridoxal and nitrogen-rich medium; hence, CadA activity was increased to 60 g-cadaverine/g-dry cell weight/h. By reutilizing the whole-cell biocatalysts in two repeated reactions with the supplementation of fresh cells, a total cadaverine of 576 g/L was obtained even without additional PLP. Notably, PLP decoy augment the enzymatic activities of 5-aminolevulinic acid synthase and glutamate/lysine/arginine decarboxylases by over 100 %. Finally, a conserved PLP-binding pocket, Ser-His-Lys, was identified as a vital PLP sponge site that simultaneously improved protein quality and quantity.

Enhancing pH stability of lysine decarboxylase via rational engineering and its application in cadaverine industrial production

Cadaverine, an important C5 platform compound, is the raw material of polyamides. Under a carbon neutral context, cadaverine bio-production using lysine decarboxylase as a catalyst from the sustainable resource L-lysine is more attractive than chemical synthesis from fossil resources because it is environmentally-friendly and highly efficient. However, the alkaline conditions caused by accumulation of cadaverine decreases lysine decarboxylase activity, which limits its industrial applications. Herein, we aimed to improve cadaverine enzymatic production via enhancing stability of lysine decarboxylase from Escherichia coli (EcCadA) under alkaline pH by rational engineering. Mutations of interfacial disulfide bonds between subunits in the EcCadA decamer, M1 (L89C/L442C), M2 (F102C/L547C), and M3 (V12C/D41C) were chosen. M3 exhibited a 6-fold increase in cadaverine production at pH 10.0. Non-reduced SDS-PAGE analysis demonstrated that the proportion of decamers was greatly increased compared to wild-type enzyme. In addition, molecular dynamics simulations showed that the distance between subunits increased with increased pH, especially in region B. Finally, the fed-batch bioconversion of cadaverine from L-lysine in a 5 L fermenter using M3 by whole-cell catalysis led to 418 g/L cadaverine, which is the highest titer produced to date. This study provided a more efficient enzyme to industrially produce cadaverine with reduced acid use.

Effect of an Environment Friendly Heat and Relative Humidity Approach on γ-Aminobutyric Acid Accumulation in Different Highland Barley Cultivars.

In this study, heat and relative humidity (HRH) treatment was applied in highland barley for γ-aminobutyric acid (GABA) accumulation. Tibetan highland barley cultivars (25) were selected for comparison and analysis. HRH treatment could accumulate GABA in several hours with low moisture content and high temperature, and the grains were treated for 2.5 h at 65 °C in this study. The GABA content of processed grains under HRH optimal condition ranged from 26.91 to 76.28 mg·100 g−1, which was significantly higher than the initial content (12.78–43.00 mg·100 g−1). The highest GABA accumulation capacity was observed in two-row yellow cultivars (YT1), increasing from 36.52 to 76.28 mg·100 g−1. Correlation analysis showed that the accumulation of GABA after HRH treatment was positively and significantly (p < 0.05) correlated with the contents of protein (0.52), total free amino acids (0.68), threonine (0.53), serine (0.51), glutamate (0.69), glycine (0.49), alanine (0.46), cysteine (0.57), tyrosine (0.50), lysine (0.53), proline (0.40), and glutamate decarboxylase (GAD) activity (0.62), which were closely related to GABA-shunt pathway. The polyamines contents, diamine oxidase (DAO) and polyamine oxidase (PAO) activities, as the substrates and critical enzymes of polyamine degradation pathway, showed no significant correlation with GABA accumulation. The results suggested that the main pathway of GABA accumulation in highland barley under HRH treatment was GABA-shunt pathway.

Acid resistance system CadBA is implicated in acid tolerance and biofilm formation and is identified as a new virulence factor of Edwardsiella tarda

Edwardsiella tarda is a facultative intracellular pathogen in humans and animals. The Gram-negative bacterium is widely considered a potentially important bacterial pathogen. Adaptation to acid stress is important for the transmission of intestinal microbes, so the acid-resistance (AR) system is essential. However, the AR systems of E. tarda are totally unknown. In this study, a lysine-dependent acid resistance (LDAR) system in E. tarda, CadBA, was characterized and identified. CadB is a membrane protein and shares high homology with the lysine/cadaverine antiporter. CadA contains a PLP-binding core domain and a pyridoxal phosphate-binding motif. It shares high homology with lysine decarboxylase. cadB and cadA are co-transcribed under one operon. To study the function of the cadBA operon, isogenic cadA, cadB and cadBA deletion mutant strains TX01ΔcadA, TX01ΔcadB and TX01ΔcadBA were constructed. When cultured under normal conditions, the wild type strain and three mutants exhibited the same growth performance. However, when cultured under acid conditions, the growth of three mutants, especially TX01ΔcadA, were obviously retarded, compared to the wild strain TX01, which indicates the important involvement of the cadBA operon in acid resistance. The deletion of cadB or cadA, especially cadBA, significantly attenuated bacterial activity of lysine decarboxylase, suggesting the vital participation of cadBA operon in lysine metabolism, which is closely related to acid resistance. The mutations of cadBA operon enhanced bacterial biofilm formation, especially under acid conditions. The deletions of the cadBA operon reduced bacterial adhesion and invasion to Hela cells. Consistently, the deficiency of cadBA operon abated bacterial survival and replication in macrophages, and decreased bacterial dissemination in fish tissues. Our results also show that the expression of cadBA operon and regulator cadC were up-regulated upon acid stress, and CadC rigorously regulated the expression of cadBA operon, especially under acid conditions. These findings demonstrate that the AR CadBA system was a requisite for the resistance of E. tarda against acid stress, and played a critical role in bacterial infection of host cells and in host tissues. This is the first study about the acid resistance system of E. tarda and provides new insights into the acid-resistance mechanism and pathogenesis of E. tarda.

Burkholderia cepacia Complex Taxon K: Where to Split?

The objective of the present study was to provide an updated classification for Burkholderia cepacia complex (Bcc) taxon K isolates. A representative set of 39 taxon K isolates were analyzed through multilocus sequence typing (MLST) and phylogenomic analyses. MLST analysis revealed the presence of at least six clusters of sequence types (STs) within taxon K, two of which contain the type strains of Burkholderia contaminans (ST-102) and Burkholderia lata (ST-101), and four corresponding to the previously defined taxa Other Bcc groups C, G, H and M. This clustering was largely supported by a phylogenomic tree which revealed three main clades. Isolates of B. contaminans and of Other Bcc groups C, G, and H represented a first clade which generally shared average nucleotide identity (ANI) and average digital DNA-DNA hybridization (dDDH) values at or above the 95–96% ANI and 70% dDDH thresholds for species delineation. A second clade consisted of Other Bcc group M bacteria and of four B. lata isolates and was supported by average ANI and dDDH values of 97.2 and 76.1% within this clade and average ANI and dDDH values of 94.5 and 57.2% toward the remaining B. lata isolates (including the type strain), which represented a third clade. We therefore concluded that isolates known as Other Bcc groups C, G, and H should be classified as B. contaminans, and propose a novel species, Burkholderia aenigmatica sp. nov., to accommodate Other Bcc M and B. lata ST-98, ST-103, and ST-119 isolates. Optimized MALDI-TOF MS databases for the identification of clinical Burkholderia isolates may provide correct species-level identification for some of these bacteria but would identify most of them as B. cepacia complex. MLST facilitates species-level identification of many taxon K strains but some may require comparative genomics for accurate species-level assignment. Finally, the inclusion of Other Bcc groups C, G, and H into B. contaminans affects the phenotype of this species minimally and the proposal to classify Other Bcc group M and B. lata ST-98, ST-103, and ST-119 strains as a novel Burkholderia species is supported by a distinctive phenotype, i.e., growth at 42°C and lysine decarboxylase activity.

Isolation and Characterization of &lt;i&gt;Escherichia albertii&lt;/i&gt; from Environmental Water in Akita Prefecture, Japan

Escherichia albertii is a newly emerging food-borne pathogen. However, infection routes to patients remain unclear. In the present study, we aimed to isolate and characterize E. albertii from environmental water in Akita Prefecture of Japan. E. albertii was detected in 16 of 52 samples by real-time polymerase chain reaction analysis, and a total of 13 E. albertii strains were isolated from 9 samples. None of isolates showed the properties of biogroup 1 or 2 described previously on indole production and lysine decarboxylase activity. Susceptibility testing against 11 antimicrobial agents showed that one isolate was resistant only to ampicillin (7.7%). All isolates were positive for eae and cdt as virulence genes, but were negative for stx, including stx2f. Pulsed-field gel electrophoresis analysis showed that E. albertii isolates were genetically heterogeneous, but two isolates from environmental water had the similarity of >80% to some E. albertii strains from humans. These results suggest that E. albertii derived from environmental water has the potential to infect humans.

Technological properties of autochthonous Lactobacillus plantarum strains isolated from sucuk (Turkish dry-fermented sausage).

Five Lactobacillus strains isolated from sucuk (Turkish dry-fermented sausage) were studied for their genetic and technological properties. For genotypic identification, strains 16S rRNA gene sequences were used. To determine the antimicrobial activity of strains, seven foodborne pathogens were tested. Strains technological properties were characterized. These strains were identified as Lactobacillus plantarum by 16S rRNA gene sequence analysis and the phylogenetic tree obtained by neighbor-joining method allowed grouping of these strains into three subgroups. L. plantarum strains showed antagonistic activities against Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Micrococcus luteus strains. PCR assay, using specific primers, showed the presence of bacteriocin (plantaricin) encoding genes in all L. plantarum strains tested. Antimicrobial metabolite production of these strains started at log phase and reached the maximum level at the end of the stationary phase. Regarding their technological properties, better growth was observed at 25 °C compared with 15 °C and 45 °C. The isolates which grown well within the pH scale pH 4.5-6.5 range additionally showed a decent growth at 6.5% salt concentration. It has been found that strains do not exhibit lipolytic and proteolytic activities nor have lysine, ornithine, and arginine decarboxylase activity. On the other hand, one strain showed weak nitrate reductase activity, and four strains produced acetoin from glucose. In addition, all strains were DL-lactic acid producers. Consequently, L. plantarum strains isolated exhibited some biochemical properties required for a starter culture in sucuk and similar products. All identified strains may be a protective culture in the production of fermented meat products. In particular, L. plantarum S51 was distinguished from other isolates due to the inability to form acetoin from glucose. Further work will be needed to characterize L. plantarum strains as starter culture.

Development of an integrated process for the production of high‐purity cadaverine from lysine decarboxylase

AbstractBACKGROUNDCadaverine is a natural polyamine that can replace fossil‐derived hexamethylene diamine to produce bio‐based polyamide. Despite huge developments in cadaverine bioproduction, an econo"mically attractive purification process is a highly important part of the production of high‐purity cadaverine.RESULTSThis study utilized l‐lysine hydrochloride as a substrate for the production of high‐purity cadaverine via a litre‐scale integrated process incorporating fermentation, bioproduction, deprotonation, extraction and rectification. First, 8.24 U mg–1 lysine decarboxylase activity and 205 g L–1 cadaverine with a yield of 91.61% were achieved through the fermentation of low‐cost industrial culture by engineered Escherichia coli and the bioproduction of cadaverine by lysine decarboxylase. Secondly, four integrated purification methods – deprotonation‐evaporation, pH adjustment‐deprotonation‐evaporation, deprotonation‐extraction‐evaporation and deprotonation‐extraction‐rectification – were explored, and the conditions of each unit operation in the chosen method (deprotonation‐extraction‐rectification) were optimized. Then the optimized method was applied to a 2‐L purification system, resulting in 99% purity of cadaverine with a yield of 87.47%. Furthermore, the n‐butanol recovery ratio reached 91.25%, effectively avoiding the waste of resources and environmental pollution, and thus reducing purification costs.CONCLUSIONThe proposed experimental approach proves that this integrated process leads to the production of high‐purity cadaverine. In the 2‐L‐scale integrated process, 358.64 g cadaverine of 99% purity was produced at a cost of US$3.99 (US$1.11 per 100 g), which is far below its market price (US$784.71 per 100 g). This integrated process provides a potential way for the industrial‐scale production of bio‐based cadaverine. © 2020 Society of Chemical Industry

바이오제닉 아민 생성 및 분해균의 혼합 배양으로 제조한 청국장의 품질 특성

The objective of this study was to evaluate biogenic amine (BA) formation and degradation abilities of Bacillus strain and lactic acid bacteria (LAB) isolated from meju and doenjang and to determine the microbiological and physicochemical qualities of Cheonggukjang prepared by mixed culture of these strains. Accumulation of cadaverine (421.7 ± 11.0 mg/L) and histamine (628.7 ± 24.1 mg/L) was assessed in the culture media of Bacillus subtilis MJ226 that displayed lysine and histidine decarboxylase activities. No ability to form cadaverine, histamine, putrescine, tryptamine, and tyramine by Lactobacillus acidophilus D11, L. acidophilus D38, Lactobacillus sakei D44, Lactobacillus plantarum D70, which exhibited high BA degradation ability. The amino nitrogen and free amino acid contents were significantly (p&lt;0.05) increased in Cheonggukjang prepared by the mixed culture of LAB (L. sakei D44, L. plantarum D70, or L. pentosus D74) and B. subtilis MJ226, whereas the ammonia nitrogen and BA (cadaverine and histamine) contents were significantly (p&lt;0.05) decreased. Consequently, it was confirmed that BA formation in Cheonggukjang prepared by starter culture of B. subtilis was effectively inhibited by the certain strains of LAB having BA-degrading ability.

Non-biogroup 1 or 2 Strains of the Emerging Zoonotic Pathogen Escherichia albertii, Their Proposed Assignment to Biogroup 3, and Their Commonly Detected Characteristics.

Escherichia albertii, a zoonotic enteropathogen, is responsible for outbreaks of disease in humans. Identifying strains of E. albertii by phenotypic characterization tests is difficult because of its poorly defined properties. Screening its phenotypic characteristics is, nevertheless, a necessary prerequisite for further genetic analysis of its properties, and species-specific polymerase chain reaction (PCR) analysis can be used to type the pathogen. While two E. albertii biogroups (1 and 2) have been described, strains with characteristics divergent from both biogroups have been reported worldwide. The aim of the present study was to evaluate the characteristics of non-biogroup 1 or 2 strains, and discern the characteristics common to all of the E. albertii strains from this study. Altogether, 107/414 field isolates were selected for examination based on pulsed-field gel electrophoresis analysis. The 107 strains were isolated from 92 sources, including humans and pigeon feces, other wild birds, and retail chicken livers. All strains were then examined using various culture-based, biochemical (API 50CHE tests, API Zym test, and others) and molecular (virulence gene screening, multi-locus sequence analysis) testing methods. Our results revealed that all field strains (n = 107) showed non-biogroup 1 or 2 characteristics, with multiple sequence differences. Variations in indole production and the lysine decarboxylase activity profiles among the isolates made identification of E. albertii very difficult. Therefore, we propose that non-biogroup 1 or 2 of E. albertii should be assigned to biogroup 3 to make screening of them easier in public health and clinical laboratory settings. Clearly, having group criteria for indole-negative/lysine-positive, indole-positive/lysine-negative, and indole-positive/lysine-positive E. albertii biogroups 1, 2, and 3 strains, respectively, should provide for more accurate identification of E. albertii isolates. Based on our findings, we recommend that isolates displaying phenotype mobility-negativity (sulfide-indole-motility medium, 37°C), hydrogen sulfide production-negativity (triple sugar iron medium), acid production-negativity from xylose, negative β-glucuronidase activity properties, and showing indole production and lysine decarboxylase activity profiles in accordance with one of the three biogroups, should be further assessed using an E. albertii-specific PCR assay.

Reversible Shape-Morphing and Fluorescence-Switching in Supramolecular Nanomaterials Consisting of Amphiphilic Cyanostilbene and Cucurbit[7]uril.

We demonstrate a reversible shape-morphing with concurrent fluorescence switching in the nanomaterials which are complexed with cucurbit[7]uril (CB[7]) in water. The cyanostilbene derivative alone forms ribbon-like two-dimensional (2D) nanocrystals with bright yellow excimeric emission in water (λem =540 nm, ΦF =42 %). Upon CB[7] addition, however, the ribbon-like 2D nanocrystals immediately transform to spherical nanoparticles with significant fluorescence quenching and blue-shifting (λem =490 nm, ΦF =1 %) through the supramolecular complexation of the cyanostilbene and CB[7]. Based on this reversible fluorescence switching and shape morphing, we could demonstrate a novel strategy of turn-on fluorescence sensing of spermine and also monitoring of lysine decarboxylase activity.

The effect of leaf galls of Cynipidae on accumulation and biosynthesis of plant amines in oak trees

Polyamines (PAs) are involved in plant response to abiotic and biotic stresses, however, their role in biochemical insect-plant interactions is not clear. Therefore, we compared the involvement of polyamines and key enzymes of their biosynthesis in gall formation process. The present study had used galls on oak leaves caused by asexual generation (♀♀) of three Cynipidae species, namely Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and N. quercusbaccarum L., as a model. The obtained results indicate that gall formation on oak leaves affected amine content, but intensity of the changes in their levels were strongly dependent on the insect species. Nevertheless the downward trend was dominant among those changes. Changes in the activity of lysine decarboxylase (LDC), tyrosine decarboxylase (TyDC) and ornithine decarboxylase (ODC) usually corresponded with the direction of changes in polyamine contents. Several cases of divergence between changes in amine levels and the rate of their biosynthesis may suggest the involvement of other regulation mechanisms such as: arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) as well as amine oxidases involved in its catabolic pathways. Thus, the future studies on biochemical mechanism of regulation of PAs accumulation during galls formation should be focused on importance of these enzymes.

Sphingobacterium bovisgrunnientis sp. nov., isolated from yak milk.

A novel Gram-negative, rod shaped, non-motile bacterium, designated strain YK2T, was isolated from yak milk from Leh, India. The strain was positive for oxidase- and catalase-activities and negative for starch hydrolysis, nitrate reduction, citrate utilization, urease, lysine decarboxylase and ornithine decarboxylase activities. The predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c and/or iso-C15 : 0 2-OH (summed feature 3). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid and six unidentified lipids. The DNA G+C content of the strain was 38.9 mol%. The 16S rRNA gene sequence analysis indicated that strain YK2T was a member of the genus Sphingobacterium and closely related to Sphingobacterium alimentarium and Sphingobacterium composti with pair-wise sequence similarity of 98.3 and 97.9 %, respectively. The sequence similarity to other members of the genus Sphingobacterium was between 92.6 to 96.3 %. Phylogenetic analysis showed that strain YK2T clustered with Sphingobacterium alimentarium and together clustered with Sphingobacterium composti. DNA-DNA hybridization of strain YK2T with Sphingobacterium alimentarium WCC 4521T and Sphingobacterium composti T5-12T showed a relatedness of only 38 and 54 %, respectively. Based on the phenotypic characteristics and on phylogenetic inference, it appears that strain YK2T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium bovisgrunnientis sp. nov. is proposed. The type strain of Sphingobacterium bovisgrunnientis sp. nov. is YK2T (=MTCC 12631T=KCTC 52685T=JCM 31951T).

High Phenotypic Variability among Representative Strains of Common Salmonella enterica Serovars with Possible Implications for Food Safety

Salmonella is an important foodborne pathogen, whose ability to resist stress and survive can vary among strains. This variability is normally not taken into account when predictions are made about survival in foods with negative consequences. Therefore, we examined the contribution of variable phenotypic properties to survival under stress in 10 Salmonella serovars. One strain (Typhimurium 10) was intentionally RpoS-negative; however, another strain (Heidelberg) showed an rpoS mutation, rendering it inactive. We assessed an array of characteristics (motility, biofilm formation, bile resistance, acid resistance, and colony morphology) that show major variability among strains associated with a 10- to 19-fold difference between the highest and the lowest strain for most characteristics. The RpoS status of isolates did not affect variability in the characteristics, with the exception of resistance to NaCl, acetic acid, lactic acid, and the combination of acetic acid and salt, where the variability between the highest and the lowest strain was reduced to 3.1-fold, 1.7-fold, 2-fold, and 1.7-fold, respectively, showing that variability was significant among RpoS-positive strains. Furthermore, we also found a good correlation between acid resistance and lysine decarboxylase activity, showing its importance for acid resistance, and demonstrated a possible role of RpoS in the lysine decarboxylase activity in Salmonella.

Burkholderia puraquae sp. nov., a novel species of the Burkholderia cepacia complex isolated from hospital settings and agricultural soils

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and β-galactosidase activities.

The effect of Tetraneura ulmi L. galling process on the activity of amino acid decarboxylases and the content of biogenic amines in Siberian elm tissues.

Tetraneura ulmi (L.), a member of Eriosomatinae subfamily, is one of the gall-forming aphids occurring on elms. Sap-sucking behaviour of founding mothers results in the formation of new plant organs. This study documents the changes in the content of plant biogenic amines (putrescine, cadaverine, spermidine, tryptamine, spermine and histamine) and key enzymes of their biosynthesis: lysine decarboxylase (LDC), tyrosine decarboxylase and ornithine decarboxylase (ODC) in galls and other parts of Siberian elm (Ulmus pumila L.) leaves during the galling process. The direction and intensity of these changes for particular amines and enzymes were dependent on the stage of gall development and part of the galling leaf. Generally, the amine content tended to increase in gall tissues during the 1st and 2nd period of the galling process and decreased in later phases. LDC and ODC activities were markedly enhanced, especially in gall tissues at the initial stage of the galling process.

Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution.

Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.

Crystal Structure and Pyridoxal 5-Phosphate Binding Property of Lysine Decarboxylase from Selenomonas ruminantium.

Lysine decarboxylase (LDC) is a crucial enzyme for acid stress resistance and is also utilized for the biosynthesis of cadaverine, a promising building block for bio-based polyamides. We determined the crystal structure of LDC from Selenomonas ruminantium (SrLDC). SrLDC functions as a dimer and each monomer consists of two distinct domains; a PLP-binding barrel domain and a sheet domain. We also determined the structure of SrLDC in complex with PLP and cadaverine and elucidated the binding mode of cofactor and substrate. Interestingly, compared with the apo-form of SrLDC, the SrLDC in complex with PLP and cadaverine showed a remarkable structural change at the PLP binding site. The PLP binding site of SrLDC contains the highly flexible loops with high b-factors and showed an open-closed conformational change upon the binding of PLP. In fact, SrLDC showed no LDC activity without PLP supplement, and we suggest that highly flexible PLP binding site results in low PLP affinity of SrLDC. In addition, other structurally homologous enzymes also contain the flexible PLP binding site, which indicates that high flexibility at the PLP binding site and low PLP affinity seems to be a common feature of these enzyme family.

Development of a continuous l-lysine bioconversion system for cadaverine production

Abstract Cadaverine, a five carbon diamine (1,5-diaminopentane), plays a role as a building block of polyamides and it can be made by fermentation or direct bioconversion. To improve its production by increasing reusability of immobilized enzyme and avoid separation of enzyme in bioconversion, a continuous l -lysine bioconversion process for cadaverine production has been developed. Various divalent cations, alginate concentrations, cell density with alginate and flow rate of feed were examined to maximize the lysine decarboxylase activity of the whole-cell immobilized beads. Under the selected conditions, 123 h of continuous cadaverine production has been performed and 5.5 L of 819 mM cadaverine were produced with 14 mL reactor resulting in 466.5 g of cadaverine. Cadaverine production was possible with small volume reactor maintaining relatively high concentration of substrate.