Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD ₁ and nylE ₁ , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD₁) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE₁) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP⁺ as a cofactor. Phylogenic analysis revealed that NylD₁ should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE₁ should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD₁/NylE₁ coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

     

Rational protein design for thermostabilization of glycoside hydrolases based on structural analysis

Glycosidases are used in the food, chemical, and energy industries. These proteins are some of the most frequently used such enzymes, and their thermostability is essential for long-term and/or repeated use. In addition to thermostability, modification of the substrate selectivity and improvement of the glycosidase activities are also important. Thermostabilization of enzymes can be performed by directed evolution via random mutagenesis or by rational design via site-directed mutagenesis; each approach has advantages and disadvantages. In this paper, we introduce thermostabilization of glycoside hydrolases by rational protein design using site-directed mutagenesis along with X-ray crystallography and simulation modeling. We focus on the methods of thermostabilization of glycoside hydrolases by linking the N- and C-terminal ends, introducing disulfide bridges, and optimizing β-turn structures to promote hydrophobic interactions.

     

Agroinfiltration: a rapid and reliable method to select suitable rose cultivars for blue flower production

Rose cultivars with blue flower color are among the most attractive breeding targets in floriculture. However, they are difficult to produce due to the low efficiency of transformation systems, interactive effects of hosts and vectors, and lengthy processes. In this study, agroinfiltration-mediated transient expression was investigated as a tool to assess the function of flower color genes and to determine appropriate host cultivars for stable transformation in Rosa hybrida. To induce delphinidin accumulation and consequently to produce blue hue, the petals of 30 rose cultivars were infiltrated with three different expression vectors namely pBIH-35S-CcF3′5′H, pBIH-35S-Del2 and pBIH-35S-Del8, harbouring different sets of flower color genes. The results obtained showed that the ectopic expression of the genes was only detected in three cultivars with dark pink petals (i.e. ‘Purple power’, ‘High & Mora’ and ‘Marina’) after 6–8 days. The high performance liquid chromatography analyses confirmed delphinidin accumulation in the infiltrated petals caused by transient expression of CcF3′5′H gene. Moreover, there were significant differences in the amounts of delphinidin among the three cultivars infiltrated with the three different expression vectors. More specifically, the highest delphinidin content was detected in the cultivar ‘Purple power’ (4.67 µg g^?1 FW), infiltrated with the pBIH-35S-Del2 vector. The expression of CcF3′5′H gene in the infiltrated petals was also confirmed by real time PCR. In conclusion and based on the findings of the present study, the agroinfiltration could be regarded as a reliable method to identify suitable rose cultivars in blue rose flower production programs.     

Conversion of iodine to fluorine-18 based on iodinated chalcone and evaluation for β-amyloid PET imaging

In the amyloid cascade hypothesis, β-amyloid (Aβ) plaques is one of the major pathological biomarkers in the Alzheimer’s disease (AD) brain. We report the synthesis and evaluation of novel radiofluorinated chalcones, [¹⁸F]4-dimethylamino-4′-fluoro-chalcone ([¹⁸F]DMFC) and [¹⁸F]4′-fluoro-4-methylamino-chalcone ([¹⁸F]FMC), as Aβ imaging probes. The conversion of iodine directly introduced to the chalcone backbone into fluorine was successfully carried out by ¹⁸F-labeling via the corresponding boronate precursors, achieving the direct introduction of fluorine-18 into the chalcone backbone to prepare [¹⁸F]DMFC and [¹⁸F]FMC. In a biodistribution study using normal mice, [¹⁸F]DMFC and [¹⁸F]FMC showed a higher initial uptake (4.43 and 5.47% ID/g at 2?min postinjection, respectively) into and more rapid clearance (0.52 and 0.66% ID/g at 30?min postinjection, respectively) from the brain than a Food and Drug Administration (FDA)-approved Aβ imaging agent ([¹⁸F]Florbetapir), meaning the improvement of the probability of detecting Aβ plaques and the reduction of non-specific binding in the brain. In the in vitro binding studies using aggregates of recombinant Aβ peptides, [¹⁸F]DMFC and [¹⁸F]FMC showed high binding affinity to recombinant Aβ aggregates at the K_d values of 4.47 and 6.50?nM, respectively. In the in vitro autoradiography (ARG) experiment with AD brain sections, [¹⁸F]DMFC and [¹⁸F]FMC markedly accumulated only in a region with abundant Aβ plaques, indicating that they clearly recognized human Aβ plaques in vitro. These encouraging results suggest that [¹⁸F]DMFC and [¹⁸F]FMC may be promising PET probes for the detection of an amyloid pathology and the early diagnosis of AD with marked accuracy.     

Effects of bedtime periocular and posterior cervical cutaneous warming on sleep status in adult male subjects: a preliminary study

Sleep and Biological Rhythms - Appropriate warming of the periocular or posterior cervical skin has been reported to induce autonomic or mental relaxation in humans. To clarify the effects of...     

Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons

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Large‐scale culture of a megakaryocytic progenitor cell line with a single‐use bioreactor system

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single‐use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large‐scale production and feasibility of meeting clinical‐grade standards. The current work evaluated the capacity of a single‐use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k_La) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h^?1, respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7‐fold with a total cell number of 1.2 ± 0.2 × 10⁹ cells L^?1. In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single‐use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362–369, 2018     

Stabilization by GroEL, a Molecular Chaperone, and a Periplasmic Fraction, as Well as Refolding in the Presence of Dithiothreitol, of Acid-Unfolded Dimethyl Sulfoxide Reductase, a Periplasmic Protein of Rhodobacter sphaeroides f. sp. denitrificans

The mechanisms of folding of a periplasmic protein was studiedin vitro using dimethyl sulfoxide reductase (DMSOR), a periplasmicenzyme of Rhodobacter sphaeroides f. sp. denitrificans. WhenDMSOR was denatured by acidification to pH 2 at 30°C, themolybdenum cofactor was immediately released and unfolded formsof DMSOR appeared within 2 min. When the acid-unfolded DMSORhas been incubated in refolding buffer (pH 8.0) at 20°Cfor 2 h, it became almost undetectable after electrophoresison a non-denaturing gel. This result suggests that the acid-unfoldedDMSOR might have aggregated after incubation. The aggregationwas suppressed by incubation in the presence of commercial GroEL,a molecular chaperone. When reduced dithiothreitol (DTT) wasadded to the acid-unfolded forms in the presence of GroEL, someof the DMSOR was converted to the native form, which had thesame mobility on a non-denaturing gel as the active emzyme.Non-reducing SDS-polyacrylamide gel electrophoresis of the acid-unfoldedforms of DMSOR indicated that the unfolded forms were a mixtureof heterogeneously folded or misfolded forms and that theirforms were converted by DTT to the fully reduced form. The periplasmicfraction of the phototroph was also able to suppress the aggregationof the acid-unfolded DMSOR, and a protein(s) with a molecularmass of about 40 kDa in the periplasm was revealed to have stabilizingactivity. It appears that there exists a mechanism whereby theunfolded DMSOR that is secreted into the periplasm is maintainedin a non-aggregated and reduced form during folding to the nativeform. (Received November 4, 1995; Accepted February 8, 1996)     

Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome

A physical map of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome has been constructed with restriction endonucleases PmeI, SwaI, and an intron-encoded endonuclease I-CeuI. The estimated size of the genome is 2.7 Mb. On the genome 49 genes or operons have been mapped. Two rRNA operons are separated by 600 kb and transcribed oppositely.     

A cytokinin-binding protein complex from tobacco leaves

A cytokinin binding protein complex (CBP130) has been purified from tobacco leaves (Nicotiana sylvestris). It contains two protein species of 57 and 36 kDa (CBP57 and CBP36). The cDNAs encoding CBP57 have been isolated from a tobacco cDNA library. Their predicted amino acid sequences showed significant homology between CBP57 and S-adenosyl-L-homocysteine (SAH) hydrolase, which catalyzes the reversible hydrolysis of SAH, a methyltransferase inhibitor. A combination of gel filtration an western blot analysis revealed that both CBP57 and benzyladenine (BA)-binding activity were eluted at a peak of 130 kDa. A purified CBP130 fraction contains SAH hydrolase activity. We discuss possible CBP57 as a cytokinin receptor subunit and its possible role as a regulator of methylation.