As a novel cofactor of oxidoreductase, pyrroloquinoline quinone (PQQ) has a great potential of application in medicine, food industries. In order to improve the efficiency of the PQQ production by Methylobacterium extorquens AM1, the strain was treated by atmospheric and room temperature plasma (ARTP). Positive mutants with changes in PQQ yield were obtained based on a high-throughput screening approach. After ARTP treatment, analysis data show that the positive mutation rate was 31.6%. Furthermore, we obtained an excellent positive mutant M. extorquens AM1 (E-F3) with the yield of 54.0 mg/L PQQ, which was approximately 3 times as much compared with that of the wild-type strain. The robust high-throughput screening method for mutagenesis by ARTP improves PQQ production. In addition, this method also provides a new strategy for further strain improvement.
Bacterial Proteins ; biosynthesis ; High-Throughput Screening Assays ; Methylobacterium extorquens ; enzymology ; genetics ; Mutagenesis ; PQQ Cofactor ; biosynthesis ; Plasma Gases ; Temperature

OBJECTIVETo investigate the effect of pyrroloquinoline quinone (PQQ) on nerve regeneration of transected sciatic nerve in animal models.

METHODSForty SD rats weighing 220-240 g were randomized into a PQQ group (n = 20) and a control group (n = 20). Each animal underwent sciatic nerve transection operation. After the operation, PQQ 0.5 ml (250 microg/Kg) was injected at the operation site in the PQQ group, while the same volume of normal saline was delivered in the control group. Nerve functional evaluation, electrophysiological index recording were carried out according to the experimental design. Newly generated nerve specimens were harvested 12 weeks postoperatively for morphological studies.

RESULTSIn the PQQ group there was a good nerve regeneration and the sciatic nerve function, sciatic nerve function index, electrophysiological index and morphological appearance were superior to the control group (P < 0.05).

CONCLUSIONSPQQ has a remarkable effect in enhancing nerve regeneration of transected peripheral nerve.

Animals ; Male ; Nerve Regeneration ; drug effects ; PQQ Cofactor ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; injuries ; pathology ; physiology

Pyrroloquinoline quinone (PQQ), an important redox enzyme cofactor, has many physiological and biochemical functions, and is widely used in food, medicine, health and agriculture industry. In this study, PQQ production by recombinant Gluconobacter oxydans was investigated. First, to reduce the by-product of acetic acid, the recombinant strain G. oxydans T1 was constructed, in which the pyruvate decarboxylase (GOX1081) was knocked out. Then the pqqABCDE gene cluster and tldD gene were fused under the control of endogenous constitutive promoter P0169, to generate the recombinant strain G. oxydans T2. Finally, the medium composition and fermentation conditions were optimized. The biomass of G. oxydans T1 and G. oxydans T2 were increased by 43.02% and 38.76% respectively, and the PQQ production was 4.82 and 20.5 times higher than that of the wild strain, respectively. Furthermore, the carbon sources and culture conditions of G. oxydans T2 were optimized, resulting in a final PQQ yield of (51.32±0.899 7 mg/L), 345.6 times higher than that of the wild strain. In all, the biomass of G. oxydans and the yield of PQQ can be effectively increased by genetic engineering.
Fermentation ; Gene Knockout Techniques ; Gluconobacter oxydans ; genetics ; metabolism ; Industrial Microbiology ; methods ; Multigene Family ; genetics ; Organisms, Genetically Modified ; PQQ Cofactor ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics

OBJECTIVETo investigate the effects of pyrroloquinoline quinine (PQQ) on proliferation and expression of c-fos, c-jun, CREB and PCNA in cultured Schwann cells.

METHODSSchwann cells were cultured and purified in vitro. The purity of Schwann cells was identified by immunofluorescence of S-100. After synchronization of cell cycle by serum-free medium, different concentration of PQQ (0,1, 10, 100, 1,000, 10,000 nmol/L) were added into culture medium for 72 h. Flow cytometry was used to determine cell cycle. The content of c-fos, c-jun, and CREB mRNA were detected by RT-PCR, and the expression of PCNA protein was detected by Western blot.

RESULTSAfter PQQ treatment, the percentage of cells in G0/G1 phase decreased and the percentage of cells in S and G2/M phase increased. After treated by PQQ at concentration of 1-10,000nmol/L, content of c-fos,c-jun,CREB mRNA was increased by 0.33,0.42 and 0. 52 fold (P < 0. 05). However, at concentration of 1 000 nmol/L, there was no difference in mRNAs content when compare to control (P >0.05). And it showed a decline at concentration of 10,000 nmol/L (P < 0.05). PCNA protein expression was up-regulated at PQQ concentration of 1-100 nmol/L. At 100 nmol/L, the expression increased by 1.17 fold (P < 0.05); However, at 1,000 nmol/L, there was no difference in PCNA expression when compared to control. And 10,000 nmol/L of PQQ inhibited the expression of PCNA (P < 0.05).

CONCLUSIONSWhen treated with PQQ at concentration of 10-100 nmol/L, the proliferation of Schwann cells increased and the expression of c-fos,c-jun, CREB and PCNA was up-regulated.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Genes, fos ; Genes, jun ; PQQ Cofactor ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; drug effects ; metabolism

OBJECTIVETo investigate the roles of PI3K/Akt signal pathway in Schwann cells proliferation promoted by pyrroloquinoline quinone (PQQ).

METHODSSchwann cells were cultured and purified in vitro. The purity was identified by immunofluorescence of S-100; the expression of Akt and phosphorylated-Akt (p-Akt) were detected by western blot, and the expression of p-Akt after blocking the PI3K signal transduction pathway by PI3K inhibitor wortmannin was detected by western blot.

RESULTSMorphological change was observed in PQQ-treated Schwann cells, p-Akt was detected 30 min after PQQ treated, reached the peak at 4 h, and disappeared 12 h later. 1-100 nmol/L PQQ could up-regulate the expression of p-Akt; this up-regulated expression of p-Akt was inhibited by wortmannin (P<0.05).

CONCLUSIONSPQQ could affect morphology of Schwann cells and activation of Akt. It indicates that PI3K/Akt signal pathway might be involved in Schwann cells proliferation promoted by PQQ.

Androstadienes ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; PQQ Cofactor ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; drug effects ; metabolism ; Signal Transduction