Cytidine production of screening strains in 96-well deep-hole culture plate.

<p>Cytidine production of screening strains in 96-well deep-hole culture plate.</p

(A). Effect of different cytidine concentrations on CDA assay in H₂O.

<p>Cytidine standard curves were conducted under standard reaction conditions in section 2.5 in H₂O (B), LB medium (C), M9 medium (D) and 10-fold diluted LB medium (E), respectively. Each plot represents the average of three samples. Absorbance was measured using a microplate reader.</p

High-Efficiency Secretion of β‑Mannanase in <i>Bacillus subtilis</i> through Protein Synthesis and Secretion Optimization

The manno endo-1,4-mannosidase (β-mannanase, EC. 3.2.1.78) catalyzes the random hydrolysis of internal (1 → 4)-β-mannosidic linkages in the mannan polymers. A codon optimized β-mannanase gene from <i>Bacillus licheniformi</i>s DSM13 was expressed in <i>Bacillus subtilis</i>. When four Sec-dependent and two Tat-dependent signal peptide sequences cloned from <i>B. subtilis</i> were placed upstream of the target gene, the highest activity of β-mannanase was observed using SP_<i>lipA</i> as a signal peptide. Then a 1.25-fold activity of β-mannanase was obtained when another copy of <i>groESL</i> operon was inserted into the genome of host strain. Finally, five different promoters were separately used to enhance the synthesis of the target protein. The results showed that promoter P<i>_mglv</i>, a modified maltose-inducible promoter, significantly elevated the production of β-mannanase. After 72 h of flask fermentation, the enzyme activity of β-mannanase in the supernatant when using locust bean gum as substrate reached 2207 U/mL. This work provided a promising β-mannanase production strain in industrial application

Summary of parameters of the cytidine assay by CDA.

<p>Summary of parameters of the cytidine assay by CDA.</p

(A) Cytidine concentrations in fermentation flasks were determined using the CDA assay and HPLC.

<p>Samples were collected from the fermentation flasks every 8 h from 8 h to 40 h. (B). The correlation between cytidine added in fermentation broths and that detected by CDA assay.</p

Substrate specificity of the CDA.

<p>Substrate specificity of the CDA.</p

Screening high cytidine-producing strains using CDA assay.

<p>(A) Production of cytidine in a 96-well deep-hole culture plate from randomly picked mutation strains. The letters and numbers represented row and column number of the 96-well deep-hole culture plate respectively. C: Control, A1 represented for cytidine concentration of parent strain. (B) Top five samples (F12, E7, H3, B8 and H4) determined using CDA assay was chosen to detect cytidine by HPLC. CDA assay and HPLC had a positive correlation on cytidine concentration. Each experiment runs in triplicate.</p

Effect of medium components on the cytidine assay.

<p>Note: Values reported in the table were the average of three parallel determinations. The absorbance was reported as a percentage of that obtained with cytidine, (2 mM) dissolved in water, i.e., ((absorbance with cytidine + test compound)/absorbance with cytidine alone) × 100%. A value of 100 means no interference; a value of 0 means total interference, i.e., no color formation at all, and values greater than 100 mean the test compound enhances the absorbance of the solution.</p><p>Effect of medium components on the cytidine assay.</p

UV-visible absorption spectra of products in the tandem enzyme assay.

<p>Precorrin-2 was produced from ALA by the tandem-enzyme reaction system containing purified, recombinant PBGS, PBGD, UROS, SUMT, and SAM (dotted line); precorrin-2 was then converted into sirohydrochlorin by precorrin-2 dehydrogenase in the presence of NAD (solid line).</p

Synthesis pathway of tetrapyrroles.

<p>ALA, 5-aminolevulinic acid; PBGS, porphobilinogen synthase; PBGD, porphobilinogen deaminase; UROS, uroporphyrinogen III synthase; SUMT, S-adenosyl-l-methionine-dependent urogen III methyltransferase; SAM, S-adenosyl-l-methionine; SAH, S-adenosylhomocysteine</p