Isolation and identification of high-yielding alkaline phosphatase strain: a novel mutagenesis technique and optimization of fermentation conditions

Isolating and screening enzyme-producing strains from microorganisms and the commercial production of ALPs from microorganisms are of increasing interest. In this work, isolation and identification of high-yielding alkaline phosphatase strain were carried out using atmospheric and room temperature plasma mutagenesis (ARTP) for optimization of fermentation conditions. A strain of alkaline phosphatase-producing bacteria was screened from soil and identified by 16S rRNA gene sequencing as Bacillus amyloliquefaciens and named S-1. This strain had an alkaline phosphatase activity of 2594.73 U/L. Later, mutagenesis breeding of the alkaline phosphatase-producing S-1 strain was conducted using (ARTP), from which a higher alkaline phosphatase-producing positive mutant strain S-52 was screened. A central combination of five factors, including corn starch, yeast extract, metal ions, fermentation temperature and inoculum ratio, was then used to influence the activity of alkaline phosphatase. Results from the response surface methodology showed that the maximum enzyme activity of alkaline phosphatase was 12,110.6 U/L at corn starch, yeast extract and magnesium ions concentrations of 17.48 g/L, 18.052 g/L and 0.744 g/L, respectively; fermentation temperature of 37.192 °C; and inoculation ratio of 5.59%. This study is important for further exploring ARTP mutagenesis in B. amyloliquefaciens and the commercialization of ALPs.</p

Information of primers used in PCR amplification for <i>NOTCH3</i> gene.

<p>Information of primers used in PCR amplification for <i>NOTCH3</i> gene.</p

A novel G149V mutation found in a CADASIL family.

<p>The pedigree of this Chinese CADASIL family was shown (A). The proband is indicated with an arrow. MscI digestion results corresponding to each individual were shown below the pedigree. The affected showed a digested band (131 bp) with an undigested band (149 bp), while the normal controls showed only one digested band at 131 bp. III1 and III3 carried the mutation but are asymptomatic so far. Electropherograms of the sequence showed the nucleotide change in the affected individual with respect to the normal control bp. III1 and III3 carried the mutation but are asymptomatic so far. Electropherograms of the sequence showed the nucleotide change in the affected individual with respect to the normal control (B). P.G149V (c.446G>T) was indicated with an arrow.</p

Brain MRI images of the proband showing characteristic diffuse leukoencephalopathy.

<p>T2-weighted MRI images (A–C) showed diffuse hyperintensities in the periventricular white matter with a symmetrical distribution, the centrum semiovale, and the basal ganglia. Fluid-attenuated inversion recovery (FLAIR) T2 sequences (D–F) revealed confluent white-matter lesions in multiple brain regions. Diffusion-weighted imaging (DWI) sequence (G) demonstrated acute ischemic stroke in the centrum semiovale. MR angiography (H) revealed no intracranial arterial stenosis or occlusion.</p

Alignment of EGF-like repeat domain of NOTCH3.

<p>Alignment of C4–C6 in EGF-like domains of NOTCH3 was shown in the upper panel (A). Conserved G36 in human EGF is shown in the most EGF-like domains of NOTCH3. Alignment of C4–C6 in EGF-like repeat domain 3 homolog from different species was shown in the middle panel of the figure (B). Amino acid sequence alignment from various species showed the conservation of this glycine residue. The reported CADASIL mutations within this EGF-like repeat domain were indicated in the lower panel (C).</p

Table_1_Luminescent Polynuclear Zn- and Cd-Ln Square-Like Nanoclusters With a Flexible Long-Chain Schiff Base Ligand.DOC

<p>Two series of Zn-Ln and Cd-Ln nanoclusters [Ln₄Zn₈L₂(OAc)₂₀(OH)₄] [Ln = Nd (1), Yb (2), and Sm (3)] and [Ln₂Cd₂L₂(OAc)₂(OH)₂(OCH₃)₂] [Ln = Nd (4), Yb (5), and Sm (6)] were prepared using a long-chain Schiff base ligand with a flexible (CH₂)₂O(CH₂)₂O(CH₂)₂ chain. All these clusters show square-like structures. The Schiff base ligands show “linear” configurations in the structures of 1-6, and the metric dimensions of Zn-Ln and Cd-Ln clusters measure ~8 × 14 × 21 and 8 × 12 × 12 Å, respectively. The study of luminescence properties shows that the Zn/L and Cd/L chromophores can effectively transfer energy to the lanthanide ions, and 1-6 show visible and NIR emissions.</p

Data_Sheet_1_Luminescent Polynuclear Zn- and Cd-Ln Square-Like Nanoclusters With a Flexible Long-Chain Schiff Base Ligand.DOC

<p>Two series of Zn-Ln and Cd-Ln nanoclusters [Ln₄Zn₈L₂(OAc)₂₀(OH)₄] [Ln = Nd (1), Yb (2), and Sm (3)] and [Ln₂Cd₂L₂(OAc)₂(OH)₂(OCH₃)₂] [Ln = Nd (4), Yb (5), and Sm (6)] were prepared using a long-chain Schiff base ligand with a flexible (CH₂)₂O(CH₂)₂O(CH₂)₂ chain. All these clusters show square-like structures. The Schiff base ligands show “linear” configurations in the structures of 1-6, and the metric dimensions of Zn-Ln and Cd-Ln clusters measure ~8 × 14 × 21 and 8 × 12 × 12 Å, respectively. The study of luminescence properties shows that the Zn/L and Cd/L chromophores can effectively transfer energy to the lanthanide ions, and 1-6 show visible and NIR emissions.</p

Data_Sheet_2_Luminescent Polynuclear Zn- and Cd-Ln Square-Like Nanoclusters With a Flexible Long-Chain Schiff Base Ligand.DOC

<p>Two series of Zn-Ln and Cd-Ln nanoclusters [Ln₄Zn₈L₂(OAc)₂₀(OH)₄] [Ln = Nd (1), Yb (2), and Sm (3)] and [Ln₂Cd₂L₂(OAc)₂(OH)₂(OCH₃)₂] [Ln = Nd (4), Yb (5), and Sm (6)] were prepared using a long-chain Schiff base ligand with a flexible (CH₂)₂O(CH₂)₂O(CH₂)₂ chain. All these clusters show square-like structures. The Schiff base ligands show “linear” configurations in the structures of 1-6, and the metric dimensions of Zn-Ln and Cd-Ln clusters measure ~8 × 14 × 21 and 8 × 12 × 12 Å, respectively. The study of luminescence properties shows that the Zn/L and Cd/L chromophores can effectively transfer energy to the lanthanide ions, and 1-6 show visible and NIR emissions.</p