Investigation of γ-polyglutamic acid production via asynchronous saccharification and fermentation of raw corn starch.

Starch, a crucial raw material, has been extensively investigated for biotechnological applications. However, its application in γ-polyglutamic acid (γ-PGA) production remains unexplored. Based on γ-PGA output of Bacillus subtilis SCP010-1, a novel asynchronous saccharification and fermentation process for γ-PGA synthesis was implemented. The results revealed that a starch concentration of 20%, α-amylase dosage of 75 U/g, liquefaction temperature of 72℃, and γ-PGA yield of 36.31 g/L was achieved. At a glucoamylase dosage of 100 U/g, saccharification 38 h at 60℃, the yield of γ-PGA increased to 48.88 g/L. The contents of total sugar, glucose, maltose and oligosaccharide in saccharified liquid were determined. Through batch fermentation of saccharified liquid in fermentor, the γ-PGA output was elevated to 116.08 g/L. This study can offer a potential cost reduction of 40%, which can be a promising advancement in industrial γ-PGA production. Moreover, our approach can be applied in other starch-based fermentation industries.

Biochemical and molecular characterization of a novel porphobilinogen synthase from Corynebacterium glutamicum.

Corynebacterium glutamicum porphobilinogen synthase (PBGS) is a metal enzyme with a hybrid active site metal binding sequence. In this study, the porphobilinogen synthase gene of C. glutamicum was cloned and heterogeneously expressed in Escherichia coli. C. glutamicum PBGS was purified, and its enzymatic characteristics were analyzed. The results showed that C. glutamicum PBGS is a Zn2+-dependent enzyme, and Mg2+ has allosteric regulation. The allosteric Mg2+ plays a vital role in forming the quaternary structure of C. glutamicum PBGS. Based on the ab initio predictive structure modeling of the enzyme and the molecular docking model of 5-aminolevulinic acid (5-ALA), 11 sites were selected for site-directed mutagenesis. When the hybrid active site metal binding site of C. glutamicum PBGS is converted into a cysteine-rich motif (Zn2+-dependent) or an aspartic acid-rich motif (Mg2+/K+-dependent), the enzyme activity is basically lost. Four residues, D128, C130, D132, and C140, in the metal binding site, were the binding sites of Zn2+ and the active center of the enzyme. The band migration, from the native PAGE, of five variants with mutations in the center of enzyme activity was the same as that of the variant enzymes as purified, individually adding two metal ion chelating agents. Their Zn2+ active center structures were abnormal, and the quaternary structure equilibrium was altered. The destroyed active center affects the construction of its quaternary structure. The quaternary structural balance between octamer and hexamer through dimers was regulated by the allosteric regulation of C. glutamicum PBGS. The enzyme activity was also affected by the change of the active site lid structure and (α ß)8-barrel structure caused by mutation. Structural changes in the variants were analyzed to understand C. glutamicum PBGS better.