Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11691.

Information about the inulosucrase of nonlactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the Gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0-9.0 and 50-55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multiangle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3-27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

A unique Zn(II)2-Cys6-type protein, KpeA, is involved in secondary metabolism and conidiation in Aspergillus oryzae.

Aspergillus oryzae is an important microorganism in the bio- and food industries; therefore, understanding the mechanism underlying its secondary metabolism regulation is important for ensuring its safe use. Here, we screened a novel Zn(II)2-Cys6-type protein-encoding gene, AO090003001186, designated as kpeA (kojic acid production enhancement A), from an A. oryzae disruption mutant library of transcriptional regulators. kpeA is highly conserved among filamentous fungi and encodes a protein with Zn(II)2-Cys6 motif located in the middle of the sequence. Phylogenetic analysis revealed that KpeA was classified into a distal group compared to other fungal Zn(II)2-Cys6-type transcriptional regulators. A Cys to Ala substitution mutant of KpeA showed identical phenotype to the kpeA disruption strain, confirming that KpeA is novel type Zn(II)2-Cys6 binding protein. Colonies of the kpeA disruption strain (ΔkpeA) had longer aerial hyphae and showed decreased conidia production. Microscopic analysis suggested that the reduced vesicle size and conidial head formation in ΔkpeA strain account for the decreased conidia production. Transcriptional levels of brlA and downstream abaA and wetA were decreased in ΔkpeA strain. Moreover, ΔkpeA strain produced 6-fold more kojic acid than the control strains, and the expression of kojR and kojA was increased in ΔkpeA strain. Therefore, KpeA is a novel Zn(II)2-Cys6-type protein likely involved in conidiation and kojic acid production at the transcriptional level.

Application of multiple ultra-high-pressure homogenization to the pasteurization process of Japanese rice wine, sake.

Hiire is a pasteurization process in the production of Japanese rice wine (sake), which stabilizes the quality of product; however, it also generates the carcinogen ethyl carbamate (EC). In this study, we investigated the application of ultra-high-pressure homogenization (UHPH) as an alternative sterilization method for sake production. Microbiological analysis revealed that multiple UHPH treatments sterilized hiochi lactobacilli (Lactobacillus fructivorans, L. homohiochii, L. casei, and L. hilgardii) and Saccharomyces cerevisiae. Enzyme activity assays revealed that α-amylase, glucoamylase, and acid-carboxypeptidase activities were reduced to less than 1% of the levels in non-pasteurized sake after four-time UHPH treatment. These results show that UHPH treatment meets the two requirements of the sake sterilization process sterilization and enzyme inactivation. The UHPH-processed sake did not show any significant changes in general properties but had reduced organic acid and aromatic component contents, with ethyl caproate content showing the most significant reduction of approximately 20%. Interestingly, EC was detected in pasteurized sake but not in UHPH-processed sake. These findings indicate that the UHPH technology could be used to inactivate microorganisms and enzymes in sake without generating EC.

New role of a histone chaperone, HirA: Involvement in kojic acid production associated with culture conditions in Aspergillusoryzae.

Kojic acid (KA) is a representative secondary metabolite of Aspergillus oryzae, but the underlying molecular mechanisms that regulate KA production are unknown. This study tried to find a genetic factor of KA production in A. oryzae, with a special focus on liquid cultures. We screened a gene predicted to encode HirA, a subunit of the histone chaperon, the HIR complex. A gene disruption strain of hirA showed decreased KA production in liquid culture, whereas it showed increased KA production in plate culture. We confirmed that a decrease/increase of KA production observed by hirA disruption was caused by altered expression of kojA and kojR. These observations suggested the regulatory role of histone chaperon in secondary metabolism in filamentous fungi. So far as we know, this report is the first showing that disruption of a gene resulted in the opposite effect on KA production in liquid and plate cultures in A. oryzae.