Aims:  The present study was conducted to screen for psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to examine the cold‐active pectinolytic enzymes produced by the isolated psychrophilic yeasts. Methods and Results:  Psychrophilic yeasts, which grow on pectin as a sole carbon source, pectinolytic–psychrophilic yeast (PPY) strains PPY‐3, 4, 5 and 6, were isolated from soil from Abashiri (Hokkaido, Japan). The sequences of 28S rDNA D1/D2 of strains PPY‐3 and 4 indicated a taxonomic affiliation to Cryptococcus cylindricus and Mrakia frigida, respectively, strains PPY‐5 and 6 belonged to Cystofilobasidium capitatum. The isolated strains were able to grow on pectin at below 5°C, and showed the activities of several cold‐active pectinolytic enzymes. Conclusion:  The findings of this study indicate the possibility that the isolated strains produce novel pectinolytic enzymes that are able to degrade pectin compounds at low temperature. Significance and Impact of the Study:  It is possible that the cold‐active pectinolytic enzymes from the isolated strains can be applied to the food industry, e.g. the clarification of fruit juice below 5°C.

Aims:  The present study was conducted by screening zein‐degrading bacteria in an attempt to obtain zein‐degrading protease. Methods and Results:  Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein‐degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS‐1 strain. The strain produced two different types of extracellular proteases, BPP‐A and BPP‐B. In this study, we purified and characterized BPP‐A because it exhibited a higher ability to hydrolyze zein than BPP‐B. When casein was used as the substrate, the optimal pH for BPP‐A was 11·0. In BPP‐A, zein was better substrate than casein at pH 13·0, whereas casein was better one than zein at pH 11·0. The bppA gene encoded a 383‐amino acid pre‐pro form of BPP‐A, and mature BPP‐A contained 275 amino acid residues. It was concluded that BPP‐A belonged to the subtilisin family. Conclusion:  A zein‐degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP‐A and BPP‐B. BPP‐A exhibited an ability to hydrolyze zein in an extreme alkaline condition. Significance and Impact of the Study:  This is a first report on screening for zein‐degrading micro‐organisms. The subtilisin‐like protease BPP‐A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2. The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD-encoding gene, MOD2. A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%). The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase-depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1. The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction. The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range. Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level. These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p.

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, P MOD1 and P MOD2 , were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5 ) as a reporter. Heterologous gene expression driven by the P MOD1 and P MOD2 promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. P MOD1 was induced not only by methanol but also by glycerol. P MOD2 was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that P MOD2 is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by P MOD1 and P MOD2 . P MOD1 expression was optimal at low methanol concentrations, whereas P MOD2 was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both P MOD2 and P MOD1 should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using P MOD1 and P MOD2 in the same host cell and in the same flask.

A pectinolytic and psychrophilic yeast was isolated from soil from Abashiri, Hokkaido, Japan. The phenotype and sequencing of the 28S rDNA of the isolated strain (PPY-1) indicated a taxonomic affiliation to the basidiomycetous yeast Cystofilobasidium capitatum. C. capitatum strain PPY-1 was able to grow on two pectic compounds, polygalacturonate and pectin, at below 5 degrees C. Moreover, the extracellular fraction of the strain exhibited pectin methylesterase, pectin lyase and polygalacturonase activities at 5 degrees C. Thus strain PPY-1 may produce novel enzymes that are able to degrade pectin at low temperature, although the strain has isozymes of these enzymes.