Jun Hitomi

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like cyrstals.

Purification of alkaline proteases from a Bacillus strain and their possible interrelationship

Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55°C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60°C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5°C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.

Stress Tolerance of Methylobacterium Biofilms in Bathrooms

A comprehensive survey of microbial flora within pink biofilms in bathrooms was performed. Pink biofilms develop relatively rapidly in bathrooms, can be difficult to remove, and are quick to recur. Bacterium-sized cells were found to be predominant in 42 pink biofilms in Japan using a scanning electron microscope. Methylobacterium strains were detected from all samples in bathrooms by an isolation method. To explain this predominance, 14 biofilm samples were analyzed by fluorescence in situ hybridization. Methylobacterium was indicated to be the major genus in all biofilms. The isolated Methylobacterium survived after contact with 1.0% cleaning agents, including benzalkonium chloride for 24 h. Their tolerance did not differ under biofilm-like conditions on fiber reinforced plastics (FRP), a general material of bath tubs, floors, and walls. Also, the strains exhibited higher tolerance to desiccation than other isolated species on FRP. Some Methylobacterium survived and exhibited potential to grow after four weeks of desiccation without any nutrients. These specific characteristics could be a cause of their predominance in bathrooms, an environment with rapid flowing water, drying, low nutrients, and occasional exposure to cleaning agents.

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from the Alkalophilic Bacillus sp. KSM-K16

Abstract The gene for an alkaline protease, suitable for use in both powder and liquid detergents, from the alkalophilic Bacillus sp. KSM-K16 was cloned and sequenced. Its nucleotide sequence included an open reading frame of 1,143 bp (380 amino acids) that encoded a pre-pro-peptide (111 amino acids) and a mature protein (269 amino acids, 26,723 Da). The deduced amino acid sequence of the enzyme exhibited high homology to those of alkaline proteases from alkalophilic strains of Bacillus and moderate homology to those of subtilisins reported to date. The catalytic triad (Asp32, His64 and Ser221) and the subsite sequence (Ser125-Leu126-Gly127) of subtilisin BPN′ were well conserved as Asp32, His62 and Ser215 and as Ser123-Leu124-Gly125, respectively. However, the third (“wobble”) positions of sense codons revealed some marked differences between our protease and subtilisins (and related alkaline proteases).

Moraxella Species Are Primarily Responsible for Generating Malodor in Laundry

ABSTRACT Many people in Japan often detect an unpleasant odor generated from laundry that is hung to dry indoors or when using their already-dried laundry. Such an odor is often described as a “wet-and-dirty-dustcloth-like malodor” or an “acidic or sweaty odor.” In this study, we isolated the major microorganisms associated with such a malodor, the major component of which has been identified as 4-methyl-3-hexenoic acid (4M3H). The isolates were identified as Moraxella osloensis by morphological observation and biochemical and phylogenetic tree analyses. M. osloensis has the potential to generate 4M3H in laundry. The bacterium is known to cause opportunistic infections but has never been known to generate a malodor in clothes. We found that M. osloensis exists at a high frequency in various living environments, particularly in laundry in Japan. The bacterium showed a high tolerance to desiccation and UV light irradiation, providing one of the possible reasons why they survive in laundry during and even after drying.

Method for identifying heat-resistant fungi of the genus Neosartorya.

Species of the genus Neosartorya are heat-resistant fungi that cause the spoilage of heat-processed acidic foods due to the formation of heat-resistant ascospores, and they produce mycotoxins, such as fumitremorgins and gliotoxin. Their anamorphs are phylogenetically and morphologically very close to Aspergillus fumigatus, which has never been reported as a spoilage agent in heat-processed food products. Therefore it is important to discriminate between the species of Neosartorya and A. fumigatus in the food industry. In the present study, we examined β-tubulin and calmodulin genes to identify Neosartorya and A. fumigatus at the species level and found a region for specifically detecting these species. We succeeded in developing the PCR method of differentiating and identifying Neosartorya and A. fumigatus using specific primer sets. Moreover, we developed specific primer sets to identify Neosartorya species, N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri, and N. spinosa-complex, which are important in food spoilage; these fungi vary in heat resistance and productivity of mycotoxins, depending on the species. PCR using these primer sets did not detect other fungi involved in food spoilage and environmental contamination. These identification methods are rapid and simple with extremely high specificity.

Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp

Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

Genus Enhydrobacter Staley et al. 1987 should be recognized as a member of the family Rhodospirillaceae within the class Alphaproteobacteria

The genus Enhydrobacter, first reported as a member of the family Vibrionaceae, has been placed in the family Moraxellaceae, but as a genus incertae sedis in Bergeys Manual of Systematic Bacteriology 2nd edition. During our taxonomic investigation of Enhydrobacter‐like organisms, we observed that the 16S rRNA sequences of E.   aerosaccus‐type strain versions NCIMB 12535T, ATCC 27094 T and CCUG 58314T were very different from the accessible data (accession no. AJ550856). Phylogenetic analysis of our 16S rRNA sequence data revealed that these organisms were located within the family Rhodospirillaceae. The genera Inquilinus, Oceanibaculum, Skermanella and Nisaea were closely related (sequence similarities were 88.3∼87.0%), but Enhydrobacter could be distinguished from these genera by growth characteristics, fatty acid profiles (C19:0 cyclo ω8c; 38.4% C18:1ω7c; 32.2%, and C16:0; 8.9% were major components), in being non‐flagellated, and differing in enzymatic activities, including trypsin and β‐glucosidase. From these data, we conclude that the genus Enhydrobacter should be recognized as an independent genus of the family Rhodospirillaceae within the class Alphaproteobacteria.

Effects of disinfectants against norovirus virus–like particles predict norovirus inactivation

Human noroviruses (NoVs) are a major cause of epidemic and sporadic acute gastroenteritis worldwide. Public and personal hygiene is one of the most important countermeasures for preventing spread of NoV infection. However, no a practicable cell culture system for NoV had been developed, initial tests of the virucidal effectiveness of anti‐NoV disinfectants and sanitizers have been performed using surrogate viruses. In this study, NoV virus‐like particles (VLPs) were used as a new surrogate for NoVs and a method for evaluating NoV inactivation using them developed. This method is based on morphological changes in VLPs after treatment with sodium hypochlorite. VLP specimens were found to become deformed and degraded in a concentration‐dependent manner. Based on these results, the effects of sodium hypochlorite on VLPs were classified into four phases according to morphological changes and number of particles. Using the criteria thus established, the efficacy of ethanol, carbonates and alkali solutions against VLPs was evaluated. Deformation and aggregation of VLPs were observed after treatment with these disinfectants under specific conditions. To determine the degradation mechanism(s), VLPs were examined by SDS‐PAGE and immunoblotting after treatment with sodium hypochlorite and ethanol. The band corresponding to the major capsid protein, VP1, was not detected after treatment with sodium hypochlorite at concentrations greater than 500 ppm, but remained after treatment with ethanol. These results suggest that VLPs have excellent potential as a surrogate marker for NoVs and can be used in initial virucidal effectiveness tests to determine the mechanism(s) of chemical agents on NoVs.