3.1 Description of strain ZHDP1 and its genomic information
A colony with a clear zone was obtained during the isolation process, indicating the protease activity of this strain named ZHDP1. The sequencing depths were ~230.57X and ~204.67X for the reads from Illumina and Nanopore platforms, respectively. A complete genome of ZHDP1 without any gap was assembled. The size of this genome was 4,917,748 bp and the GC content was 35.95%. A total of 4,478 coding genes was annotated in the genome, and 5 rRNA genes, 26 sRNA genes, and 89 tRNA genes were predicted. Nine gene islands and 1 prophage were found in the genome. Most genes were assigned into “Global and overview maps” (241 genes), “Carbohydrate metabolism” (252 genes), and “Amino acid metabolism” (235 genes) pathways according to the results of KEGG annotation (Figure 1A). A total of 160 and 138 genes were annotated into “Translation, ribosomal structure and biogenesis” and “Amino acid transport and metabolism” COG groups, respectively (Figure 1B). Both of the annotation results showed the genes related to amino acid metabolism were abundant in the genome, indicating that strain ZHDP1 had a developed metabolic system that was used to treat the hydrolysates of proteases, which endows this strain with the potential in the food industry (Neis, Dejon et al. 2015, Lin, Liu et al. 2017). The general characteristics of the genome were listed in Table 1 and Figure 1C.
3.2 Identification of strain ZHDP1 based on the genomic information
Based on the 16S rRNA gene sequences, strain ZHDP1 had a maximum identity of 99.07% and was phylogenetically closed to C. gambrini DSM 18014 (Figure 2). Therefore, strain ZHDP1 was assigned into genus Chryseobacterium. The colony of strain ZHDP1 was circular, orange, and raised, which is similar with the former species (Kämpfer, Dreyer et al. 2003, Park, Jung et al. 2006, del Carmen Montero-Calasanz, Göker et al. 2013). Nevertheless, the maximum values of ANI and DDH of ZHDP1 genome that calculated with other genomes from genus Chryseobacterium were 91.39 and 47.8, respectively, which were lower than the thresholds (ANI < 95%-96%; DDH < 70%) (Goris, Konstantinidis et al. 2007, Yoon, Ha et al. 2017) for a new genome (Table 2).
3.3 Protease genes and activity optimization of strain ZHDP1
A total of 27, 22, 23 protease genes, including the genes of metalloprotease, serine protease, lon protease, thiol protease, tail-specific protease, rhomboid protease, and alkaline protease, were annotated in the genome of this potential new species by using Uniprot, Pfam, and Refseq databases, respectively, indicating that strain ZHDP1 is a novel protein-degrading member in genus Chryseobacterium in addition to previously reported strains. Except for the analysis at genome level, the activity of protein degradation by ZHDP1 was verified and optimized in this study. The factors of temperature, pH, and metal ions could affect the protease activity in the fermentation broth of ZHDP1. The results showed that the optimum temperature and pH were 60°C and 7.0, respectively. Fe3+ could mostly increase the protease activity in the broth. Finally, the maximum activity reached 25.7 U/mL under the optimum conditions (Figure 3). The protease genes annotated in the genome not only are beneficial for the related industries but also are possible participants in glycoprotein link (Parente, Casabuono et al. 2014), immune response (Lad, Yang et al. 2007), biofilm formation (Marr, Overhage et al. 2007), and pathogenesis (Miyoshi, Shinoda 2000). Therefore, the protease genes make strain ZHDP1 become a potential novel member of genus Chryseobacterium with values for industrial production, medical treatment, ecological research, and molecular biology.