Complete Genome Sequence and Characterization of a Protein-Glutaminase Producing Strain, Chryseobacterium proteolyticum QSH1265

Recently, an enzyme named protein-glutaminase (PG) has been identified as a new type of enzyme with significant potential for deamidation of food proteins. The enzyme is shown to be expressed as a pre-pro-protein with a putative signal peptide of 21 amino acids, a pro-sequence of 114 amino acids, and a mature PG of 185 amino acids. The microbial enzyme PG specifically catalyzes deamidation of proteins without protein hydrolysis pretreatment and only reacts with glutamine residues in the side-chains of proteins or long peptides. All these attributes suggest that it has a great potential for food industrial applications. However, until recently, there have been relatively few studies of the PG-producing strains. A strain named Chryseobacterium proteolyticum QSH1265 which can produce PG was isolated from a soil sample collected in Songjiang, Shanghai, China. Its enzyme activity was about 0.34 ± 0.01 U/mL when using carboxybenzoxy-Gln-Gly as a substrate. The strain can produce acid from D-glucose, maltose, L-arabinose sucrose, glycerol, and mannitol but not fructose, and it is also positive for indole production and urease. Here we describe the complete genome sequence of this strain via PacBio RSII sequencing. The C. proteolyticum QSH1265 genome consists of a circular chromosome with total length of 4,849,803 bp without any plasmids. All of 4563 genes were predicted including 4459 genes for protein-coding and 104 RNA-relative genes with an average G+C content of 36.16%. The KEGG and COG annotation provide information for the specific function of proteins encoded in the genome, such as proteases, chromoproteins, stress proteins, antiporters, etc. A highly conserved hypothetical protein shares a promoter with the gene encoding the protein-glutaminase enzyme. The genome sequence and preliminary annotation provide valuable genetic information for further study of C. proteolyticum

Preparation of PGA–PAE-Micelles for Enhanced Antitumor Efficacy of Cisplatin

Poly-γ-l-glutamic acid (PGA) is an outstanding drug carrier candidate owning to its excellent biodegradability and biocompatibility. The PGA carrier may shield toxic drugs from the body and enable the delivery of poorly soluble or unstable drugs and thereby minimize the side effects and improve drug efficacy. However, the limitation of PGA as a drug carrier is low drug loading efficiency (DLE), which is usually below 30%. In this study, we reported a chemical modification method using l-phenylalanine ethyl ester (PAE). PGA–PAE construct was amphiphilic, which could form micelles in aqueous solution. Cisplatin (CDDP), a commonly used chemotherapy drug whose side effect is well-known, was used as a model molecule to test the drug-loading efficiency of PGA–PAE. In this paper, two sizes of CDDP-loaded PGA–PAE micelles (M­(Pt)-1 and M­(Pt)-2) were prepared, the average diameter of M­(Pt)-1 was 106 ± 6 nm and M­(Pt)-2 was 210 ± 9 nm. The DLE of M­(Pt)-1 and M­(Pt)-2 was 52.8 ± 2.2 and 55.8 ± 1.2%, respectively. Both exhibited excellent biocompatibility, stability, and drug-retaining capability in physiological condition. The in vitro accumulative drug-releasing profile, IC₅₀ for different tumor cell lines HeLa, A549, and HCCC9810, and in vivo pharmacokinetics were similar between these two micelles; however, M­(Pt)-1 showed higher tumor tissue retention and longer efficient cancer cell internalization time (up to 20 d). Our results suggested PGA–PAE micelle carriers reduced the toxicity of CDDP and its size at around 100 nm was the better for CDDP high-efficacy