Yongjun Xia

Common Non-classically Secreted Bacterial Proteins with Experimental Evidence.

The non-classically secreted proteins are the proteins that are located in the extracellular milieu despite the absence of known signal peptides or other targeting peptides. Numerous experimental studies have discovered many non-classically secreted proteins, but there are many inconsistencies in these reports even on a specific organism. In order to reduce the impact of artefactual contamination of protein samples and cell lysis, this review listed 45 common non-classically secreted proteins. The list of non-classically secreted proteins was complied from studies carried out by different groups in various bacterial species. The complied list can provide a reliable experimental material for further research on non-classical protein secretion. It is noticeable that some of these proteins are hyper-secreted when expressed in specific species or under some induction conditions. So we also discussed the possibilities of using the non-classical secretion pathway for the production of recombinant proteins in the review.

A new potential secretion pathway for recombinant proteins in Bacillus subtilis

BackgroundSecretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable ‘cell factory’ for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium.ResultsAll four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides.ConclusionOur results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (k(cat)/K(m)). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.

Bioactive exopolysaccharides from a S. thermophilus strain: Screening, purification and characterization.

A lactic acid bacteria strain AR333 with high-production of exopolysaccharides (EPS) was screened out from 350 bacteria strains isolated from naturally fermented dairy products. It was identified as Streptococcus thermophilus by morphological observation, physiological and biochemical characteristics, and 16S rDNA sequence determination. The EPS from AR333 were purified through DEAE-Sepharose Fast Flow and Sepharose CL-6B, and the purified fraction was designated as EPS333. In vitro test showed that EPS333 could stimulate macrophage RAW 264.7 to release NO significantly (p<0.05). The further work tried to elucidate the structural features of EPS333 via FT-IR spectrum, high-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography (HPSEC). The results showed that EPS333 was a pure neutral polysaccharide with monosaccharide composition of galactose and glucose in a molar ratio of 6:5. A certain amount of acetyl groups might exist in EPS333 according to the FT-IR spectrum. The molecular weight (Mw) was estimated to be 332 kDa. Current study suggested that the lactic bacteria strain AR333 could be a potential source of immunoregulatory polysaccharide.

Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression

Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.

Use of a Novel Report Protein to Study the Secretion Signal of Flagellin in Bacillus subtilis.

Flagellin (also called Hag) is the main component of bacterial flagellum and is transported across the cytoplasmic membrane by flagellar secretion apparatus. Because flagella play an essential role in the pathogenesis of numerous pathogens, the flagellins of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Campylobacter jejuni, and Vibrio cholerae have been intensively studied; however, very few studies have focused on the flagellin of Bacillus subtilis, which is considered to be a model organism with which to study the secretion of bacteria and is used on an industrial scale for the secretion of proteins. The signal of B. subtilis flagellin is still debated. This study was performed to seek the export signals of flagellin from B. subtilis. The naturally nonsecretory, intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was used as the reporter protein. Our results demonstrate that the export signal is contained within the first 50 amino acids of B. subtilis flagellin. Nsp is easily degraded inside the cell and can be exported into culture medium with the aid of the signal of flagellin. This method provides a new potential strategy for the expression of proteins with high proteolytic susceptibility via fusion to export signals.

Comparison of oenological property, volatile profile, and sensory characteristic of Chinese rice wine fermented by different starters during brewing

ABSTRACT Oenological properties, volatile profiles, and sensory characteristics between Chinese Hong Qu rice wine (HQW) and Chinese Wheat Qu rice wine (WQW) were investigated. Forty and thirty-five volatile compounds in HQW and WQW were, respectively, identified. According to the door activity values (OAVs > 1), isoamyl acetate, ethyl octanoate, ethyl tetradecanoate, nonanal, and 2-methoxy-phenol were the major flavour contributors for HQW and WQW. Principal component analysis (PCA) showed that middle-chain fatty-acid (C6-C12) ethyl esters were the dominant representative flavour in HQW and WQW. Sensory evaluation indicated that the aroma of esters and alcohols differed largely between HQW and WQW. By partial least squares regression (PLSR), middle-chain fatty-acid-ethyl-esters (FAEEs) had a strong impact on the sensory attributes of fruit aroma, continuation, and full body in HQW and WQW.