Junzhong Liu

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.

Isolation and characterization of marine Brevibacillus sp. S-1 collected from South China Sea and a novel antitumor peptide produced by the strain.

A Gram-positive, rod-shaped bacterium, designated as S-1, was isolated from a marine sediment sample collected from South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence showed that S-1 belongs to the genus Brevibacillus. A novel cytotoxic peptide was isolated from the fermentation broth of the marine-derived bacterium Brevibacillus sp. S-1, using ion-exchange chromatography and reverse-phase HPLC chromatography. The molecular weight of this peptide was determined as 1570 Da by MALDI-TOF mass spectrometry, and its structure was proposed as a cyclic peptide elucidated by MALDI-TOF/TOF mass spectrometry and de novo sequencing. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that this peptide exhibited cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, U251 human glioma cells and MCF-7 human breast carcinoma cells. Additionally, SBP exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells. The result suggested that the cytotoxic effect of the peptide is specific to tumor cells.

Identification of a Marine Bacillus Strain C5 and Parathion-Methyl Degradation Characteristics of the Extracellular Esterase B1

A bacterial strain C5 that can produce new type of marine esterase was isolated and screened from marine sludge. According to 16S rRNA sequence analysis and physiological and biochemical experiments, the strain was identified as Bacillus subtilis. A single isozyme with a molecular weight of 86 kDa was observed by SDS-PAGE and native-PAGE. On this basis, the mechanism of esterase B1 secreted by strain C5 degrading parathion-methyl was explored, and the effects of temperature and pH on the degradation rate were investigated. From the results, p-nitrophenol was one of the degradation products of B1 degrading parathion-methyl, and the best degradation effect could be achieved at the temperature of 40°C and the neutral pH value.

Hymenobacter profundi sp. nov., isolated from deep-sea water

A Gram-stain-negative, rod-shaped, red-pigmented, aerobic bacterium, strain M2T, was isolated from a seawater sample collected from the western Pacific Ocean at a depth of 1000 m and characterized using polyphasic taxonomy. Strain M2T was catalase-positive and oxidase-negative. Cells grew at 4-33 °C (optimum, 25 °C), at pH 6-9 (optimum, 7) and with 0-4 % (w/v) (optimum, 1 %) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain M2T was associated with the genus Hymenobacter. Strain M2T showed the highest 16S rRNA gene sequence similarities to Hymenobacter actinosclerus CCUG 39621T (95.7 %), Hymenobacter tibetensis XTM003T (95.6 %) and Hymenobacter psychrotolerans Tibet-IIU11T (95.2 %). The DNA G+C content was 59.98 mol%. Strain M2T contained C16 : 1ω5c (25.0 %), iso-C15 : 0 (23.9 %) and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c, 20.4 %) as major cellular fatty acids. The major quinone of strain M2T was menaquinone 7 and the major polar lipid was phosphatidylethanolamine. The major polyamine of strain M2T was sym-homospermidine. The phylogenetic analysis and physiological and biochemical data showed that strain M2T should be classified as representing a novel species of the genus Hymenobacter, for which the name Hymenobacter profundi sp. nov. is proposed. The type strain is M2T (=CCTCC AB 2017185T=KCTC 62120T).

Pseudomonas profundi sp. nov., isolated from deep-sea water

A Gram-stain-negative, aerobic bacterium, strain M5T, was isolated from a seawater sample collected from the western Pacific Ocean at a depth of 1000 m and characterized by using polyphasic taxonomy. Cells of the strain were rod-shaped and motile by a single polar flagellum. Cells grew at 4-40 °C (optimum, 25 °C), at pH 7-10 (optimum, 9) and with 0-10 % NaCl (optimum, 1-2 %). Phylogenetic trees based on 16S rRNA gene sequences showed that strain M5T was associated with the genus Pseudomonas, and showed highest similarities to Pseudomonas pelagia CL-AP6T (97.8 %) and Pseudomonas salina XCD-X85T (97.5 %) and Pseudomonas sabulinigri J64T (96.4 %). The average nucleotide identity scores for strains CL-AP6T and XCD-X85T were 74.6 % and 73.7 %, the Genome-to-Genome Distance Calculator scores were 15.8-19.5 % and 15.4-19.7 %, and the species identification scores were 92.3 % and 92.4 %. The major isoprenoid quinone of strain M5T was ubiquinone (Q-9) and the major cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 33.2 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 22.8 %) and C16 : 0 (13 %). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipid and some unidentified lipids. The phylogenetic analysis and physiological and biochemical data showed that strain M5T should be classified as representing a novel species in the genus Pseudomonas, for which the name Pseudomonas profundi sp. nov. is proposed. The type strain is M5T (=CCTCC AB 2017186T=KCTC 62119T=CICC 24308T).

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium Alteromonas sp. L82

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni2+ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a Km of 0.55 mM, a Vmax of 83.6 U/mg, a kcat of 74.3 s-1 and kcat/Km of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.