Screening and Characterization of the Alkaline Protease Isolated from PLI-1, a Strain of Brevibacillus sp. Collected from Indonesia's Hot Springs

A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia's hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70℃ and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70℃ and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.

Effect of thermophilic bacterium HB27 manganese superoxide dismutase in a rat model of chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS)

We investigated the therapeutic effects of superoxide dismutase(SOD)from thermophilic bacterium HB27 on chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS)and its underlying mechanisms.A Sprague–Dawley rat model of CP/CPPS was prepared and then administered saline or Thermus thermophilic(Tt)-SOD intragastrically for 4 weeks.Prostate inflammation and fibrosis were analyzed by hematoxylin and eosin staining,and Masson staining.Alanine transaminase(ALT),aspartate transaminase(AST),serum creatinine(CR),and blood urea nitrogen(BUN)levels were assayed for all animals.Enzyme-linked immunosorbent assays(ELISA)were performed to analyze serum cytokine concentrations and tissue levels of malondialdehyde,nitric oxide,SOD,catalase,and glutathione peroxidase.Reactive oxygen species levels were detected using dichlorofluorescein diacetate.The messenger ribonucleic acid(mRNA)expression of tissue cytokines was analyzed by reverse transcription polymerase chain reaction(RT-PCR),and infiltrating inflammatory cells were examined using immunohistochemistry.Nuclear factor-κB(NF-κB)P65,P38,and inhibitor of nuclear factor-κBα(I-κBα)protein levels were determined using western blot.Tt-SOD significantly improved histopathological changes in CP/CPPS,reduced inflammatory cell infiltration and fibrosis,increased pain threshold,and reduced the prostate index.Tt-SOD treatment showed no significant effect on ALT,AST,CR,or BUN levels.Furthermore,Tt-SOD reduced inflammatory cytokine expression in prostate tissue and increased antioxidant capacity.This anti-inflammatory activity correlated with decreases in the abundance of cluster of differentiation 3(CD3),cluster of differentiation 45(CD45),and macrophage inflammatory protein 1α(MIP1α)cells.Tt-SOD alleviated inflammation and oxidative stress by reducing NF-κB P65 and P38 protein levels and increasing I-κBαprotein levels.These findings support Tt-SOD as a potential drug for CP/CPPS.