Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29.

The environmental threat posed by disposal of plastic wastes has drawn extensive attention in recent years wherein polyethylene terephthalate (PET) constitutes one of the major plastic materials in the wastes. Recycling of PET wastes into reusable materials effectively overcomes its accumulation in the environment and can be achieved by mechanical, chemical, and biological processes. In comparison to the other methods, enzymatic treatment utilizing PET hydrolyzing enzymes (PETases) is environmental-friendly which avoids the use of hazardous chemicals. In this study, we report on the secretory expression in Bacillus subtilis a PETase (BhrPETase) from the bacterium HR29, a close homologue of the leaf-branch compost cutinase (LCC) with 94 % sequence identity. The expression titer of BhrPETase reached 0.66 g/L in an engineered chaperone-overexpression Bacillus subtilis strain, and the biochemical characterization of BhrPETase for the first time revealed its high hydrolyzing activity towards amorphous PET in comparison to two reported PET hydrolyzing enzymes LCC and IsPETase, which were expressed under the same expression conditions in Bacillus subtilis in our study. Most intriguingly, purified BhrPETase displayed a melting temperature as high as 101 °C. To our knowledge it is the most thermostable bacterial PETase characterized so far. The superior activity and thermostability of BhrPETase rendered it one of the most promising PETases for plastic waste recycling and bioremediation applications in the future.

Co-expression of Pseudomonas alcaligenes lipase and its specific foldase in Pichia pastoris by a dual expression cassette strategy.

Lipomax is a commercialized foldase-dependent Pseudomonas lipase that was previously expressed only in Pseudomonas strains. Here, using Pichia pastoris as the host, we report a new co-expression method that leads to the successful production of Lipomax. The active Lipomax is extracellularly co-expressed with its cognate foldase (LIM); and the purified enzyme mix has the optimum pH at pH 8.0 and an optimal temperature around 40 °C. N-glycosylation was observed for Pichia produced Lipomax, and its reduction was shown to increase the lipolytic activity. With different p-nitrophenyl esters as the substrates, the substrate profiling analyses further indicate that Lipomax prefers esters with middle-long chain fatty acids, showing the highest specific activity to p-nitrophenyl caprylate (C8). The extracellular co-expression of Lipomax and LIM in Pichia will not only increase our ability to investigate additional eukaryotic hosts for lipase expression, but also be of considerable value in analyzing other foldase-dependent lipases.

Identification and Characterization of a New Alkaline Thermolysin-Like Protease, BtsTLP1, from Bacillus thuringiensis Serovar Sichuansis Strain MC28.

Thermolysin and its homologs are a group of metalloproteases that have been widely used in both therapeutic and biotechnological applications. We here report the identification and characterization of a novel thermolysin-like protease, BtsTLP1, from insect pathogen Bacillus thuringiensis serovar Sichuansis strain MC28. BtsTLP1 is extracellularly produced in Bacillus subtilis, and the active protein was purified via successive chromatographic steps. The mature form of BtsTLP1 has a molecule mass of 35.6 kDa as determined by mass spectrometry analyses. The biochemical characterization indicates that BtsTLP1 has an apparent Km value of 1.57 mg/ml for azocasein and is active between 20°C and 80°C. Unlike other reported neutral gram-positive thermolysin homologs with optimal pH around 7, BtsTLP1 exhibits an alkaline pH optimum around 10. The activity of BtsTLP1 is strongly inhibited by EDTA and a group of specific divalent ions, with Zn(2+) and Cu(2+) showing particular effects in promoting the enzyme autolysis. Furthermore, our data also indicate that BtsTLP1 has potential in cleaning applications.

Preparation of monomeric B27 Lys destripeptide insulin by intein mediated expression in Escherichia coli.

The B27K-DTrI insulin (human insulin with B28-30 removed and B27 Thr replaced by Lys) was reported to have superior monomeric property with 80% insulin activity in vivo. It has potential use as a new fast-acting analog of insulin. We cloned the monomeric insulin B27 DTrI precursor (MIP) into the pTWIN1 vector, and prepared by intein mediated expression in Escherichia coli. After tryptic digestion, the MIP was converted to B27K-DTrI insulin. The product was purified by HPLC. The mass spectrometry showed that the molecular mass of purified B27K-DTrI was consistent with the theoretical value.