Sequence-Structure Based Comparison of Structurally Homologous Thermophilic and Mesophilic Polyethylene Terephthalate (PET) Hydrolases

Protein structure has a direct impact on thermostability. Deviations in the primary sequence can affect structural changes, leading to alterations in thermostability properties. However, the molecular basis of protein thermostability is unspecified; thus, elucidation of key factors that role particular protein thermostability is required when engineering proteins to be thermostable. To address this challenge, the amino acid composition, hydrophobicity/hydrophilicity ratio, cysteine bridges, and intrinsic features of two structurally homologous but different thermostability, poly(ethylene terephthalate) hydrolase (PETase) were compared. According to the findings, thermostable and thermolabile PETases have similar folds, compactness, and disulfide bridges. Interestingly, an abundance gap of aromaticity, hydrophobic cluster area, polar amino acid and hydrogen bond network compositions demonstrated dominant trends of variations for both PET hydrolases, indicating a pivotal role of these features in the thermostability of PET hydrolase. Furthermore, increased hydrophobic amino acid frequency in the inner surface of thermostable proteins contributed significantly to thermostability by forming more internal hydrophobic interactions and a less hydrophobic patch. There are no consistent trends in insertions and deletions between both PETases. Taken together, these observations demonstrate that hydrophobicity and hydrogen bond networks are essential factors in thermostability of thermostable PETase

Coproduction of alkaline protease and xylanase from genetically modified Indonesian local Bacillus halodurans CM1 using corncob as an inducing substrate

The production of corn generates a substantial amount of agro-industrial waste, with corncob accounting for a significant portion of this waste. In this study, we focused on utilizing corncob as a carbon source and inducer to simultaneously produce two valuable industrial enzymes, protease, and xylanase, using a recombinant strain of B. halodurans CM1. Interestingly, xylan-rich corncob not only enhanced the xylanase activity but also induced protease activity of the modified B. halodurans CM1 strain. The effect of corncob concentration on the coproduction of protease and xylanase was investigated. Corncob with 6 % concentration induced protease activity of 1020.7 U/mL and xylanase activity of 502.8 U/mL in a 7 L bioreactor under the condition of 1 vvm aeration, 250 rpm agitation, 37 °C temperature, initial pH 9.0, and 40 h incubation period. The protease produced was an alkalothermophilic enzyme whose highest activity was at pH 12 and 50 °C, and it belonged to a serine protease family. This alkalothermophilic protease’s activity to some degree was reduced by Co2+, Mg2+, Fe2+, Zn2+, and K+, but enhanced by Ca2+ and Ni2+ (at 5 mM). The protease was stable even under the presence of a 15 % concentration of acetone, DMSO, ethanol, and isopropyl alcohol. The protease activity at 30 °C was not considerably changed by the presence of detergent, indicating excellent potential as a washing detergent additive. According to these findings, corncob has the potential to be a substrate for the coproduction of protease and xylanase, which have a wide range of industrial uses