Search

Abstract Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

Kelulut honey was dehydrated at 40, 55, and 70°C up to 84 h in a dehydrator. The changes of its properties and qualities in terms of moisture content, water activity, hygroscopicity, moisture adsorption isotherm, colour intensity, total phenolic content (TPC), viscosity, glass transition temperature (Tg), surface stickiness, hydroxymethylfurfural (HMF) content, and diastase activity were evaluated. The dehydration process for 18 h between temperatures of 55 and 70°C can safely produce Kelulut honey product with less than 8% moisture content and water activity below 0.6. Similar quality of Kelulut honey dehydrated at lower temperature between 40 and 55°C requires up to 36 h of dehydration. These recommended dehydration conditions were able to increase TPC of honey from 7.86% from its original value for the shorter duration of 18 h and lower dehydration temperature of 40°C and up to 70.9% for the longer duration of 36 h and higher temperature of 70°C. Dehydrated honey was darker, more viscous, and stickier. The increase of HMF content in dehydrated honey at 40 and 55°C up to 36 h was not significant which are at 0 and 5.81 mg/kg honey, respectively, and at 70°C, it was about 80 mg/kg honey. The honey was found to have very low diastase activity ranging from 0 to 0.75 DN, thereby causing its changes to be insignificant during dehydration.