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Alcohol oxidase (AOX) oxidizes alcohols to produce carbonyl compounds and peroxides. Its promoter (PAOX1) is widely used in methylotrophic yeasts. A promising yeast expression system (Pichia sp. strain SO) was developed for bacterial lipase expression regulated by PAOX1 of Komagataella phaffii (previously known as Pichia pastoris). However, its unidentified AOX gene and the protein structure have deterred the search for the best inducer. This study was aimed to identify the yeast species and determine the best inducer for PAOX1 upregulation using in silico AOX protein analysis. Morphological (scanning and transmission electron microscopies) and carbon assimilation analyses confirmed isolate SO as Meyerozyma guilliermondii (previously known as Pichia guilliiermondii). Using Hidden-Markov model and degenerate PCR, the LCAO gene (2091 bp) was discovered in M. guilliermondii strain SO. The enzyme, MgFAO1 shared 14% similarity to K. phaffii AOX1 protein (KpAOX1). Molecular docking of MgFAO1 three-dimensional structure predicted using AlphaFold2 showed its preference toward long-chain 1-dodecanol as the substrate unlike KpAOX1 (short-chain methanol). While the alcohol-binding pocket in MgFAO1 was more hydrophobic compared to KpAOX1, 1-dodecanol could be a better inducer for protein expression in M. guilliermondii strain SO. Thus, in silico pipeline employed in this study can help identify homologous proteins in other expression hosts and their preferred substrates for promoter upregulation. However, the computational analyses were merely predictions and further wet-lab validation is required. Yet, this strategy allows cost-efficient screening of potential inducers for microbe-based protein production in the industries, reducing the production cost and offering cheaper options for consumers. [ABSTRACT FROM AUTHOR]

Industrial and sand mining activities have severely degraded the water quality of the Kelantan River, as well as the river ecosystem. However, there have been inadequate studies on the effect of industrial and sand mining on organisms in the Kelantan River. Therefore, this study assessed the concentrations of Pb, Cd, Zn and Cu in the bivalves of genus (Polymesoda expansa) marsh clams from the Kelantan River tributaries, namely the Geting River and the Semarak River, which are located near to an industrial and a sand mining area, respectively. More than 50 marsh clams were collected at these sites. Heavy metals in marsh clams were extracted using aqua regia methods and analyzed using a Flame Atomic Absorption Spectrometer (FAAS). The mean concentrations of Pb, Cd, Zn and Cu at the Semarak River were 4.61, 2.16, 194 and 6.20 mg/kg, respectively. Accordingly, the concentrations at the Geting River were 4.43, 2.50, 196 and 7.90 mg/kg, respectively. The pattern of metal concentrations within the whole tissue of marsh clams from the Geting River and the Semarak River were in the following order: Zn > Cu > Pb > Cd (concentration in mg/kg). The mean concentration of selected metals (Zn, Cd, Pb) in the studied samples exceeded the permissible limit set by the Malaysia Food Safety and Food Regulation 1985, the Food and Agricultural Organization, the World Health Organization, the Ministry of Health Thailand, and the Food and Drug Administration of United States (USFDA). Therefore, continuous monitoring of these bioindicators is essential to minimize the increasing health risk for aquatic life, as well as in food sources for human consumption, in the Kelantan River Basin. [ABSTRACT FROM AUTHOR]