Glycerol

Glucose, xylose, arabinose, sucrose, lactose, and glycerol were supplied from Bio Basic Canada Inc. Furfural, 5-hydroxymethyl Furfural (HMF), and 3,5-dinitrosalycilic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agroindustrial by-products (wheat bran and sugarcane bagasse) were kindly provided by a local food processing industry (White-rose group, Sfax, Tunisia). Almond shell, date palm leaves, and Posidonia oceanica balls were collected locally. All lignocellulosic residues are conserved at 4°C until use.
The new oleaginous yeast strain Y-MG1, isolated from rotten fruit, was identified in our previous work based on its internal transcribed spacer (ITS) sequence [21 (link)]. Y-MG1 strain was identified as being Rhodotorula mucilaginosa and was submitted in the National Strains Collection of Centre of Biotechnology of Sfax, CBS, Tunisia, under the accession number CTM-30138. The ITS sequence of Y-MG1 was also submitted to GenBank under the accession number ID: KX347596.1. This strain was stored at −80°C in sterilized glycerol-enriched solution containing 2% (w/v) Glucose, 1% (w/v) yeast extract, 1% (w/v) bacto-peptone, and 20% (w/w) glycerol.

All chemicals procured were either analytical or laboratory grade and were used as received without further purification. Reagent and stock/standard solutions were prepared in deionized water (pH 7.0) purified by Barnstead Water Purification Systems (Thermo Scientific, Waltham, MA, USA). NaCl and sodium carbonate were purchased from Junsei (Japan). Standard PAs, Put, Spd, and Spm were obtained from Sigma-Aldrich, Saint Louis, MO, USA. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid, phenol, chloroform, trimethylamine, perchloric acid, and most other chemicals were obtained from Sigma-Aldrich, Saint Louis, MO, USA. Glycerol and lactic acid were purchased from Biobasic (Canada).

β-lactamase activity was assessed spectrophotometrically by measuring the hydrolysis of nitrocefin as previously described with little modification [47 (link)]. Briefly, the reaction mixture consisted of 83 μg of nitrocefin (EMD Millipore Corp., Burlington, MA, USA), 10% glycerol (Bio Basic Inc., Ontario, Canada), 167 mg of bovine serum albumin (Sigma-Aldrich Corp, Burlington, MA, USA), and 0.02 mg/mL of β-lactamase obtained from Bacillus cereus 569H (EMD Millipore Corp., Burlington, MA, USA). The activity of β-lactamase was determined by kinetically measuring the absorbance at 490 nm for 60 sec using UV-Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan). To evaluate the inhibitory effects, inhibitors including tBLIP-II, tBLIP-II-CPP, and CPP-tBLIP-II were pre-incubated with the assay mixture without nitrocefin for 15 min at 30°C before adding nitrocefin as the substrate. The concentrations of the inhibitors ranged from 0.0078 μM to 2 μM. The IC₅₀ values were determined through direct competition between nitrocefin and protein inhibitors under the aforementioned conditions. The IC₅₀ values were obtained by plotting the percent residual enzyme activity on nitrocefin against the inhibitor concentration. The IC₅₀ was defined as the concentration of the inhibitor that inhibited the hydrolytic activity of the enzyme by 50%.