The hypothesis was that drying method would affect the solidification of latex laticifer and the entrapment of lipase. Using freeze-drying to obtain papaya latex powder could improve the lipase isolation. The zwitterion sodium lauroyl sarcosinate was expected as an effective agent in latex solubility to produce protein fractions with high lipase activity.

Sodium lauroyl sarcosinate (purity 97%), p-nitrophenyl palmitate (purity 98%), p-nitrophenol (purity 99%), NaCl (purity 99.5%), n-hexane, 2-propanol (purity 99.8%) and ammonium sulfate (purity 99%) were purchased from Sigma–Aldrich, Singapore; Tris base (purity 99.9%) and Triton X-100 were provided by Bio Basic, Canada. All other chemicals used were of analytical grade.

Papaya latex was collected between 6 am and 8 am from unripe fruits grown in Quang Nam province, Vietnam. The 4 – 6 incisions were made along with the fruits by stainless steel knife and the latex was let drain into plastic bottles and stored in cold box with ice and frozen within 4 hours for freeze-drying. The CPL powder was obtained after grinding of dried matter. The latex was also sun dried and dried in an oven at 40 °C until constant weight and then ground into CPL powders. These powders were compared for their specific lipase activity and the most active powder was used for other experiments.

The 3 grams of CPL powder were weighed into 100 mL of distilled water and mixed for 3 minutes. The solution was centrifuged at 6000 rpm at 4 °C in 20 minutes using Hettich universal 320R centrifuge (Germany). The supernatant was disposed, and the precipitate was collected. These steps were repeated for 3 times and the final precipitate was freeze-dried in Alpha 1-2 LDplus freeze dryer (Germany) at -43 °C, 24 h to obtain crude CPL lipase.

One gram of crude CPL lipase was suspended in the 50 mL of the mixture of n-hexane and 2-propanol (1:1 v/v) and shaken in 30 minutes at 4 °C to extract lipid from the latex. It was then centrifuged at 7500 rpm in 20 minutes. The solvent residue was eliminated from the precipitate in vacuum rotary evaporator to obtain the lipid-free CPL lipase. This residue was then suspended into 50 mL solution of Tris–HCl 0.1M pH 8, prior mixed with the 0.5% (w/v) sodium lauroyl sarcosinate. The suspension was shaken for 30 minutes and then centrifuged at 7500 rpm in 20 minutes at 4 °C to obtain both supernatant and precipitate. These 2 fractions were used in other experiments.

Protein in supernatant collected after solubilizing crude CPL lipase in SLS was fractionally precipitated by using ammonium sulfate crystal. The precipitation was separated by ammonium sulphate saturation in the range of 0 – 90% (v/v). Precipitation was allowed to occur at 4 °C for 1 h and followed by centrifugation at 7500 rpm for 20 mins to obtain protein precipitate. All of the precipitate samples were dissolved in the solution of Tris HCl 0.1M, pH 8 and lipase activity was measured (Burgess, 2009). The fraction with the highest activity was subjected to dialysis using cellophane to eliminate salts at 4 °C for 48h. The obtained solution was used for chromatographic and electrophoretic analysis.

Samples after dialysis were loaded on the HiTrap Q Sepharose Fast flow column (5.0 mL, 1.6 cm × 2.5 cm) of the flash protein liquid chromatography system (GE Akta Purifier 100, Sweden). The column was firstly equilibrated with starting buffer of Tris-HCl pH 9. The sample was then eluted with an increase of NaCl concentration from 0 to 1 M at rate 0.1 M NaCl.min_-1. The flow rate was set at 1 mL.min_-1. The eluent was measured for absorbance at 280 nm and collected in fraction tubes of 1 mL volume.

The molecular mass of the purified lipase was determined by SDS-PAGE as described by Minaev and Makhova (2019) using 15% acrylamide gel, prestained dual colour protein molecular weight marker (10 – 170 kDa).

The Lineweaver–Burk plots were used to determine the Michaelis–Menten constant (K_m) and the maximum velocity for the reaction (V_max) of lipase for p-nitrophenyl palmitate at pH 8.0, using a spectrophotometric method. Briefly, assays with lipases were performed in 0.1M Tris–HCl buffer, pH 8.0 at 40 °C with increasing concentrations of p-nitrophenyl palmitate from 0.397 mM to 3.973 mM, to calculate K_m and V_max (Holme and Peck, 1998).

Lipase activity was measured spectrophotometrically using an assay based on the enzymatic hydrolysis of p-nitrophenyl palmitate (pNPP) to form p-nitrophenol (pNP) whose absorbance was measured at 410 nm (Jenway 6305, UK) (Palacios et al., 2014). One activity unit (U) of lipase was defined as 1 μmol p-nitrophenol produced per minute under the assay condition.

Briefly, solution A containing 30 mg pNPP in 10 mL propan-2-ol and solution B containing 180 mL Tris-HCl buffer (0.1 M; pH 8.0), 720 μL Triton X-100 and 180 mg gum Arabic were prepared. The mixture of 4860 μL of solution B and 540 μL of solution A were incubated at 40 °C, then added with 0.0015 g enzyme for the reaction to take place. The absorbance was measured spectrophotometrically at 410 nm after 10 minutes and the amount of hydrolysed p-nitrophenol was determined from the standard curve prior built with pNP standard.

Proximate composition of papaya latex was analysed using appropriate methods: AOAC 927.05 for moisture, AOAC 425.06 for ash, AOAC 2011.04 for protein and AOAC 948.15. for lipid.

Experiments were done in triplicates and the results were expressed as mean values. ANOVA and the Duncan Test was applied to assess significant difference at a significant level of α 0.05 using software Minitab 16.0.

Papaya latex collected from the fruit was analysed for its proximate composition. The results were shown in Table 1. The fresh latex contained a high amount of water, protein and low lipid. High protein content could be linked to the presence of various enzymes and the high water content may explain for its flowability. This result was in accordance with Macalood et al. (2013).

However, the papaya latex tends to solidify quickly in the air, and this may entrap lipase and affect the lipolytic activity of the latex. In this study, the papaya latex was dried in 3 different methods until its constant mass as described in 2.2.1. The dried latex was ground to powder and measured for their lipolytic activity. The results were presented in Table 2.

The results showed that long time of air exposure at high temperature had a significant negative impact on the lipase activity (p <0.001). The freeze-drying gave the highest quality powder, which achieved four-time higher of specific activity than those from sun-drying or Oven-drying. Freeze-drying was also confirmed to be a good method for preserving activity of lipase from Yarrowia lipotica (Darvishi et al., 2012).