Extraction and identification of ananain-like protease from pineapple crown and stem

Abstract

Pineapple (Ananas comosus) is the major tropical fruits and belongs to the family Bromeliaceae. The pineapple waste from pineapple industry has contributed to an increase of waste in Malaysia and around the world every year. Hence, there is a demand need to study the utilization of pineapple wastes such as pulp, peel, core, leaves and crown for industrial applications instead of disposing them, which may result in the loss of important biomass and nutrients. The pineapple contains endopeptidase enzymes that constitute the major components of bromelain extract which are stem bromelain, fruit bromelain, ananain and comasain. This study was conducted to extract and purify protease from crown and stem of MD2 pineapple. Protease was extracted and purified using anion exchange chromatography (IEX), gel filtration, desalting method before being identified using LC/MS. Proteolytic activity was determined using Casein Digestion Unit (CDU) and well diffusion method, whereas fibrinolytic activity was determined using fibrin suspension. In the present study, proteolytic assay showed 1 kg crown of MD2 cultivar produced the activity of 126.0 ± 3.86 U/mL, the specific activity of 3937.50 U/mg and total activity of 3.94 x 109 U. In another assessment, 1 kg stem from MD2 cultivar showed the proteolytic activity, specific activity and total activity of 118.5 ± 1.19 U/mg, 5925 U/mg and 5.925 x 109 U, respectively. The proteolytic activity and fibrinolytic activity of purified enzyme from the crown extract were 144.02 U/mL and 0.51 x 10"6 U/mL, respectively. The molecular weight of the purified enzyme was in the range of 25 to 35 kDa at the optimum condition of pH 7 at 37 °C. Meanwhile, a purified enzyme from stem extract was observed to contain high proteolytic activity and fibrinolytic activity which were 20.76 U/mL and 0.000272 U/mL, respectively. Purification of the extract yielded a band at molecular weight of between 20-25 kDa at the optimum conditions of pH 3 and 9 at 60 °C. From LC/MS analysis, the purified enzyme from the crown extract was similar to ananain under accession number A0A199VSS3 (according to Uniprot). It had seven unique peptides and covered 164/356 amino acids (44.9 percent coverage). The ananain (EC 3.4.22.31) is classified in the subfamilies of cysteine protease C1A (clan CA, family C1) that is peptidase family related to papain. In conclusion, protease was successfully extracted and identified as ananain-like protease from crown. However, protease extracted from the stem of MD2 cultivar was not determined. Therefore, further study on this enzyme needs to be explored in the near future.