First report on Chlorella vulgaris collagenase production and purification by aqueous two-phase system

Highlights

• First report on microalgae true collagenase production and purification.

• Chlorella vulgaris was cultivated in autotrophic and mixotrophic conditions.

• A 2⁴-full factorial design established the best purification conditions by ATPS.

• A purification factor of 10.79 was obtained for a partition coefficient of 23.74

• Optimum activity was observed under pH 9 and 37 °C, was inhibited by EDTA.

Abstract

Collagen is triple helical structured protein, which is undegradable by most proteases and reacts only with collagenases. These enzymes are applied and important in several commercial segments and the interest for new collagenases has only been increasing. Microalgae are not a very prominent source of enzymes but are extremely commercially attractive forms of life due to photosynthetic growth. Yet, the literature is absent on reports about algae collagenases and this work is the first report on Chlorella vulgaris collagenolytic enzyme production, purification and characterization. C. vulgaris was cultivated in autotrophic and mixotrophic conditions and the biomass used to obtain the cell extract. The autotrophic extract displayed a higher collagenolytic activity and was used for ATPS experimental design. Through 2⁴-full factorial design, high collagenolytic activity was observed at pH 8.0, PEG molar mass of 1500 g/mol, PEG concentration of 12.5% (w/w) and phosphate buffer concentration of 15% (w/w). In these conditions, a purification factor of 10.79 was obtained for a partition coefficient of 23.74 with the collagenase retained in top PEG rich phase. Purified collagenase optimum activity was observed under pH 9 and 37 °C, displayed less gelatinolytic and caseinolytic activity, with higher collagenolytic activity with azocoll than crude extract, attesting this enzyme is a true collagenase. The collagenolytic activity was inhibited by EDTA, an indicative of a metallocollagenase.

Introduction

Collagen is triple helical structured protein, found in the extracellular matrices of animals and represents 30% of the total proteins in mammals. Because of its extensive distribution in the earth's animal kingdom, is an important nitrogen source in global nitrogen cycle. Due to its rigid structure, native collagen is undegradable by most proteases and reacts only by few types, the collagenases (Bhattacharya et al., 2019). The importance of collagenolytic enzymes is visible in medical, pharmaceuticals, food, cosmetics and textiles segments. Given its potential and high demand, the interest for new collagenases has only been increasing (Wanderley et al., 2016).

In addition to the various chemical and chemical properties (Daboor, 2012), collagenases have been widely used in human - and veterinary medicine as well - with the aim of cleaning necrotic wounds, bedsores, postoperative healing, psoriasis and pediculosis treatment, implants and injuries. wounds, nipple injuries of lactating women, treatment of hypertrophic scarring, treatment of heart ischemia, ulcer debridement of diabetic people (Takahashi et al., 1999; Watanabe, 2004; Arakawa et al., 2012; Tallis et al., 2013), preparation for diagnostic tests (Lima et al., 2013), production of peptides with antioxidant, antimicrobial (Lima et al., 2014) and antiproliferative activities (Bu et al., 2017).

Up to now, microalgae was not very prominent potential source of enzymes. This group represents extremely commercially attractive forms of life, presenting potential exploitation as commercial sources of a wide range of biomolecules. Their importance has been increasing in cosmeceuticals, nutraceuticals, functional foods, pharmaceuticals, medicine and biofuels fields. Commercial microalgae production have been growing in the last few decades and they have already been established as commercial sources of high-value products such as β-carotene, astaxanthin, phycobilin pigments and algal extracts for use in cosmetics (Borowitzka, 2013; Sathasivam et al., 2019).

There is a rising interest in using algae also as a source of commercial enzymes, due to low-cost production, their ability to convert solar energy to chemical energy by fixing CO₂, to adapt to adverse conditions and their relatively simple genetic background, allowing genetic manipulation. Several enzymes have been isolated from algae, as Carbonic anhydrase, Hydrogenase, Laccase, β-Lactamase, Nitrogenase, Superoxide dismutase (Mogharabi et al., 2017; Sathasivam et al., 2019). However, very few reports on microalgae protease purification and characterization are found in literature. The reported microalgae proteases show high activities in the neutral and alkaline ranges, differing from most organisms and higher plants proteases (Homaei et al., 2016).

Recently, biotechnological application is focused on four major groups of microalgae: Spirulina (Arthrospira), Chlorella vulgaris, Dunaliella salina, and Haematococcus pluvialis. Chlorella species are characterized as single-cell, spherical shaped and photoautotrophic green microalgae without flagella. Chlorella multiplies rapidly requiring only CO₂, water, sunlight, and a small amount of minerals. This makes the cultivation and production of biocompounds cheaper when compared to other organisms such as fish, fungi or bacteria. Besides not presenting biological risk. (Mobin and Alam, 2017). It has be stated that Chlorella vulgaris approved by the U.S. Food and Drug Administration (FDA) as GRAS (Generally Recognized as Safe), is one of the most interesting and abundant microalgae species, which extracts promotes collagen synthesis in skin, tissue regeneration, also have been reported to make soft and flexible both skin and hair (Ariede et al., 2017; de Melo et al., 2019). Various compounds could be extracted from Chlorella biomass and used as antioxidants, antimicrobials, antivirals, food supplements, for biofuels and bioremediation, among others (Silva et al., 2018). Yet, the literature is scant on reports about algae collagenases and there are no reports on Chlorella vulgaris collagenolytic protease purification and characterization.

Aqueous two-phase system (ATPS) has been widely used to purify enzymes quickly and cheaply (Wanderley et al., 2017). The choice polymer−salt ATPS's is mainly driven by the recovery yield and selectivity for the target solute and price of the ATPS constituents (Silvério et al., 2012). The choice of variables was based on previous authors who had already applied the SDFA for purification of collagenase produced by Penicillium (Lima et al., 2013; Wanderley et al., 2017).

This work is a report on Chlorella vulgaris collagenolytic activity, never before reported and studied. Including enzyme production, factorial design to obtain the best conditions of its purification using aqueous two-phase system (polyethileneglycol/phosfate buffer) and enzyme characterization on optima pH and temperature, substrate specificity and inhibitors.