*2.2. Enzymes for Healthcare Application*

Enzymes for healthcare applications can include: (1) Enzymes used directly as "drugs", or (2) enzymes involved in the biosynthetic pathway of bioactive compounds (Figure 4). Regarding the first group, the most studied enzyme is l-asparaginase. l-asparaginase is an l-asparagine amidohydrolase enzyme used for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin's lymphoma [49]. Its hydrolytic effect reduces asparagine availability for cancer cells that are unable to synthesize l-asparaginase autonomously [50] l-asparaginase was historically first discovered and then produced in bacteria (e.g., *Escherichia coli*, *Erwinia aroideae*, *Bacillus cereus*) [51–53]. However, in order to overcome some of the economical and safety limits associated with marketing the enzyme [54,55], increased efforts began to focus on the identification and characterization of the enzyme in microalgae strains.

**Figure 4.** Enzymes for Healthcare Applications. Enzymes for healthcare applications can include: (**a**) Enzymes used directly as "drugs", such as the l-asparaginase (**b**) enzymes involved in the biosynthetic pathway of active compounds, such as polyketides, carotenoids, or oxylipins. In the synthesis of polyketides, the enzymes studied are polyketide synthases and nonribosomal peptide synthases. For the synthesis of carotenoids, the most studied enzymes are phytoene synthase (PSY), phytoene decarboxylase (PDS) and zeaxanthin epoxidase (ZEP). For the synthesis of oxylipins the studied enzymes are lipoic acid hydrolases (LAH) and PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin)/LH2 (Lipoxygenase homology). An example of molecules and their roles for each pathway is also outlined.

Paul [56] first purified an l-asparaginase in*Chlamidomonas* spp. with limited anticancer activity, and tested it in an in vivo anti-lymphoma assay. Ebrahiminezhad and coworkers screened 40 microalgal isolates via activity assays and reported on *Chlorella vulgaris* as a novel potential feedstock for l-asparaginase production [57].

Regarding enzymatic pathways involved in the synthesis of bioactive compounds, many studies have focused on polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS produce polyketides, while NRPS produce nonribosomal peptides. Both classes of secondary metabolites are formed by sequential reactions operated by these "megasynthase" enzymes [58,59]. Polyketides and nonribosomal peptides have been reported to have antipredator, allelopathic, anticancer, and antifungal activities [58,60–62]. PKS can be multi-domain enzymes (Type I PKS), large enzyme complexes (Type II), or homodimeric complexes (Type III) [63]. Genes potentially encoding these first two types' of PKSs have been identified in several microalgae (e.g., *Amphidinium carterae*, *Azadinium spinosum*, *Gambierdiscus* spp., *Karenia brevis* [64–67]). Similarly, NRPSs have a modular organization similar to type I PKSs, and genes potentially encoding NRPSs have been found in different microalgae [68]. Moreover, metabolites that are likely to derive from hybrid NRPS/PKS gene clusters have been reported from *Karenia brevis* [69]. However, to our knowledge, there are no studies reporting the direct correlation of a PKS or NRPS gene from a microalga with the production of a bioactive compound.

Other microalgal enzymes which have been widely studied are those involved in the synthesis of compounds with nutraceutical and cosmeceutical applications, such as those involved in carotenoid synthesis (e.g., astaxanthin, β-carotene, lutein, and canthaxanthin). Carotenoids are isoprenoid pigments, which have many cellular protective effects, such as antioxidant effects occurring via the chemical quenching of O2 and other reactive oxygen species [70–72]. Their antioxidant properties can potentially protect humans from a compromised immune response, premature aging, arthritis, cardiovascular diseases, and/or certain cancers [72]. Among microalgae, the most studied for the industrial production of carotenoids are the halophile microalga *Dunaliella salina* and the green alga *Haematococcus pluvialis*, which naturally produce high amounts of carotenoids [73]. Moreover, *D.* *salina* is a particularly versatile feedstock, and many researchers have focused on obtaining maximum carotenoid yields without impeding its growth [74–76]. In addition, *D. salina* has been successfully transformed via different approaches, such as microparticle bombardment [77] or via *Agrobacterium tumefaciens* [78], increasing the feasibility of its use for biotechnological applications.

The most studied enzymes involved in carotenoid synthesis are: β-carotene oxygenase, lycopene-β-cyclase, phytoene synthase, phytoene desaturase, β-carotene hydroxylase, and zeaxanthin epoxidase [79]. In order to improve the production of carotenoids, different metabolic engineering approaches have been employed. The initial method used was to induce random or site directed mutations in an attempt to improve the activity of enzymes involved in the carotenoid metabolic pathway. Increased production of carotenoids can also be achieved by changing culturing conditions or by employing genetic modifications [79]. For example, mRNA levels of β-carotene oxygenase, involved in the biosynthesis of ketocarotenoids [80], increased in *Chlorella zofengiensis* under combined nitrogen starvation and high-light irradiation, and an increase canthaxanthin, zeaxanthin, and astaxanthin was observed [81]. Couso et al. [82] reported an upregulation in lycopene-β-cyclase, which converts lycopene to β-carotene [83] in *C. reinhardtii* under conditions of high light.

Regarding genetic modifications, Cordero [84] transformed the green microalga *C. reinhardtii* by overexpressing a phytoene synthase (which converts geranylgeranyl pyrophosphate to phytoene) isolated from *Chlorella zofingiensis*, resulting in a 2.0- and 2.2-fold increase in violaxanthin and lutein production, respectively. A phytoene desaturase, which transforms the colorless phytoene into the red-colored lycopene [85], was mutated in *H. pluvialis* by Steinbrenner and Sandmann [86], resulting in the upregulation of the enzyme and an increase in astaxanthin production. Galarza and colleagues expressed a nuclear phytoene desaturase in the plastidial genome of *H. pluvialis*, resulting in a 67% higher astaxanthin accumulation when the strain was grown under stressful conditions [87]. The insertion of a β-carotene hydroxylase from *C. reinhardtii* in *Dunaliella salina* resulted in a 3-fold increase of violaxanthin and a 2-fold increase of zeaxanthin [78]. The inhibition of *D. salina* phytoene desaturase using RNAi technology [88] resulted in an increase in phytoene content, but also a decrease in photosynthetic efficiency and growth rate.

More modern methods which have been used include the use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for precise and highly efficient "knock-out" of key genes [89]. For example, Baek et al. have used CRISPR/Cas9 to knock-out the zeaxanthin epoxidase (ZEP) gene in *C. reinhardtii* [90]. This enzyme is involved in the conversion of zeaxantin to violaxantin [91], and with its knock-out they obtained a 47-fold increase in zeaxanthin productivity. The current state-of-art involved in metabolic engineering for carotenoid production in microalgae is further discussed in other reviews [72,92].

Other studies have focused on enzymes involved in the synthesis of oxylipins, which are secondary metabolites that have previously been shown to have antipredator and anticancer activities [93–95]. Although oxylipin chemistry and putative biosynthetic pathways have been extensively studied in both plants and microalgae [96–98], the related enzymes and genes have only recently been identified and characterized in microalgae. Adelfi and coworkers have studied genes involved in the biosynthesis of oxylipins in *Pseudo-nitzchia multistriata* and performed transcriptome analysis on these genes in *Pseudo-nitzchia arenysensis* [99]. In diatoms, they characterized, for the first time, two patatin-like lypolitic acid hydrolases (LAH1) involved in the release of the fatty acid precursors of oxylipins and tested their galactolipase activity in vitro. Transcriptomic analysis also revealed three of seven putative patatin genes (g9879, g2582, and g3354) in *N. oceanica* and demonstrated that they were u-regulated under nitrogen-starvation conditions [100]. Similarly, Lauritano and coworkers analyzed the transcriptome of the green alga *Tetraselmis suecica* and reported three PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin)/LH2 (Lipoxygenase homology) domain transcripts [68]. The group also performed in silico domain assessment and structure predictions. The enzymes discussed in this section are described in Table 2.

**Table 2.** Enzymes from Microalgae for Healthcare Applications. Marine, freshwater, and soil strain sources are abbreviated as M, F, or S, respectively. Algal classes of *Chlorophyceae*, *Trebouxiophyceae*, *Bacillariophyceae*, *Dinophyceae*, and *Chlorodendrophyceae*, are abbreviated as CH, TR, BA, DY, and CR respectively.



**Table 2.** *Cont*.

#### *2.3. Enzymes for Bioremediation*

Bioremediation is the use of microorganisms and their enzymes for the degradation and/or transformation of toxic pollutants into less dangerous metabolites/moieties. The potential, which microalgae possess to proliferate in environments that are rich in nutrients (e.g., eutrophic environments) and to biosequestrate heavy metal ions, makes them ideal candidate organisms for bioremediation strategies [101,102]. The optimal goal in this area is to combine bioremediation activities with the possibility of extracting lipids and other high-value added compounds from the biomass that is produced [103–106] in order to reduce overall costs and to recycle materials. In this section, the focus will be on enzymatic bioremediation, which is a novel approach involving the direct use of purified or partially purified enzymes from microorganisms, and in this case, from microalgae, in order to detoxify a specific toxicant/pollutant [107]. This method has recently started to demonstrate promising results through the use of bacterial enzymes [108,109]. Examples are the use of enzymes for the bioremediation of industrial waste and, in particular, the recent use of chromate reductases found in chromium resistant bacteria, known to detoxify the highly toxic chromium Cr(VI) to the less-toxic Cr(III) [110].

In microalgae, a recent study focused on Cr(VI) reduction involving *C. vulgaris* [111]. This activity was suggested to involve both a biological route, through the putative enzyme chromium reductase, and a nonbiological route: Using the scavenger molecule glutathione (GSH). With respect to chromium removal, several strains of microalgae have been reported to be capable of achieving Cr(IV) removal from water bodies, including *Scenedesmus* and *Chlorella* species [112–114]. In the aforementioned transcriptome study on the green algae *Tetraselmis suecica*, a transcript for a putative nitrilase was reported [68]. Given that nitrilases are enzymes that catalyze the hydrolysis of nitriles to carboxylic acids and ammonia [115] and that this enzyme has recently been used for cyanide bioremediation in wastewaters [116], this nitrilase in *T. suecica* may prove to be useful in the treatment of cyanide contaminated water bodies.

Other enzymes have been reported to be overexpressed in microalgae when they are exposed to contaminants, but it is not clear whether or not they are directly involved in their degradation or whether they are produced as a stress defensive response in the cell in order to help balance cellular homeostasis (e.g., to detoxify reactive oxygen/nitrogen species produced after exposure to contaminants). Examples of these enzymes include peroxidases (Px), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR). SOD, Px, and CAT typically function in helping detoxify the cell from oxygen reactive species [117,118], while GR replenishes bioavailable glutathione, catalyzing the reduction of glutathione disulfide (GSSG) to the sulfhydryl form (GSH) [119]. Regarding

the detoxification of reactive nitrogen species, the most studied enzymes in microalgae are the nitrate and nitrite reductases. The first enzyme reduces nitrate (NO3 <sup>−</sup>) to nitrite (NO2 −), while the second subsequently reduces nitrite to ammonia (NH4 <sup>+</sup>). NH4 <sup>+</sup> is then assimilated into amino acids via the glutamine synthetase/glutamine-2-oxoglutarate amino-transferase cycle [120] (Figure 5).

**Figure 5.** Enzymes for Bioremediation. Enzymes for Bioremediation can be: (**a**) Enzymes directly used for the degradation of toxicant compounds to less or non toxic versions (e.g., the hexavalent Chromium is converted to the less toxic trivalent Chromium due to the activity of Chromium Reductase); (**b**) Enzymes involved in cellular stress response mechanisms, such as peroxidases (Px), superoxide dismutase (SOD), and catalase (CAT) that detoxify reactive oxygen species (ROS), nitrate reductase (NR), and nitrite reductase (NiR) that detoxify reactive nitrogen species (RNS) in ammonium, and GR, that catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH).

For example, peroxidase activity has been reported in extracts from the green alga *Selenastrum capricornutum* (now named *Raphidocelis subcapitata* [121]), which was highly sensitive to very small concentrations of copper (Cu) (0.1 mM), and the authors proposed that the enzyme could be employed as a sensitive bioindicator of copper contamination in fresh waters [122]. Levels of Px, SOD, CAT, and GR have been reported to be upregulated following Cu contamination in *P. tricornutum* and following lead (Pb) contamination in two lichenic microalgal strains from the *Trebouxia* genus (prov. names, TR1 and TR9) [123,124]. In Morelli's work, an increase of 200% in CAT activity indicated its important role in Cu detoxification. In contrast, Alvarez and coworkers reported that Px, SOD, CAT, and GR activity was higher in TR1 than in TR9 under control conditions (with the exception of CAT), while prolonged exposure to Pb resulted in the enzymatic activities of the two microalgae changing to similar levels, reflecting the different physiological and anatomical adaptations of the two organisms. TR1 possesses a thinner cell wall, thereby requiring it to have a more efficient basal enzymatic defence system, while TR9 has a thicker cell wall and induces the expression of intracellular defense mechanisms when the contaminant concentrations are high and physical barriers are no longer effective. Further studies will be required to assess whether these TR1 enzymes are more efficient than enzymes from other microalgal sources and the potential applications that these enzymes may have. All of the enzymes discussed in this section are reported in Table 3.


**Table 3.** Enzymes from Microalgae with utility in Bioremediation applications Marine, freshwater, and lichenic strain sources are abbreviated as M, F, and L respectively. Algal classes of *Trebouxiophyceae*, *Chlorodendrophyceae*, *Chlorophyceae*, and *Bacillariophyceae* are abbreviated as TR, CR, CH, and BA, respectively.

#### **3. Conclusions and Future Perspectives**

Among aquatic organisms that have recently received attention as potential sources of industrially relevant enzymes [125,126], microalgae, in particular, stand out as a new sustainable and ecofriendly source of biological products (e.g., lipids, carotenoids, oxylipins, and polyketides). This review summarized the available information on enzymes from microalgae with possible biotechnological applications, with a particular focus on value-added lipid production, together with healthcare and bioremediation applications.

The promise of microalgae as potential sources of novel enzymes of interest is reflected in the abundance of recent reports in the literature in this area. However, the biotechnological exploitation of their enzymes in comparison to other potential sources has only become more feasible quite recently, primarily due to the implementation of novel isolation and culturing procedures, together with an increase in the availability of -omics data. This data has facilitated the use of a broader array of approaches, such as site-specific mutagenesis, bioinformatics-based searches for genes of interest, and/or the use of genome editing tools (e.g., CRISPR/Cas9 and TILLING), resulting in promising results particularly with respect to high-performance lipid [46] and carotenoid [89] production in different microalgae.

The majority of studies to date have focused on enzymes involved in pathways for lipid synthesis in order to increase their total production or to direct cellular production to lipid classes with applications as nutraceuticals, cosmeceuticals, or as a feedstock for biodiesel production. For this reason, several recent studies have focused on the improvement of lipid production in oleaginous microalgae. In addition, algal biomass is often used for the extraction of both lipids and other value-added products, such as pigments and proteins, in order to maximize the production of useful products such as these at the lowest possible cost [127–129].

Future approaches to maximize the enzymatic potential of microalgae are likely to focus on three different approaches: (1) The use of ever-increasing amounts of available -omics data to optimize microalgal strains for the production of valuable products, through the overexpression of one or more enzymes through the use of genome editing tools; (2) identification and subsequent characterization of metabolic pathways involved in the production of specific bioactives (e.g., polyketides), many of which are still poorly characterized; (3) the search for genes with direct biotechnological applications (e.g., l-asparaginase, chromate reductase, nitrilase) in microalgal genomes and transcriptomes datasets. A common element in all three approaches is the potential use of next generation sequencing based approaches (NGS) [130], the price of which is declining rapidly [131].

The feasibility of employing any of the aforementioned three approaches will be directly influenced by progress in methods to decrease the costs of growth and genetic manipulation of microalgae. The ultimate aim would be to mimic what has happened in the area of bacterial enzymology, where robust pipelines for enzyme discovery have been established. If this could be achieved, then it is clear that microalgae are likely to meet our expectations as a promising source of novel enzymes with utility in a variety of different biotechnological applications.

**Author Contributions:** G.M.V., P.D.L., A.I., A.D.W.D. and C.L. co-wrote the review.

**Funding:** G.M.V. was supported by a Stazione Zoologica Ph.D. fellowship via the Open University.

**Acknowledgments:** Authors thank Servier Medical Art (SMART) website (https://smart.servier.com/) by Servier for the elements of Figure 3. SMART is licensed under a Creative Commons Attribution 3.0 Unported License.

**Conflicts of Interest:** The authors declare no conflict of interest.
