Application-Oriented Marine Isomerases in Biocatalysis

The class EC 5.xx, a group of enzymes that interconvert optical, geometric, or positional isomers are interesting biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. This class, named “isomerases,” can transform cheap biomolecules into expensive isomers with suitable stereochemistry useful in synthetic medicinal chemistry, and interesting cases of production of l-ribose, d-psicose, lactulose, and d-phenylalanine are known. However, in two published reports about potential biocatalysts of marine origin, isomerases are hardly mentioned. Therefore, it is of interest to deepen the knowledge of these biocatalysts from the marine environment with this specialized in-depth analysis conducted using a literature search without time limit constraints. In this review, the focus is dedicated mainly to example applications in biocatalysis that are not numerous confirming the general view previously reported. However, from this overall literature analysis, curiosity-driven scientific interest for marine isomerases seems to have been long-standing. However, the major fields in which application examples are framed are placed at the cutting edge of current biotechnological development. Since these enzymes can offer properties of industrial interest, this will act as a promoter for future studies of marine-originating isomerases in applied biocatalysis.


Introduction
In 2010, one of the first comprehensive review articles about enzymes of marine origin especially suitable for future applications in biocatalysis reported an account of the knowledge in the field. As stated in the conclusion, the enormous pool of marine biodiversity is an excellent natural reservoir for acquiring an inventory of enzymes that is one of the focal points of the potential of blue biotechnology. The importance of the examples reported, picked up from the different classes of enzymes, supported the view that the marine environment is to be considered as an additional source of new enzymes for the biocatalysis. Important examples are among oxidoreductases and carbohydrate-active enzymes. Their novel chemical and stereochemical properties are included in the list of useful habitat-related characteristics possessed by these enzymes, adding value to the often-observed usual resistance of these proteins to high salt concentration and/or organic solvent. Other enzymes, characterized by a potent chemical action on nonactivated carbon atoms are also of extreme interest. Details on the characteristics of other marine representatives belonging to other classes of enzymes (lipid active hydrolases, novel esterases, and other hydrolytic activities) further supported the conclusion [1]. Isomerases are included only with a few cases, in particular, an alanine racemase from the hepatopancreas of the black tiger prawn, Penaeus mondon [2] and a fatty acid isomerase from marine alga Ptilota filicina [3].

Literature Search
The search for articles in this review has been preliminarily conducted using the words "isomerase" and "marine" in titles, abstracts, and keywords without interval time limit in the Science Direct database with access to 3800 scientific journals in major scientific disciplines. However, the limited range of articles retrieved (31 hits) prompted us to use the more generic PubMed database with the same words thus retrieving 172 hits. Besides, trivial names of these enzymes (as epimerase, racemase, cis-trans isomerases, cycloisomerase, and tautomerase) were also coupled with "marine" in an accessory search using Web of Science database (Table 1). Science alert mailing lists were used, up to the manuscript submission to update the analysis with the most recent results. In this review, an analysis of reports about enzymatic activities already oriented to applications is firstly compiled in the central Section 3. In Sections 4-6, tabulated lists of the remaining articles according to types of molecules on which these isomerases can catalyze their reactions are discussed. Isomerases acting on carbohydrate molecules ( Table 2)  are listed in the first of three tables following with lipids (Table 3) [39][40][41][42][43][44][45][46] and amino acids and peptides (Table 4) . Each chronologically ordered line reports the name of the enzyme, as indicated in the reference, the reaction catalyzed, and a short description of the intent and scientific field of the work.

Databases Search Statement Hits
Science Direct Isomerase * and marine in titles, abstracts and keywords 31 PubMed Isomerase * and marine 172 WoS 1 Marine epimerase * in All fields 53 WoS Marine racemase * in All fields 44 WoS Marine cis-trans isomerase * in All fields 56 WoS Marine cycloisomerase * in All fields 3 WoS Marine tautomerase * in All fields 26 WoS Marine mutase * in All fields 45

Application-Oriented Biocatalysts
A few applicative examples of works using marine isomerase are collected in this paragraph. A gene encoding for d-xylose isomerase from a marine bacterium, Vibrio sp. strain XY-214, has been expressed in E. coli, and the production of d-xylulose from β-1,3-xylan was carried out. This paper concerns the growing attention to green technologies for the conversion of biomass and saccharification of carbohydrate polymers, in particular, the polysaccharide β-1,3-xylan of the invasive green alga Caulerpa taxifolia. Marine Vibrio sp. strain can grow on β-1,3-xylan as a sole carbon source; the rationale for the work is based on the synergistic action of two types of enzymes enabling the complete degradation of β-1,3-xylan to d-xylose, i.e., 1,3-βd-xylan xylanohydrolase and a β-1,3-xylosidase.
d-xylulose is then formed by the marine d-xylose isomerase. d-xylulose can be used for ethanol fermentation thus allowing the use of the algal polymer β-1,3-xylan of C. taxifolia as a base for ethanol production. The article is a preliminary study for a possible real application of the total saccharification of the polymer. The work has been conducted by purified enzymes, and 2.62 g/L reducing sugars were released by the action of the two β-1,3-xylan degrading activities, the subsequent isomerization of d-xylose thus producing d-xylulose. Time-course experiments analyzing reaction mixtures by HPLC were reported, and different reaction conditions were analyzed also in presence of sodium tetraborate for possible complexes with xylulose-borate shifting the equilibrium [75].
Yarrowia lipolytica is a marine microorganism of industrial interest for the physiological ability to utilize different substrates for growth (polyalcohols, organic acids, and long-chain hydrocarbons). In a recent short communication [76], the isomaltulose production using an engineered Yarrowia lipolytica strain is reported. Sucrose isomerase catalyzes the enzymatic rearrangement of the α-1,2 linkage between glucose and fructose to an α-1,6 linkage (producing isomaltulose) or α-1,4 linkage (producing trehalulose). Marine origin examples of sucrose isomerase and its use for biological isomaltulose production were not known up to a review of 2014 [77]. In fact, the sucrose isomerase overexpressed from Pantoea dispersa but the high and efficient process of isomaltulose production was based on enzyme production and enzymatic catalysis during fermentation, thus reducing costs and simplifying the bioprocess. The maximum isomaltulose production was 572.1 g/L, with a yield of 0.96 g/g of sucrose.
A very recent report demonstrates the use of l-arabinose isomerase for production of D-tagatose, a rare sugar of importance in the food industry that has been approved as GRAS drug by the US Food and Drug Administration and used as a substitute for sucrose in low-calorie sweeteners. As in the above case, although the enzyme is from Lactobacillus sakei 23K and converts d-galactose from agar into d-tagatose, it is worth to mention this research effort here to show the interest for the profitable application of red algae carbohydrate polymers as a substrate for d-tagatose production ( Figure 1) [78].
Mar. Drugs 2020, 18, 580 10 of 21 Figure 1. Reaction scheme of L-arabinose isomerase. The enzyme can also convert D-galactose to Dtagatose with lower efficiency. The enzyme is also present in the marine Geobacillus stearothermophilus (see Table 2).
Ribose-5-phosphate isomerase is another enzyme of interest in the field of rare sugars that are used as sweeteners and for production of interesting building blocks for fine chemistry as reported in the study of ribose-5-phosphate isomerase from an Ochrobactrum sp. [7] to increase reaction rate in isomerizing L-rhamnose to L-rhamnulose already mentioned. Although the microorganism was  Table 2).
Ribose-5-phosphate isomerase is another enzyme of interest in the field of rare sugars that are used as sweeteners and for production of interesting building blocks for fine chemistry as reported in the study of ribose-5-phosphate isomerase from an Ochrobactrum sp. [7] to increase reaction rate in isomerizing l-rhamnose to l-rhamnulose already mentioned. Although the microorganism was isolated from soil samples, marine examples are also known and could be potential alternatives. Substrate specificity and reaction properties were explored, the results encouraged for the application of ribose-5-phosphate isomerase as a biocatalyst in preparation of rare sugars.
A report already present in literature in 1994 was the first describing a novel isomerase for the biosynthesis of conjugated triene-containing fatty acids in the red alga Ptilota filicina [79]. At the time, in fact, many hypotheses and studies on the biosynthetic pathway of conjugated polyenes in marine organisms were already present in literature, coming from studies centered on natural products of marine origin. The main interest is focused on pharmacological studies indicating a role for these bioactive molecules in the treatment of tumors, against weight gain, and as enhancers of the immune system. In particular, the biosynthesis of a conjugated triene (4,5Z,7E,9E,14Z,17Z)-eicosapentaenoic acid from eicosapentaenoic acid was indicated. The product is present among natural products of the red alga Ptilota filicina. The enzyme was isolated from alga tissues and assayed with arachidonic acid forming a triene structure, evidenced by UV absorption. The product of arachidonate incubation was also identified as the corresponding conjugated triene metabolite. A substrate specificity investigation revealed that the eicosapentaenoic structure was the best substrate for the enzyme. Incorporation of deuterium at C11 position of arachidonate was demonstrated by 1 H NMR spectroscopy and mass spectrometry for the reaction conducted in the deuterated buffer. Intramolecular and intermolecular hydrogen transfers using stereospecifically deuterated substrates and oxygen sensibility of reaction were also studied. These authors were able to show that unlike the well-characterized aerobic reaction of lipoxygenases, molecular oxygen was not required by their isomerase with no net desaturation occurring during the reaction, thus providing useful insights for the use of these biocatalysts as tools for the synthesis of novel compounds. Later in time, the P. filicina enzyme was purified to electrophoretic homogeneity, and the cloning and functional expression including the study of other important characteristics such as molecular weight, subunit structure, and glycosylation were reported [3]. The ability of this enzyme in the isomerization of methylene interrupted olefins led the authors to try the reaction with anandamide, the well-known N-ethanolamide of arachidonic acid, the first endogenous ligand of cannabinoid receptor. The conjugated triene anandamide product was shown to possess high-affinity binding for the receptor [80] (Figure 2). A 33% yield was obtained in preparative experiments for the full spectroscopic chemical characterization of the reaction product. As the same authors speculated, they were able to show the use of these marine enzymes in the synthetic production of novel compounds for pharmacological probes. The same group already studied, in 1991, the oxylipin metabolism. In this work, the conversion of arachidonic acid into the vicinal diol fatty acid 12R,13S-dihydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid by an acetone powder of the marine red alga, Gracilariopsis lemaneiformis, occurred via intermediate formation of hydroperoxide 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid, postulating the existence of a hydroperoxide isomerase in this red alga. The broad substrate specificity and the high stereospecificity of the product formed in the step of oxygen insertion were of interest in the application in biocatalysis [81].
By studying how cultured fish cells derived from turbot (Scophthalmus maximus), gilthead seabream (Sparus aurata), and Atlantic salmon (Salmo salar) metabolize all-cis octadecapentaenoic acid, some authors discovered the action of an isomerase acting on all-cis 18:5n-3 acids producing 2-trans 18:5n-3 acids thought to be common intermediates in the β-oxidation of these acids by marine animals [82]. Similar isomerases acting on double bonds of different compounds have been hypothesized in a study on biodegradation of alkenones and related compounds of the marine microalgae Emiliania huxleyi by microbial mats collected in large ponds. Among products, authors found cis/trans or trans/cis alkene and alkenone isomers and suggested that the formation of these isomeric compounds is likely due to extracellular bacterial cis/trans isomerases [83].
work, the conversion of arachidonic acid into the vicinal diol fatty acid 12R,13S-dihydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid by an acetone powder of the marine red alga, Gracilariopsis lemaneiformis, occurred via intermediate formation of hydroperoxide 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid, postulating the existence of a hydroperoxide isomerase in this red alga. The broad substrate specificity and the high stereospecificity of the product formed in the step of oxygen insertion were of interest in the application in biocatalysis [81]. By studying how cultured fish cells derived from turbot (Scophthalmus maximus), gilthead seabream (Sparus aurata), and Atlantic salmon (Salmo salar) metabolize all-cis octadecapentaenoic acid, some authors discovered the action of an isomerase acting on all-cis 18:5n-3 acids producing 2trans 18:5n-3 acids thought to be common intermediates in the β-oxidation of these acids by marine animals [82]. Similar isomerases acting on double bonds of different compounds have been hypothesized in a study on biodegradation of alkenones and related compounds of the marine microalgae Emiliania huxleyi by microbial mats collected in large ponds. Among products, authors found cis/trans or trans/cis alkene and alkenone isomers and suggested that the formation of these isomeric compounds is likely due to extracellular bacterial cis/trans isomerases [83].
In a study centered on searching for marine microorganisms capable of carbazole remediation schematically represented in Figure 3, the marine bacterium Neptuniibacter sp. strain CAR-SF has been found utilizing carbazole as its sole carbon and nitrogen sources [84]. Among the enzymes involved in the degradation pathway, 4-oxalocrotonate tautomerase (and others) is indicated, and Escherichia coli cells transformed in this work required ferredoxin and ferredoxin reductase for necessary initial dioxygenation of carbazole. The authors indicated that this was the first report of genes involved in carbazole degradation isolated from a marine bacterium, however, only the conversion product of carbazole through dioxygenation by dioxygenase was found (2′aminobiphenyl-2,3-diol).
Various enzymes have been analyzed in an interesting report [85] on strategies for the deracemization of a racemate into a single stereoisomeric product; these include mandelate racemase, lactate racemase, or alkyl sulfatases from the actinomycete Rhodococcus ruber DSM 44541, the marine planctomycete Rhodopirellula baltica DSM 10,527, and others, known to possess a rich inorganic sulfur metabolism. Although not belonging to isomerases, it is worth mentioning that alkyl sulfatases display not only enantioselectivity but also stereoselectivity for retention or inversion of the configuration of the formed product during sulfate hydrolysis and these authors report about a scheme devised to produce single stereoisomer from racemate. The (±)-sec-alkyl sulfate ester is subjected to inverting alkyl sulfatase producing a mixture of hydrolyzed ester with the same configuration of the remaining unreacted ester. The latter is then hydrolyzed in a chemical step with In a study centered on searching for marine microorganisms capable of carbazole remediation schematically represented in Figure 3, the marine bacterium Neptuniibacter sp. strain CAR-SF has been found utilizing carbazole as its sole carbon and nitrogen sources [84]. Among the enzymes involved in the degradation pathway, 4-oxalocrotonate tautomerase (and others) is indicated, and Escherichia coli cells transformed in this work required ferredoxin and ferredoxin reductase for necessary initial dioxygenation of carbazole. The authors indicated that this was the first report of genes involved in carbazole degradation isolated from a marine bacterium, however, only the conversion product of carbazole through dioxygenation by dioxygenase was found (2 -aminobiphenyl-2,3-diol).
Mar. Drugs 2020, 18, 580 12 of 21 retention of configuration producing the alcohol in this enantioconvergent process (Figure 4). The importance of marine-originating biocatalysts is clearly assessed by this example.   Various enzymes have been analyzed in an interesting report [85] on strategies for the deracemization of a racemate into a single stereoisomeric product; these include mandelate racemase, lactate racemase, or alkyl sulfatases from the actinomycete Rhodococcus ruber DSM 44541, the marine planctomycete Rhodopirellula baltica DSM 10,527, and others, known to possess a rich inorganic sulfur metabolism. Although not belonging to isomerases, it is worth mentioning that alkyl sulfatases display not only enantioselectivity but also stereoselectivity for retention or inversion of the configuration of the formed product during sulfate hydrolysis and these authors report about a scheme devised to produce single stereoisomer from racemate. The (±)-sec-alkyl sulfate ester is subjected to inverting alkyl sulfatase producing a mixture of hydrolyzed ester with the same configuration of the remaining unreacted ester. The latter is then hydrolyzed in a chemical step with retention of configuration producing the alcohol in this enantioconvergent process (Figure 4). The importance of marine-originating biocatalysts is clearly assessed by this example. Figure 3. Carbazole degradation to 2′-aminobiphenyl-2,3-diol. Remediation study is present for this compound by the marine bacterium Neptuniibacter sp. A tautomerase could be involved in the lower degradation pathway as in total cleavage pathway for the degradation of phenols, modified phenols, and catechols. Cytochromes P450 are important biocatalysts performing hydroxylation reactions in regio-and stereospecific manners operating on inactive carbon atoms; they are useful for the bioassisted synthesis of organic molecules. In an interesting paper [86], authors constructed a fusion protein of a peptidyl-prolyl cis-trans isomerase isolated from the hyperthermophilic archaeon Thermococcus sp. with the cytochrome P450 BM3 derived from Bacillus megaterium and evaluate its stability in E. coli cells in a series of bioconversion experiments with various substituted naphthalenes. It is known that peptidyl-prolyl cis-trans isomerases catalyze the cis-trans isomerization of the proline imide bond in polypeptides, which may affect the folding rate of proteins. This fusion protein exists as the predominant soluble protein and more stable than the unfused P450. Various substituted naphthalenes were converted to their monohydroxylated derivatives, and the reaction was also tested on a sesquiterpene ( Figure 5) that has physiological functions such as β-eudesmol that was found to be hydroxylated in a regio-and stereo-specific manner. Cytochromes P450 are important biocatalysts performing hydroxylation reactions in regioand stereospecific manners operating on inactive carbon atoms; they are useful for the bioassisted synthesis of organic molecules. In an interesting paper [86], authors constructed a fusion protein of a peptidyl-prolyl cis-trans isomerase isolated from the hyperthermophilic archaeon Thermococcus sp. with the cytochrome P450 BM3 derived from Bacillus megaterium and evaluate its stability in E. coli cells in a series of bioconversion experiments with various substituted naphthalenes. It is known that peptidyl-prolyl cis-trans isomerases catalyze the cis-trans isomerization of the proline imide bond in polypeptides, which may affect the folding rate of proteins. This fusion protein exists as the predominant soluble protein and more stable than the unfused P450. Various substituted naphthalenes were converted to their monohydroxylated derivatives, and the reaction was also tested on a sesquiterpene ( Figure 5) that has physiological functions such as β-eudesmol that was found to be hydroxylated in a regio-and stereo-specific manner.

Marine Isomerases Acting on Sugar Molecules
In Table 2, the articles  found on isomerases acting on carbohydrate pathways are briefly listed. As cited in the first entry about Pelvetia canaliculata in 1973 [9], the scientific interest was early generally present in literature since 1956 in investigations conducted on different organisms. For an algal polymannuronic-5-epimerase, converting polymannuronic acid to a mixed polymer containing guluronic acid, the preparation of ammonium sulfate precipitation was reported [9]. Scientific interests for the articles listed in Table 2 and experimental methodologies used reflected the successes in biochemistry, molecular biology, and genetics achieved during the last century. All these listed articles did not contain applicative results that are detailed above in the paragraph on applicationoriented biocatalysts. Very often, a basic study of the marine biocatalyst is reported as in the case [28] of xylose isomerase from Fulvimarina pelagi. It was identified by sequence analysis of the F. pelagi genome, i.e., PCR amplified, cloned, and expressed in E. coli, while the aim of the work is framed into Figure 5. β-eudesmol hydroxylation. Regio-and stereo-specific actions of cytochrome P450 [86] were studied in depth with using NMR spectroscopy; 80% yield in preparative experiments was obtained.

Marine Isomerases Acting on Sugar Molecules
In Table 2, the articles  found on isomerases acting on carbohydrate pathways are briefly listed. As cited in the first entry about Pelvetia canaliculata in 1973 [9], the scientific interest was early generally present in literature since 1956 in investigations conducted on different organisms. For an algal polymannuronic-5-epimerase, converting polymannuronic acid to a mixed polymer containing guluronic acid, the preparation of ammonium sulfate precipitation was reported [9]. Scientific interests for the articles listed in Table 2 and experimental methodologies used reflected the successes in biochemistry, molecular biology, and genetics achieved during the last century. All these listed articles did not contain applicative results that are detailed above in the paragraph on application-oriented biocatalysts. Very often, a basic study of the marine biocatalyst is reported as in the case [28] of xylose isomerase from Fulvimarina pelagi. It was identified by sequence analysis of the F. pelagi genome, i.e., PCR amplified, cloned, and expressed in E. coli, while the aim of the work is framed into the field of biofuel production ( Figure 6).
In Table 2, the articles  found on isomerases acting on carbohydrate pathways are briefly listed. As cited in the first entry about Pelvetia canaliculata in 1973 [9], the scientific interest was early generally present in literature since 1956 in investigations conducted on different organisms. For an algal polymannuronic-5-epimerase, converting polymannuronic acid to a mixed polymer containing guluronic acid, the preparation of ammonium sulfate precipitation was reported [9]. Scientific interests for the articles listed in Table 2 and experimental methodologies used reflected the successes in biochemistry, molecular biology, and genetics achieved during the last century. All these listed articles did not contain applicative results that are detailed above in the paragraph on applicationoriented biocatalysts. Very often, a basic study of the marine biocatalyst is reported as in the case [28] of xylose isomerase from Fulvimarina pelagi. It was identified by sequence analysis of the F. pelagi genome, i.e., PCR amplified, cloned, and expressed in E. coli, while the aim of the work is framed into the field of biofuel production ( Figure 6). In some cases, the interest of the article is focused on biomedical field as for the two reports [32,33] for the study of allergen function of triosephosphate isomerases from Octopus fangsiao and freshwater crayfish Procambarus clarkii. The consumption of seafood products, in fact, can be related to the high frequency of food-induced immune responses, and these studies are important to develop therapeutic and diagnostic approaches to these issues. In the case of freshwater crayfish Procambarus clarkii, the increased production and consumption can result, in fact, in allergic reactions, including life-threatening anaphylaxis. Another interesting article showing modern scientific interest is the one related to GDP-L-galactose mutase in Marinactinospora thermotolerans [34], an article dealing with a rare and interesting molecule, L-Galf, hardly found in the environment; the biosynthetic action and roles and relevant enzymes acting on L-Galf are of interest as this moiety is inserted in an aminonucleoside antibiotic possessing medicinal potential. In some cases, the interest of the article is focused on biomedical field as for the two reports [32,33] for the study of allergen function of triosephosphate isomerases from Octopus fangsiao and freshwater crayfish Procambarus clarkii. The consumption of seafood products, in fact, can be related to the high frequency of food-induced immune responses, and these studies are important to develop therapeutic and diagnostic approaches to these issues. In the case of freshwater crayfish Procambarus clarkii, the increased production and consumption can result, in fact, in allergic reactions, including life-threatening anaphylaxis. Another interesting article showing modern scientific interest is the one related to GDP-l-galactose mutase in Marinactinospora thermotolerans [34], an article dealing with a rare and interesting molecule, l-Galf, hardly found in the environment; the biosynthetic action and roles and relevant enzymes acting on l-Galf are of interest as this moiety is inserted in an aminonucleoside antibiotic possessing medicinal potential. Table 3 contains a few listed articles concerning isomerases acting on lipid molecules. The scientific interest was present as early as the beginning of the 1990s as indicated by the genetic study [39] related to the steroidogenic enzyme involved in the production of 17α-hydroxyprogesterone in the trout Oncorhynchus mykiss. The enzyme, produced after expression of cDNA in COS-1 cells, was capable of converting dehydroepiandrosterone to androstenedione. The article, aimed at the finding of necessary probes for identification of steroidogenic enzyme genes in fish species, is in the frame of investigations on the molecular evolution of vertebrate steroidogenic enzymes. As for industrial production of carotenoid pigments such as β-carotene and astaxanthin utilized as food or feed supplements, the interest for the marine bacterium Agrobacterium aurantiacum is well documented [40] in the reported review discussing the advances achieved in the field of metabolic engineering for the microbial production of these compounds at the end of 1990s'. In 1999 [42], from the same bacterium, a gene cluster was introduced into E. coli to produce astaxanthin at a value 50 times higher than previously achieved. All the aspects about the enzymes of interest from the marine alga Ptilota filicina were already discussed in the previous paragraph, and recent interest for Dunaliella salina [46] in this field is also present; the latter study provides an insight for induction of β-carotene production in optimized cultivation systems. The case of Schizochytrium, the marine fungus producing significant amounts of docosahexaenoic acid (DHA) is of very interest due to positive effects on atherosclerosis, hypertriglyceridemia, hypertension, and cancers of the compound. The paper listed reports of the mechanisms of DHA biosynthesis in Schizochytrium constructing and analyzing cDNA library with the possible interesting prospect for new tools to engineer the production of PUFAs [43]. The sterol composition and the related biosynthetic genes were also studied in Chromera velia [44], a marine alveolate although the work is framed in the field of basic studies about sterol composition for deriving chemotaxonomic relationships.

Marine Isomerases Acting on Amino Acids and Peptides
In Table 4, many articles reported concern alanine racemase found in marine organisms. This enzyme, previously discovered in 1951 in other sources was found [47] in the bivalve Corbicula japonica in 1985. The authors partially purified the protein evaluating biochemical properties in relation to those of bacterial origin and linked enzyme role to the osmoregulation in these marine organisms. Many other works related to this enzyme are present [2,48,[51][52][53] always concerning the osmoregulation action, as in the crayfish Procambarus clarkii and in the hepatopancreas of black-tiger prawn, Penaeus monodon, up to recent interest for salinipeptins, a group of natural peptides in halotolerant Streptomyces isolated from the Great Salt Lake. Salinipeptins, natural products containing D-amino acids, are subjected to extensive enzymatic post-translational modifications during biogenesis [72]. They are substrates of potentially new epimerases of interest during these bioprocesses. A study related to genomic analysis of serine racemase is also found [61]; d-serine, besides frequently found in the bacterial cell walls, lipopeptides and siderophores, also exists as a free molecule in the marine environment with Roseobacter litoralis being a special producer. The cases related to disulfide isomerase are also numerous in this Table. They are mostly related to the studies on conotoxins or conopeptides, disulfide-rich peptides found in cone snails that found application in research and possible therapy. The studies mainly based on genetics focus the attention on post-translational reactions catalyzed by these enzymes for diversification of conopeptides structures and folding. Other interests are related to mechanisms of the immune response. Another topic of great interest among these isomerases is related to the cyclophilins (peptidyl-prolyl cis-trans isomerase, PPIase activity) that catalyze the isomerization of peptide bonds from trans to cis at proline residues and facilitate protein folding. Their expression is usually enhanced in response to inflammation or malignancy and are involved in functions related to cell metabolism and energy homeostasis and are of therapeutic importance for these and other actions (antifungal, antiviral, and antioxidant activities); they are also of economic importance in oysters cultivation for their involvement in the oyster immune response against infections of Crassostrea ariakensis by pathogen rickettsia-like organisms [66].

Other Enzymes
There are other reports on different enzymatic activities belonging to isomerases that do not fit well into the sections above and are mentioned in this paragraph. The first is an interesting review on chitin metabolism in the marine environment [87]. Authors hypothesized the presence of a mutase in the chitin catabolic cascade, in a more complex system with respect to the usually simplistic accepted hydrolytic pathway based on a chitinase producing the disaccharide N,N -diacetylchitobiose, and on a beta-N-acetylglucosaminidase producing the final product GlcNAc. The mutase, as described in this review, could represent the activity that converts GlcNAc-1-P, generated from small chitin oligosaccharides and chitobiose for entering the cell membrane, into the 6-P.
Various other articles are present dealing with dopachrome tautomerases involved in the final step of the enzymically regulated melanin biogenesis for the conversion of dopachrome into dihydroxyindoles. In marine organisms, especially in bivalves, the enzymes involved in the biogenesis of melanin are recognized as the general class of phenoloxidases while less is known about the existence and functional role of dopachrome tautomerase genes [88] in mollusk or other organisms [89,90]. However, this field of investigation is quite active due to the role of D-dopachrome tautomerase as cytokine, member of the macrophage migration inhibitory factor protein superfamily. They are associated to important physiological processes such as cell recruitment and migration, tumorigenesis and cancer progress, and inflammatory and autoimmune diseases. Many of these studies on immune system of marine fish may contribute to develop better disease management strategies for fish aquaculture as for Japanese sea bass (Lateolabrax japonicus) [91] or for the clam Ruditapes philippinarum [92].

Conclusions
The study of biocatalysts on a global scale from marine environment is just starting and possesses huge potential for the development of applications with industrial benefits due to marine biological diversity and to the specificity of biological marine metabolisms. This knowledge constitutes the core of marine biotechnology and only a deep understanding of the complexity of this ecosystem will enable human beings to protect the oceans and organisms populating them and pave the way for the sustainable exploitation of marine resources. Among the many fields covered that are highly relevant to societal challenges the biorefinery value-chain, food industries and fine chemicals are included among others. However, many challenges remain, generally speaking a deep comprehension of the "marine biotechnology landscape" and a multidisciplinary approach, in education and training [93].
In two comprehensive reports on examples of the application of marine-originating biocatalysts in 2010 and 2016 abovementioned [1,4], marine isomerases were hardly discussed although other classes of enzymes cited are used in food and pharmaceutical applications. After the analysis of literature articles, a first undoubted conclusion of this in-depth review is that curiosity-driven scientific interest for these enzymes seems to be present for a long time. Most of the literature found, tabulated according to the type of molecules on which these enzymes act, indicated a general scientific interest in historical perspective in different fields. As more recent examples, the biomedical field for allergen function of triosephosphate isomerases for seafood consumption, or efforts for the elucidation of the biosynthetic action of GDP-L-galactose mutase acting on interesting and rare L-Galf, must be mentioned. As for isomerases acting on lipid molecules, both basic interest for investigations on the molecular evolution of vertebrate steroidogenic enzymes or more oriented studies for carotenoid pigments production, are present. Similar situation for isomerases acting on protein molecules was noted, e.g., of alanine racemases, in the studies related to the role of the osmoregulation in marine organisms and for new epimerases catalyzing interesting bioprocesses during post-translational modifications of natural peptides known as salinipeptins.
On the other hand, application-oriented examples of marine isomerases already applied in biocatalysis are a few confirming the general result reported in previously published reviews [1,4]. However, major fields in which these few papers are framed are depicted in a better manner in this review. Works are placed at the cutting edge of biotechnological development such as the conversion of biomasses and saccharification of carbohydrate polymers (d-xylose isomerase), in biomedicine and nutraceuticals (isomaltulose production, l-arabinose isomerase for production of d-tagatose and ribose-5-phosphate isomerase), and in bioremediation field (cytochromes P450, carbazole remediation, etc.). Therefore, despite the scarcity of direct applicative examples found, novel stability features and chemical/stereochemical properties found in general examples of marine biocatalysts will be present in the numerous studied isomerases as well. These enzymes in fact can offer properties related to the habitat, which are greatly appreciated under a general biotechnological perspective. As last conclusion, it can be said that these properties will surely act as a promoter for future studies of these marine-originating isomerases in applied biocatalysis.
Funding: This research received no external funding.

Conflicts of Interest:
The author declares no conflict of interest