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Perspective

Taking Advantage of Promiscuity of Cold-Active Enzymes

by
Sondavid K. Nandanwar
1,
Shweta Bharat Borkar
2,
Jun Hyuck Lee
3,4 and
Hak Jun Kim
2,*
1
Department of Marine Convergence Design Program, Pukyong National University, Busan 48513, Korea
2
Department of Chemistry, Pukyong National University, Busan 48513, Korea
3
Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, Korea
4
Department of Polar Sciences, University of Science and Technology, Incheon 21990, Korea
*
Author to whom correspondence should be addressed.
Appl. Sci. 2020, 10(22), 8128; https://doi.org/10.3390/app10228128
Submission received: 24 October 2020 / Revised: 12 November 2020 / Accepted: 15 November 2020 / Published: 17 November 2020
(This article belongs to the Special Issue Cold-Active Proteins and Enzymes)
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Versions Notes

Abstract

:
Cold-active enzymes increase their catalytic efficiency at low-temperature, introducing structural flexibility at or near the active sites. Inevitably, this feat seems to be accompanied by lower thermal stability. These characteristics have made cold-active enzymes into attractive targets for the industrial applications, since they could reduce the energy cost in the reaction, attenuate side-reactions, and simply be inactivated. In addition, the increased structural flexibility could result in broad substrate specificity for various non-native substrates, which is called substrate promiscuity. In this perspective, we deal with a less addressed aspect of cold-active enzymes, substrate promiscuity, which has enormous potential for semi-synthesis or enzymatic modification of fine chemicals and drugs. Further structural and directed-evolutional studies on substrate promiscuity of cold-active enzymes will provide a new workhorse in white biotechnology.

Cold-active enzymes have drawn a lot of interest from academia and industry by virtue of their unique properties [1,2,3,4,5]. Since the description of thermolability of partially purified malic dehydrogenase from psychrophilic Vibrio marinus [6], a great number of cold-active enzymes from psychrophiles have been characterized [3]. Previous reviews have dealt with the structural and catalytic features of cold-active enzymes in depth along with their biotechnological and industrial applications [1,2,3,4,5]. Therefore, in this perspective, we will not simply cover the similar topics to the previous reviews, but address the different aspect of cold-active enzymes. In general, cold-active enzymes increase their catalytic rate (kcat) by modulating their thermodynamic activation parameters: reduction of activation enthalpy (ΔH‡) and increase of activation entropy (ΔS‡) penalty [2,3,7]. To achieve the feat, the enzymes have evolved to have more conformational flexibility at low temperature, compared to mesophilic orthologs, mostly at the expense of thermal stability. The evolutionary changes are encrypted on the primary sequence of these enzymes, including, but not limited to, higher number of glycine residues, reduced frequency of proline and arginine residues, increase of lysine, reduction of hydrophobic core, increased surface hydrophobicity, and reduction of hydrogen bonds, etc. [2,8,9,10]. Higher catalytic activity at low and moderate temperature with low thermolability have made cold-active enzymes into attractive targets for industrial applications such as production of food, additives in detergents, and synthesis of fine chemicals and pharmaceuticals. They have a few advantages over mesophilic orthologs in that they could reduce the energy cost and chemical side-reactions, and they could be easily inactivated by heat, since they operate at low temperature and perish easily at higher temperature [2,8,9,10]. Some cold-active enzymes currently being used in biotechnological and industrial applications are briefly listed in Table 1 [5].
In the context of industrial applications of cold-active enzymes, the features mentioned above have been well exploited. However, less investigated has been the broadening of the substrate specificity of cold-active enzymes due to their intrinsic characteristics, which may lead to increased enzyme promiscuity, compared to mesophilic orthologs [2,3,11,12,13,14,15]. Notably, enzymes with broad substrate specificity frequently have large active site to embrace substrates of varying sizes and shapes [16,17,18]. Especially, it is likely that the hydrophobic substrate binding sites could promote binding of the non-native substrates by hydrophobic effects. The increased exposure of hydrophobic residues in cold-active enzymes may stimulate substrate promiscuity [19,20,21]. The broader substrate specificity of cold-active enzymes, another intriguing property, could expand their application to the molecular design or semi-synthesis for fine chemicals and drugs. In this article, we focus on promiscuity of cold-active enzymes with a few recent reports for the possible application in organic synthesis. Note that we mainly refer to the cold-active enzymes as a cold-adapted enzymes from psychrophilic microorganisms.
Mesophilic and/or thermophilic enzymes showing broad substrate specificity have been well reviewed [18,22,23,24]. A partial list of mesophilic/thermophilic promiscuous enzymes along with psychrophilic orthologs are listed in Table 2. The critical features of enzymes to show promiscuity are the presence of an highly mobile active site loop [25], highly reactive active sites [23], active site embedded with cofactors [23], and highly hydrophobic substrate [26]. Some of these are similar to natural characteristics of cold-active enzymes. Hence, cold-actives enzymes could be promiscuous furthermore or fortuitously, compared to their mesophilic orthologs. In addition, it is probable that the substrate promiscuity of cold-active enzymes could be escalated or arisen by thermal fluctuation compared to mesophilic orthologs, without losing their advantages mentioned above. This may occur by chance due to the experimental conditions, which are different from natural environment. In the condition of in vivo and low temperature, cold-active enzymes should have strict substrate specificity. However, cold-active enzymes examined at room temperature may undergo an increased thermal fluctuation at the flexible loop regions near the active site and it may result in an unregulated substrate binding of xenobiotic compounds. This unexpected property of cold-active enzymes could be useful for enzyme-mediated semi-synthesis of fine chemicals and drugs. However, we should consider that the structural flexibility of the active sites is very similar between cold-active and mesophilic enzymes, meaning that substrate promiscuity applies to a very limited number of cold-active enzymes, since the active site residues are highly conserved [27,28,29]. Through comparative computational studies of cold- and warm-active enzymes, Åqvist and his coworkers supposed that structural flexibility of cold-active enzymes generally located at surface loops of the protein may confer higher reaction rates at low temperature [7,27,28,29]. Except for Candida antarctica Lipase B [24,30], to date, only a handful of reports on enzyme promiscuity and possible application of cold-active enzymes have been investigated [14,15,31,32].
The cold-active lipase B (CALB) from psychrophilic Candida Antarctica is one of the most studied cold-active enzymes with broad substrate specificity [4]. CALB has a structure similar to α/β hydrolases containing both α-helices and β-strand. The catalytic triad of Ser, His, and Asp/Glu is embedded near the surface within a catalytic pocket [88]. CALB has open, large, and medium substrate-binding pockets, which imparts structural flexibility and broad substrate specificity [89]. The flexible structure of CALB allows the efficient catalysis of various industrially important reactions. CALB catalyzes the epoxidation of α,β-unsaturated aldehydes with hydrogen peroxide to give α,β-epoxy aldehydes (Figure 1A) [90]. Aldol addition of ethanethiol to α,β-unsaturated hexanal gives diastereomeric addition products 2-methyl-3-(ethylthio)pentanal by CALB mutant (Ser105Ala) (Figure 1B) [91]. CALB also efficiently catalyzes aldol addition reaction between tricyclic diketone to give bis-product within 4 h (Figure 1C) [92]. Under polar and nonpolar solvents, CALB catalyzes different reactions [93]. For example, CALB catalyzes the amidation reaction between α,β-unsaturated ester and amine to give α,β-unsaturated amide under a polar solvent, whereas Michael addition is favored under a non-polar solvent to give β-amino ester (Figure 1D) [93]. The Baeyer–Villiger oxidation reaction of α-alkyl cyclic ketone to give ethyl 5-acetoxypentanoate can also be catalyzed by CALB (Figure 1E) [94]. These results imply that CALB could accommodate various substrates, which could be useful for the catalysis of industrially important reactions.
Similar to other acylaminoacyl peptidases (AAPs), the cold-active AAP (SpAAP) from psychrophilic bacterium Sporosacrina psychrophila possesses promiscuous ester hydrolysis activity [85,95]. The SpAAP exhibited hydrolytic activity on native N-acylated amino acids p-nitroanilide substrates, and on several promiscuous p-nitrophenyl esters of fatty acids of different chain length (C4-C18). Similar characteristics in the primary sequence were found in the primary sequence SpAAP with a lack of disulfide bonds, which probably confers structural flexibility for broader substrate specificity [95]. Contrary to hyperthermophilic AAP from Aeropyrum pernix (ApAAP) [84,96,97], SpAAP had a lower Km and kcat for N-acyl amino acids, but, interestingly, higher Km and kcat for short-chain fatty acids esters. This discrepancy was supported by finding that the diameter of a tunnel in the β-propeller domain to the active site is wider in SpAAP than in mesophilic and thermophilic orthologs [98].
The cold-active acetyl xylan esterase (PbAcE) from psychrophilic soil microbe Paenibacillus sp. had a broad substrate specificity (Figure 2A) [32]. Structural analysis of PbAcE suggested that flexible subunit movements and different active site loop conformations might allow PbAcE to have these characteristics different from other mesophilic and thermophilic homologs. PbAcE forms hexamer in solution and each monomer has α/β hydrolase fold with conserved catalytic triad residues of Ser185, Asp274, and His303. Activity assay data showed that PbAcE has typical properties of cold-active enzymes with strong low-temperature activity and broad substrate specificity. PbAcE has moderate and strong deacetylation activity for glucose penta-acetate and xylan acetate as well as reversible acetylation activity for xylan. In addition, PbAcE exhibited strong deacetylation activity for lipids and tertiary alcohol esters. These results proposed that PbAcE can accommodate various substrates and it could be useful for deacetylation biocatalyst of other xenobiotic ligands. For example, acetyl group of beta-lactam antibiotics such as cefotaxime, 7-Aminocephalosporic acid (7-ACA), and cephalosporin C were successfully removed by PbAcE (Figure 2B). The enzymatic modification of antibiotics is of significant importance to overcome the antibiotic resistance issue by increasing the chemical diversity. Furthermore, the possibility of commercial application of PbAcE was assessed by ensuring that the activity was maintained more than 80% even after immobilization followed by repetitive use of 18 times.
Le et al. reported that the recombinant HaSGNH1-type lipase from Halocynthiibacter arcticus catalyzed the synthesis of biodiesel and flavor compounds [31]. HaSGNH1 had a sandwich structure containing five central parallel β-sheets between two layers of four α-helices per layer. The catalytic triad of S18, D171, and H174 was embedded near the surface within a catalytic pocket. The solvent-exposed substrate-binding pocket offered the substrate flexibility to HaSGNH1. The substrate-binding pocket encircled with five amino acids, Asn83, Met86, Arg87, Phe131, and Ile173, regulated the entry of substrates via noncovalent interactions. HaSGNH1 was applied for the hydrolysis of tert-butyl esters, carbohydrate esters, and p-nitrophenyl esters. HaSGNH1 specifically hydrolyzed butyl esters such as p-nitrophenyl butyrate, 1-naphthyl acetate, tert-butyl acetate, and glyceryl tributyrate. To improve the catalytic activity of HaSGNH1 six mutants, Asn83Leu (1), Met86Glu (2), Met86Arg (3), Arg87Leu (4), Phe131Ala (5), and Ile173Phe (6) were generated using site-directed mutagenesis. Only four showed 120% enhanced hydrolysis of p-nitrophenyl butyrate than wild-type HaSGNH1. Two showed higher activity towards carbohydrate acetates, while three showed for larger substrates like p-nitrophenyl hexanoate. The bulky phenyl side chain of six preferred bindings to short-chain fatty esters like p-nitrophenyl acetate. The immobilized HaSGNH1-CELA was applied for the synthesis of petroleum, food, and cosmetic products (butyl and oleic esters) in a nonpolar solvent. The successful synthesis of butyl acetate, butyl butyrate, and oleic acid butyl ester was confirmed by gas chromatography analysis.
Another example is Cytochrome P450 monooxygenases (CYPs). CYPs are heme-containing enzymes that catalyze the hydroxylation of a wide variety of substrate molecules [99]. Bacterial steroid hydroxylase CYPs are particularly useful for effectively introducing hydroxyl groups to a wide range of steroids in the pharmaceutical industry. Thus, these CYPs have received increasing attention in recent years for industrial applications. Kim et al. (2017) reported a newly identified CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, and its crystal structure was determined at a resolution of 2.7 Å. Furthermore, structural comparisons with other CYPs showed that the α8–α9 loop region of BaCYP106A2 was intrinsically mobile and might be important for broad substrate specificity. The hydroxylase activity of BaCYP106A2 was examined with two different kinds of steroid substrates, 4-androstenedione and nandrolone, and the reaction was analyzed with HPLC and LC-MS [99]. CYPs oxidized 4-androstenedione and nandrolone to 15β-hydroxyandrostenedione and 15β-hydroxynandrolone, respectively. Unlike the mesophilic CYP orthologs from Bacillus meagterium [100], BaCYP106A2 seemed to mainly monohydroxylate steroid substrates with high conversion yields (Figure 3). The specific modification of pharmaceutically important compounds by the cold-active CYP sheds light on drug development and production.
In conclusion, structural flexibility of cold-active enzymes could enhance or give rise to enzyme promiscuity. Consequently, cold-active enzymes have merits for the enzymatic modification and semi-synthesis of fine chemicals and medicines by virtue of their broad substrate specificity on top of their existing merits as industrial catalysts. To take advantage of the under-utilized facet of cold-active enzymes, structure-based protein engineering and directed evolution should be employed in the near future to develop enzymes with improved substrate promiscuity for various chemical reactions.

Author Contributions

Conceptualization, S.K.N., J.H.L. and H.J.K.; software, S.B.B. and J.H.L.; validation, S.B.B., and J.H.L.; investigation, S.K.N. and S.B.B.; resources, S.K.N., J.H.L. and H.J.K.; writing—original draft preparation, S.K.N., J.H.L. and H.J.K.; writing—review and editing, H.J.K.; visualization, S.B.B.; supervision, H.J.K.; project administration, J.H.L.; funding acquisition, H.J.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was a part of the project titled “Development of potential antibiotic compounds using polar organism resources (15250103, KOPRI Grant PM20030)”, funded by the Ministry of Oceans and Fisheries, Korea.

Conflicts of Interest

No conflicts of interest.

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Applsci 10 08128 g001 550
Figure 1. Reactions catalyzed by Candida antarctica lipase B. (A) Epoxidation of α,β-unsaturated aldehydes with hydrogen peroxide, (B) aldol condensation (a and b) and Michael addition (c), (C) formation of bis-product, (D) formation of Michael addition and aminolysis product, and (E) Baeyer–Villiger oxidation reaction.
Figure 1. Reactions catalyzed by Candida antarctica lipase B. (A) Epoxidation of α,β-unsaturated aldehydes with hydrogen peroxide, (B) aldol condensation (a and b) and Michael addition (c), (C) formation of bis-product, (D) formation of Michael addition and aminolysis product, and (E) Baeyer–Villiger oxidation reaction.
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Figure 2. Broad substrate specificity of cold-active acetyl xylan esterase (PbAcE) from Paenibacillus sp. (A) Relatively larger substrate binding site of PbAcE. PbAcE is displayed space-filled and its substrate-binding site is dotted in red. The substrates modified by PbAcE are listed on the right. (B) Deacetylation activity against cephalosporin antibiotics. Acetyl group is circled in red.
Figure 2. Broad substrate specificity of cold-active acetyl xylan esterase (PbAcE) from Paenibacillus sp. (A) Relatively larger substrate binding site of PbAcE. PbAcE is displayed space-filled and its substrate-binding site is dotted in red. The substrates modified by PbAcE are listed on the right. (B) Deacetylation activity against cephalosporin antibiotics. Acetyl group is circled in red.
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Figure 3. Hydroxylation of 4-androstenedione (left) and nandrolone (right) by Cytochrome P450 monooxygenases from Bacillus sp. PAMC 23377.
Figure 3. Hydroxylation of 4-androstenedione (left) and nandrolone (right) by Cytochrome P450 monooxygenases from Bacillus sp. PAMC 23377.
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Table
Table 1. List of commercially available cold-active enzymes currently used in biotechnological and industrial applications.
Table 1. List of commercially available cold-active enzymes currently used in biotechnological and industrial applications.
EnzymeSource OrganismsFunction
Alkaline phosphatase (AP)Alteromonas undina P2Removal of 3′ and 5′ phosphate groups
Antarctic bacterium TAB 5
Pandalus borealis
LipaseCandida antarcticaResolution of chiral compounds and transesterification production of biodiesel
Uracil-DNA N-GlycosylaseGadus morhuaElimination of carryover polymerase chain reaction (PCR) products by hydrolyzing N-glycoside bond between uracil base and sugar skeleton, cutting uracil from dU-containing DNA.
Marine bacterium BMTU 3346
Psychrophilic marine bacterium
NucleaseShewanella sp. strain Ac10Cleavage of phosphodiester bonds in DNA to yield oligonucleotides with 5′-phosphate and 3′-hydroxyl termini.
Pandalus borealis
ProteaseArctic marine microbialHydrolyzation of a variety of peptide bonds
Table
Table 2. Partial list of mesophilic/thermophilic and cold-active enzymes showing substrate promiscuity.
Table 2. Partial list of mesophilic/thermophilic and cold-active enzymes showing substrate promiscuity.
Mesophilic or ThermophilesPsychrophiles
Enzyme Class AbbreviationSource OrganismsSubstrateProductAbbreviationSource OrganismsSubstrateProduct
LipasePPLPorcine pancreas [33]Aldehyde b
Nitromethane b		β-nitroalcohols b (ee%–85%)SPLDasyatis pastinaca [34]Triacylglycerols aGlycerol a Acetic acid a
ANLAspergillus Niger [35]Anilines b
1,3-Diketones b		Acetanilide bZC12Psychrobacter sp. ZY124 [36]Fatty acid esters a
p-nitrophenyl esters b		(S)-1-phenylethanol (ee%–92%) p-nitrophenol b Carboxylic acid b
KM12Bacillus Licheniformis [37]Fatty acid esters a
Long chain p-nitrophenyl
Esters b		Glycerol a Fatty acid a
p-nitrophenol b Carboxylic acid b
r-CALBAspergillus oryzae [38]Fatty esters aGlycerol a Fatty acid aCALBCandida Antarctica [35]Aldehyde b
Nitromethane b Anilines b
1,3-Diketones b		β-nitroalcohols b (ee%–90%)
Acetanilide b
SGNHSinorhizobium meliloti [39]p-nitrophenyl acetate b Butyrate b Valerate b α- and β-naphthyl
acetate b (R)- and (S)-methyl-3-hydroxy-2-methylpropionate b		p-nitrophenol b Acetic acid b Butyric acid b Valeric acid b β-naphthanol b (R)- and (S)-3-hydroxy-2-methylpropionate b Methanol bSGNHHalocynthiibacter arcticus [31]Fatty acid esters a
tert-butyl acetate b
Glucose pentaacetate b
p-nitrophenyl esters b		Glycerol a Fatty acid a
tert-butyl alcohol b Acetic acid b Glucose b p-nitrophenol b Carboxylic acid b
HSLBacillus halodurans [40]p-nitrophenyl palmitate a
Unsaturated fatty acyl esters a		p-nitrophenol a Palmitic acid a Unsaturated fatty acid a Glycerol aSHLSalinisphaera sp. P7-4 [41]Fatty acid esters a
p-nitrophenyl esters b
Glyceryl tributyrate b
4-methylumbelliferyl (4 MU)-acetate b
7-aminocephalosporanic acid b
Glucose pentaacetate b
(R,S)-naproxol acetate		Glycerol a
Fatty acid a
p-nitrophenol b
Carboxylic acid b
Glycerol a
tert-butyric acid b 4-Methylumbelliferone b
Deacetylated aminocephalosporanic acid b
Acetic acid b Glucose b
(R)-naproxol (ee%–8.3%)
DehydrogenaseDm7α-HSDHDeinococcus marmoris [25]Steroids a
α-ketoesters b		Keto-steroids a α-hydroxyester bKstDsRhodococcus ruber [42]4-androstene-3,17-dione a
9α-hydroxy-4-androstene-3,17-dione a		1,4-androstadiene-3,17-dione a 9-α-hydroxy-1,4-androstadiene-3,17-dione a
Ngi1_7αHSDHMetagenome [25]
Ec7α-HSDHEscherichia coli [25]
Ca7α-HSDHClostridium absonum [25]
Hh7α-HSDHHalomonas halodenitrificans [25]
Ca7β-HSDHClostridium absonum [25]
Cae7β-HSDHCollinsella aerofaciens [25]KshABMycobacterium tuberculosis [43]1,4-androstadiene-3,17-dione a 3-hydroxy-9,10-seconandrost-1,3,5(10)-trien-9,17-dione a
Hh7β-HSDHHalomonas halodenitrificans [25]
Bsp7β-HSDHBrucella sp. [25]
Rs7β-HSDHRhodobacter sphaeroides
Csp12α-HSDHClostridium sp. [25]
Sc7β-HSDHStanieria cyanosphaera [25]
CaADHClostridium acetobutylicum [44]MoADHMoraxella sp. TAE123 [45] Carboxylic aicd
MtADHMycobacterium tuberculosis [46]N/A
IsomerasePriAActinobacteria [47]N’-[(5′-phosphoribosyl) formimino]
-5-aminoimidazole-4-carboxamide
Ribonucleotide a
Phosphoribosyl anthranilate b		N’-[(5′-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide a 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate bN/A
TIMPyrococcus furiosus [48]Cellobioside–resorufin aResorufin aTIMPseudomonas sp. [49]glyceraldehyde-3-phosphate a Triose phosphate aDihydroxyacetone phosphate a
TransferasePpATaseCHPseudomonas protegens [50]2,4- diacetylphloroglucinol a
Aniline derivatives b		1,3,5-trihydroxy-Phloroglucinol a N-acetanilide derivative bN/A
HCTSwitchgrass Arabidopsis [51]Lignin bp-Coumaroyl conjugatesN/A
hGSTT1-1Human [52]Glutathione a
7-amino-4-chloromethyl coumarinb 		rHsGSTHalomonas sp. ANT108 [53]Glutathione a Chlorodinitrobenzene aGlutathione-Chlorodinitrobenzene a conjugate
ReductaseCNRHaemophilus influenzae [54]Chloramphenicol a
4-nitrobenzene derivatives b		Reduced chloramphenicol a
Aniline derivatives b		N/A
FabIPlasmodim falciparum [55]Crotonyl Coenzyme A a,
trans-2-pentenal b
3-pentene-2-one b		Saturated Crotonyl Coenzyme A a 2-pentanal b
pentane-2-one b		Fab A, Fab B, Fab D, Fab F, Fab G, Fab H and Fab ZShewanella putrefaciens WS13 [56]Fatty acid metabolism
DHDPRMRSA [57]Dihydrodipicolinate a
NADPH a		Tetrahydrodipicolinate aPaDHDPRPaenisporosarcina sp. TG-14 [58]Dihydrodipicolinate a
NADPH a		Tetrahydrodipicolinate a
SynthasesDTSsKitasatospora griseola, Streptomyces cyslabdanicus, Salinispora arenicola, Bradyrhizobium japonicum, Erwinia tracheiphila [59]Terpentedienyl diphosphate a
copalyl diphosphate (CPP) a
ent-CPP a
(E,E,E)-geranylgeranyl diphosphate b
(Z,Z,Z)-nerylneryl diphosphate b
(E,E)-farnesyl diphosphate b
(E,E,E,E)-geranylfarnesyl diphosphate b
syn-CPP b
7-endo-CPP b, ent-7-endo-CPP b
8α-hydroxy-CPP b
8β-hydroxy-ent-CPP b
9α-hydroxy-CPP b
kolavenyl diphosphate (KPP) b
ent-KPP b
halimadienyl diphosphate (HPP) b
syn-HPP b
syn-halima-5(10) b
13E-dienyl diphosphate b
mutildienyl diphosphate b		 N/A
NalEscherichia coli K12 [60]N-acetylglucosamine (GlcNAc) a
Pyruvate a		 MvNeuBMoritella viscosa [61]N-acetylneuraminic acid a Phosphoenolpyruvate b
HydroxylaseEctDPseudomonas stutzeri [62]Ectoine a
Homoectoine b		 EctDSphingopyxis alaskensis [63]L-aspartate-β-semialdehyde a
TfdB-JLURhodococcus opacus 1G
Rhodococcus erythropolis Pseudomonas sp. NCIB934 [64,65]		2,4-dichlorophenoxyacetic acid a
Chlorophenol b
Dichlorophenol b
Trichlorophenol derivatives b		 N/A
cPAHChromobacterium violaceum [66]Phenylalanine a PAHColwellia psychrerythraea [67]Phenylalanine a
HydrolasesMhgPseudomonas fluorescens [68]γ-lactamase a
Perhydrolase a
Esters b		 HsGSTGlaciozyma antarctica [69]Dienelactone a
p-nitrophenyl esters b
DehydrataseDaDHTHerbaspirillum huttiense [70]D- altronate a
L-fuconate b
D-Arabinonate bL-xylonate b
D-idonate b
L-gluconate b		Pyruvate b L-Lactate b Glycolate b Glycerate bLsDHTPseudomonas aeruginosa [71]L-serine a
EsteraseEaGPEEnterobacter aerogenes [72]Diesters glycerol-3-Phosphoethanolamine a
4-nitrophenyl phosphate (NPP) b
Bis(4-nitrophenyl) phosphate (BNPP) b
Diethyl-nitrophenylphosphate (paraoxon) b		Glycerol a Ethanolamine Phosphoric acid a Carboxylic acid a Diethyl phosphate b p-nitrophenol bN/A
PLLSaccharolobus solfataricus [73]Paraoxon a
chemical warfare nerve agents b		Diethyl phosphate a p-nitrophenol aN/A
PLLDeinococcus radiodurans [74]Phosphotriesters a
Organophosphates b		Phosphoric acid a Alcohols a N/A
AXEBacillus subtilis [75]Glycerol triacetate b
Ethyl acetate b		Glycerol b Acetic acid b
Ethanol b		PbAcEPaenibacillus sp. R4 [32]Acetyl xylan a
p-nitrophenyl esters b
α-β-naphthyl esters b
carbohydrate esters b
tertiary alcohol esters b
lipids b, and antibiotics b		Xylanol a Acetic acid a
p-nitrophenol b Carboxylic acid b α-β-naphthanol b Carbohydrate b tert- alcohol b Fatty acid b Glycerol b
N/A SfSFGHShewanella frigidimarina [76]p-nitrophenyl esters b
glucose pentaacetate b		p-nitrophenol b Carboxylic acid b Glucose b Acetic acid b
EstBacillus subtilis DR8806 [77]p-nitrophenyl acetate ap-nitrophenol a Acetic acid aEst12Butyrivibrio proteoclasticus [78]Carbohydrate esters a
short- and middle-chain
p-nitrophenol esters b		Carbohydrate a Carboxylic acid a p-nitrophenol b
LactonaseSsoPoxSulfolobus solfataricus [79]Acyl-homoserine lactones a
Paraoxon b		Acyl-homoserine a Lactones a Diethyl phosphate b p-nitrophenol bAidPPlanococcus sp.[80]Acyl-homoserine lactonesaAcyl-homoserine a Lactones a
PeptidaseST1737Sulfolobus tokodaii [33]Aldehyde b
Nitromethane b		β-nitroalcohols (ee%–85%)ColAPColwellia psychrerythraea [81]l-alanine-4-nitroanilide hydrochloride aAlanine a p-nitroaniline a
ST0779Sulfolobus tokodaii [33]Aldehyde b
Nitromethane b		β-nitroalcohols (ee%–94%)N/A
SAtAspergillus terreus [82]Azocasein aLeucine a Glycine a p-nitroaniline a Benzyl alcohol aLsPcLysobacter sp. [83]Azocasein a
Gelatin and feather b		Leucine a Glycine a p-nitroaniline a Benzyl alcohol a Alanine b Arginine b Aspartic acid b Proline b Hydroxy Proline b
ApAAPAeropyrum pernix [84]Abz-GFEPF(NO2)RA b
Abz-GFRPF(NO2)RA b
Abz-SAVLQSGF(NO2)A b Abz-EALFQGPF(NO2)A b Ac-Phe-Nap b Gly-Phe-Nap b
Ac-Phe-Nan b 		SpAAPSporosarcina psychrophila [85]N-acetyl-L-leucine-p-nitroanilide a
Butyl-p-nitrophenyl esters b		N-acetyl-L-leucine a p-nitroaniline a Butyric acid b p-nitrophenol
GlucanaseENG16ACoprinopsis cinerea [86]β-glucan a Laminarin aShort chain carbohydratesGaEbGGlaciozyma antarctica PI12 [87]Laminarin a
Lichenin b
glucan polysaccharides b		Short chain carbohydrates
a—Natural substrate. b—Promiscuous substrate.
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Nandanwar, S.K.; Borkar, S.B.; Lee, J.H.; Kim, H.J. Taking Advantage of Promiscuity of Cold-Active Enzymes. Appl. Sci. 2020, 10, 8128. https://doi.org/10.3390/app10228128

AMA Style

Nandanwar SK, Borkar SB, Lee JH, Kim HJ. Taking Advantage of Promiscuity of Cold-Active Enzymes. Applied Sciences. 2020; 10(22):8128. https://doi.org/10.3390/app10228128

Chicago/Turabian Style

Nandanwar, Sondavid K., Shweta Bharat Borkar, Jun Hyuck Lee, and Hak Jun Kim. 2020. "Taking Advantage of Promiscuity of Cold-Active Enzymes" Applied Sciences 10, no. 22: 8128. https://doi.org/10.3390/app10228128

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