Next Article in Journal
Abatement of VOCs Using Packed Bed Non-Thermal Plasma Reactors: A Review
Previous Article in Journal
Unprecedented Multifunctionality of Grubbs and Hoveyda–Grubbs Catalysts: Competitive Isomerization, Hydrogenation, Silylation and Metathesis Occurring in Solution and on Solid Phase
Previous Article in Special Issue
Stabilization of a Lipolytic Enzyme for Commercial Application
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Catalysts
Volume 7
Issue 4
10.3390/catal7040112
catalysts-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Improving the Stability of Cold-Adapted Enzymes by Immobilization

by
ChangWoo Lee
1,*,
Sei-Heon Jang
1 and
Hye-Shin Chung
2
1
Department of Biomedical Science and Center for Bio-Nanomaterials, Daegu University, Gyeongsan 38453, Korea
2
Department of Biological Sciences and Biotechnology, Hannam University, Daejeon 34054, Korea
*
Author to whom correspondence should be addressed.
Catalysts 2017, 7(4), 112; https://doi.org/10.3390/catal7040112
Submission received: 13 January 2017 / Revised: 30 March 2017 / Accepted: 4 April 2017 / Published: 12 April 2017
(This article belongs to the Special Issue Immobilized Enzymes: Strategies for Enzyme Stabilization)
Browse Figure
Versions Notes

Abstract

:
Cold-adapted enzymes have gained considerable attention as biocatalysts that show high catalytic activity at low temperatures. However, the use of cold-adapted enzymes at ambient temperatures has been hindered by their low thermal stabilities caused by their inherent structural flexibilities. Accordingly, protein engineering and immobilization have been employed to improve the thermal stability of cold-adapted enzymes. Immobilization has been shown to increase the thermal stability of cold-adapted enzymes at the critical temperatures at which denaturation begins. This review summarizes progress in immobilization of cold-adapted enzymes as a strategy to improve their thermal and organic solvent stabilities.

1. Introduction

Access to permanently cold regions is not easy, but an increasing number of psychrophilic microorganisms has been deposited into culture collections such as the Polar and Alpine Microbial Collection (PAMC) and are available to researchers [1,2]. Cold-adapted microorganisms are largely categorized into psychrophiles (cold-loving organisms that show optimal growth at less than 15 °C) and psychrotrophs (cold-tolerant organisms that show optimal growth at 20–25 °C) [3,4]. Cold-adapted microorganisms have developed remarkable features enabling survival at low temperatures, such as increased unsaturated fatty acids content in the membrane and increased solute concentrations inside cells [5,6].
Enzymes of cold-adapted microorganisms have evolved mechanisms to avoid the rigidity of protein structures at low temperatures to maintain high catalytic activity via structural modifications, including reduced intramolecular bonds, loop extension, and increased active site accessibility [7,8,9]. As a result, cold-adapted enzymes exhibit enhanced structural flexibilities compared to their mesophilic counterparts, particularly with more local flexibility around the active site rather than global flexibility [10,11,12]. The regions distal to the active site have also been shown to affect the catalysis of active sites [13,14,15]. A lower Gibbs free energy of activation (ΔG‡) resulting from weak substrate binding enables cold-adapted enzymes to perform high catalytic activity at temperatures of approximately 0–30 °C [8,16]. Cold-adapted enzymes exhibit a lower enthalpy (ΔH) and a more negative entropy (ΔS) than their mesophilic counterparts [8]. A larger negative ΔS value indicates that psychrophilic enzymes undergo a larger conformational change, as the enzymes are more flexible without substrate binding [8]. Comparative genome analysis revealed that cold-adapted enzymes exhibit increased glycine content but reduced arginine, proline, and acidic amino acid content [17,18,19]. Meanwhile, glycine residues provide enhanced flexibility, while arginine and acidic amino acids residues participate in ionic interactions, and proline residues provide rigidity to protein structures [20,21,22]. Oligomerization, which is generally used for thermal adaptation of hyperthermophilic enzymes [23], has also been shown to be a strategy for some cold-adapted enzymes such as a GH1 β-glucosidase [24]. Each protein has adopted one or more of these structural modifications to adapt to low temperatures [25].
However, the increased flexibility of cold-adapted enzymes results in weak thermal stabilities at elevated temperatures. The active site appears to be the most heat-labile, especially for multi-domain psychrophilic enzymes [12,26,27]. Trade-offs between stability and activity are the general mechanisms of the cold-adaptation of enzymes [9,28,29].
Santiago et al. reported that, among 92 cold-adapted enzymes reported between 2010 and 2016 in the literature, hydrolases accounted for 91%, followed by oxidoreductases (4%), transferases (2%), isomerases (1%), and ligases (1%) [30]. The majority of these enzymes showed apparent optimum temperatures at 20–45 °C [30]. However, many cold-adapted enzymes do not maintain thermal stability at the apparent optimum temperatures, reflecting their actual temperature of physiological adaptation at much lower temperatures. In contrast, certain enzymes from cold-adapted yeasts, such as lipase A and B from Candida antarctica (CalA and CalB), are thermostable. CalA is extremely thermostable with an apparent optimum temperature above 90 °C [31], and CalB shows thermal stability at temperatures up to 60 °C in non-aqueous solutions [32,33].
The temperature adaptation of the catalytic properties has made cold-adapted enzymes promising biocatalysts for industrial applications, and they are now used in the synthesis of heat-labile fine chemicals, as additives in food processing at low temperatures, in detergents for cold-water laundry, and in the bioremediation of contaminated soils and waters in cold regions [34,35,36]. Thermolability is sometimes preferred, as in the selective thermal inactivation of enzymes by subtle increases in temperature in the food, dairy, and brewing industries, as well as in molecular biology [37]. However, the use of cold-adapted enzymes at ambient temperatures has been hindered by thermolability, despite cold-adapted enzymes exhibiting the potentials for industrial applications. In this review, we summarized the recent progress in improving the stability of cold-adapted enzymes, particularly via immobilization.

2. Protein Engineering to Improve the Stability of Cold-Adapted Enzymes

Many efforts have been made to improve the weak thermal stability of cold-adapted enzymes via protein engineering [38]. Protein engineering such as directed evolution and rational design has traditionally been the most popular tool to improve the activity and stability of cold-adapted enzymes [38]. Directed evolution, which mimics natural evolution, employs error-prone polymerase chain reaction and DNA shuffling [39]. Directed evolution generates a variety of mutations without any structural information [39], but is limited in that a large number of possible variant proteins are generated and that such evolution requires a high throughput screening method [40]. In contrast, rational design uses structural information and is more efficient than directed evolution when structural information is available [41,42,43].
As hot spots of mutation for activity increase are rather easily identified in active sites, rational design has been used to improve the activity of cold-adapted enzymes [38]. However, for improving thermal stability, hot spots of mutation were not easily identified, so directed evolution has been more successful than rational design [44,45].
Multiple sequence alignment of cold-adapted enzymes with homologous mesophilic and thermophilic enzymes provides valuable insight into the rational design of mutations in cold-adapted enzymes, whereby the corresponding amino acids of thermophilic enzymes are considered for mutagenesis [46]. Mutation of tryptophan 208 in the active site wall of cold-adapted Pseudomonas mandelii esterase EstK to tyrosine, in which tyrosine is highly conserved in the corresponding position of hyperthermophilic esterases, conferred catalytic site thermal stability in EstK via a strengthened hydrogen bond [47]. However, the prediction of mutation sites and proper amino acid substitutions remains a challenge [46]. It should be noted that, for cold shock proteins from the mesophile Bacillus subtilis and the thermophile Bacillus caldolyticus, which differ by 12 out of 67 amino acid residues, only 2 of these 12 residues were responsible for the difference in thermal stability in the B. caldolyticus protein [48].

3. Immobilization of Cold-Adapted Enzymes

Although an increasing number of cold-adapted enzymes has been reported, the majority of studies conducted to date have focused on the isolation, biochemical characterization, and cold-adaptation mechanism [30]. Immobilization of cold-adapted enzymes on a solid matrix is required for their industrial applications as well as removal and recovery of the enzymes for continuous use [49]. In fact, enzyme immobilization is a mature technology that was first commercialized in the 1960s [50]. Enzyme immobilization has been shown to increase (i) stability, (ii) reusability, (iii) activity, (iv) specificity, and (v) handling of the catalyst [49]. Conventional immobilization methods are divided into four categories: adsorption, covalent binding, cross-linking, and entrapment (Figure 1), each of which has advantages and disadvantages [49].
Many excellent books and review articles on enzyme immobilization are available [49,51,52,53,54,55,56]. The C. antarctica lipases CalA and CalB and lipases from mesophilic yeast Yarrowia lipolytica have been extensively used for the development of new immobilization methods [57,58,59]. Among the enzymes, CalB has received the most attention for biotechnological application and is commercially available as free enzyme or in immobilized form on different carriers, including epoxy-activated macroporous acrylic resin (brand name Novozym 435) [57,59,60]. However, these enzymes from C. antarctica are thermostable at temperatures of 60–90 °C [31,32,33] and thus beyond the scope of this review, even though they originated from cold-adapted yeasts.
When compared with investigations of the immobilization of mesophilic or thermophilic enzymes, few studies of the immobilization of cold-adapted enzymes have been conducted. Several studies have reported the thermal and storage stabilities of immobilized cold-adapted enzymes along with gene cloning and biochemical characterization [61,62,63,64,65,66]. Although temperature and organic solvents are two major considerations for industrial applications of enzymes, immobilization of cold-adapted enzymes has mainly been evaluated with regard to thermal stability. As cold-adapted enzymes have been immobilized in recent years, more new technologies, such as the use of magnetic nanoparticles [67], single-walled carbon nanotubes (SWCNTs) [68], and graphene oxide [69], have been adopted along with traditionally used matrices such as Sepharose beads. Graphene oxide, a two-dimensional carbon nanosheet with oxygen-containing functional groups (alcohols, epoxides, and carboxylic acids), has recently been used for enzyme immobilization [70]. The degree of oxidation in graphene oxide can be modulated by chemical reduction, and mesophilic enzymes immobilized on chemically reduced graphene oxide showed improved conformations via hydrophobic interactions when compared with those on graphene oxide [71]. Among the immobilization methods, covalent binding has been the most widely used for immobilization of cold-adapted enzymes followed by adsorption and entrapment. Examples of immobilized cold-adapted enzymes are listed in Table 1.

3.1. Adsorption

Physical adsorption, one of the earliest immobilization methods, is still the technique most commonly used to immobilize enzymes [49,75]. In this method, enzymes are reversibly immobilized on a solid support [49]. Enzyme activity is maintained upon adsorption, but enzymes are likely to be detached from the solid support because of the relatively weak interaction with the support [49].
A few cold-adapted enzymes have been immobilized on solid support, including diethylaminoethyl (DEAE)-Sepharose [62] or polyethylenimine (PEI)-coated agarose [63], via ionic interaction. A cold-adapted β-galactosidase, which is preferred for the degradation of lactose during transport and storage of milk at low temperatures, was immobilized on DEAE-Sepharose [62]. This Pseudoalteromonas sp. β-galactosidase from Antarctica showed storage stability at 4 °C after 1 week, but no additional thermal properties of the immobilized enzyme were measured at elevated temperatures [62]. A cold-adapted esterase EstH from the marine bacterium Zunongwangia sp. was immobilized on cellulose-coated magnetite (Fe3O4) via hydrogen bonding with cellulosic sheaths and showed enhanced thermal stability and storage stability [61]. Magnetic nanoparticles have the advantage of easy separation of the enzyme from the reaction mixture by applying magnetic fields [67]. However, magnetic nanoparticles are easily oxidized in air and likely to be clustered together [76].

3.2. Covalent Binding

Enzymes immobilized by covalent binding generally show enhanced stability and high reusability [49]. The enzyme leaching shown in adsorption is significantly reduced in covalent binding during reaction and washing [49]. Cold-adapted enzymes have been covalently immobilized on several solid supports, including agarose [63], chitosan [73], Sepharose [62], silica [66], and graphene oxide [72]. The conformational changes resulting from covalent modifications generally lead to reduced enzymatic activity.
The cold-adapted β-galactosidase (Pseudoalteromonas sp.) was also covalently immobilized on Sepharose via coupling to epoxy groups or to the PEI-coated beads via glutaraldehyde [62]. The enzyme bound to the PEI-coated support was more stable than that immobilized on epoxy-functionalized Sepharose as well as DEAE-Sepharose via ionic interaction [62]. Conversely, another cold-adapted Pseudoalteromonas sp. β-galactosidase from Antarctica was immobilized on chitosan via covalent binding using glutaraldehyde [73]. The immobilized β-galactosidase, which showed similar profiles with free enzyme but with a 10 °C shift in apparent optimum temperature, was effective at hydrolyzing lactose in milk over a temperature range of 4 to 30 °C [73].
Immobilization lead to overall improvement in the thermal stability of cold-adapted enzymes, especially at critical temperatures, at which the enzymes begin to lose their thermal stability. A psychrophilic pullulanase (Exiguobacterium sp.) was shown to lose its activity from 50 °C with almost no activity at 60 °C [66]. Pullulanase immobilized on epoxy-functionalized silica maintained its thermal stability at 50 °C and showed significantly improved thermal stability at 60 °C and 70 °C during 90 min of incubation [66]. Similarly, a cold-adapted P. mandelii esterase EstK covalently tethered to graphene oxide exhibited higher thermal stability than that of free enzyme at 20–40 °C, but immobilization did not help improve thermal stability beyond the temperatures [72]. The EstK-graphene oxide complex showed reduced catalytic efficiency of approximately 40% of that of free EstK at 40 °C, owing to distortion of the structure upon covalent attachment [72].
However, direct covalent attachment of nucleoside 2′-deoxyribosyltransferase from Bacillus psychrosaccharolyticus (BpNDT), a multimeric protein involved in nucleotide synthesis, onto glyoxyl-agarose resulted in loss of activity, possibly via distortion of the protein structure or subunit dissociation of the enzyme [63]. BpNDT immobilized on PEI-coated agarose via ionic interaction followed by cross-linking with aldehyde-dextran showed reusability at 37 °C for at least 30 cycles with 25% loss of activity [63]. The addition of glycerol (20% working concentration) as a protective agent helped maintain the tertiary structure of BpNDT with improved activity of the recovered enzyme [77]. This strategy could be used for immobilization of multimeric enzymes preventing subunit dissociation [78]. However, care should be taken to prevent the formation of extra inter-subunit covalent linkage.
Overall, cold-adapted enzymes covalently immobilized on solid support showed improved thermal stability but with reduced catalytic activity when compared with free enzymes.

3.3. Cross-Linking and Entrapment

Cross-linking is used to covalently link enzymes together [79]. Cross-linking of enzyme aggregates has been shown to be effective when a large loading of enzymes is required without support material [79]. Enzymes are precipitated, after which they are cross-linked using agents such as glutaraldehyde or aldehyde dextran [80]. On the other hand, enzymes can also be trapped or encapsulated in a polymer matrix, usually insoluble beads or microspheres [49]. The insoluble carrier may block the active site. However, only a few cold-adapted enzymes were entrapped to date. A cold-adapted cellulase from Antarctic Pseudoalteromonas sp. was covalently immobilized in sodium alginate gel beads for the ethanol fermentation of kelp cellulose [74]. The immobilized cellulase showed 58% activity after seven cycles [74]. In another study, a cold-adapted pectate lyase, which catalyzes hydrolysis of the α-1,4 linkage of homogalacturonan, was supplemented with calcium hydroxyapatite nanoparticles as a substitute for cationic activator calcium and entrapped in lipid-functionalized SWCNT [64]. The immobilized pectate lyase showed stability at a wide range of temperatures from 4 to 80 °C, as well as storage stability under repeated freeze-thaw cycles [64].

4. Stability in Organic Solvent

The use of organic solvent in enzymatic reactions is important in industrial applications to increase the solubility of non-water soluble substrates [81,82]. Organic solvents strip off water molecules from the protein surface and penetrate into the enzyme, causing denaturation of the protein structure [83]. The varying hydrophobicity of organic solvents has different effects on catalytic activity of enzymes in each organic solvent [84]. The inherent conformational flexibility of cold-adapted enzymes makes the enzymes susceptible to denaturation by heat and organic solvents [85]. Specifically, heat denatures hydrogen bonding in the protein structure, whereas organic solvents disrupt hydrophobic interactions [85]. Efforts that include the isolation of organic solvent-tolerant enzymes, the modification of enzyme structures, and the modification of the solvent environment [81,82,85] have been made to increase the stability of enzymes in organic solvent. Since the identification of the first organic solvent-tolerant lipase Lip9 from Pseudomonas aeruginosa LST-03 with high activity in n-decane, n-octane, and dimethyl sulfoxide (DMSO) [86], many organic solvent-tolerant enzymes have been cloned [87,88]. Several studies have used directed evolution to improve organic solvent tolerance of enzymes [89,90,91]. However, rational design was not efficient at improving the stability of enzymes in organic solvents. The mutation of Lip9 on the surface of the protein via directed evolution resulted in a 9- to 11-fold increase in stability in cyclohexane and n-decane [92]. However, only a few cold-adapted and organic solvent-tolerant enzymes are known [93,94,95,96,97], and fewer studies have evaluated the effects of immobilization on organic solvent stability.
Recently, the polar organic solvents, methanol and DMSO, were shown to increase the conformational flexibility of the cold-adapted and organic solvent-tolerant lipases PML (Proteus mirabilis) and LipS (P. mandelii) with increased activity at distinct organic solvent concentrations [98]. Because mesophilic organic solvent-tolerant enzymes have shown no significant changes in the structure in water and pure organic solvent [99,100,101], the effects of organic solvents on conformational flexibility were considered specific to cold-adapted enzymes displaying inherent conformational flexibility [98]. As a result of increased flexibility in polar organic solvents, both PML and LipS exhibited weaker thermal stability as indicated by reduced thermal denaturation midpoints [98]. Immobilization of cold-adapted enzymes on adequate support in the presence of organic solvents is highly sought after for the application of cold-adapted enzymes in chemical reactions requiring organic solvents or the bioremediation of organic solvent-contaminated sites.

5. Conclusions

An increasing number of cold-adapted enzymes were reported, and their cold-adaptation mechanisms are being characterized; however, the number of immobilized cold-adapted enzymes is much lower than the number of immobilized mesophilic or thermophilic enzymes. Immobilization has been shown to improve thermal stability of cold-adapted enzymes, especially at critical temperatures at which the enzymes begin to unfold. Most studies evaluated the thermal stability or storage stability of immobilized cold-adapted enzymes, but not much information is available regarding how the immobilization affected the conformations of these enzymes. More comparative studies are needed with warmer-temperature enzymes to elucidate the effects of immobilization on the stability–activity relationship. With rapid development in the field of enzyme immobilization, new support materials with enhanced functionality will improve the applications of cold-adapted enzymes at elevated temperatures and in organic solvents. Immobilization of cold-adapted enzymes will be an efficient method when enzymatic reactions at ambient temperatures with continuous use are required.

Acknowledgments

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A3A01019797 to C.L.).

Conflicts of Interest

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Lee, Y.M.; Kim, G.; Jung, Y.J.; Choe, C.D.; Yim, J.H.; Lee, H.K.; Hong, S.G. Polar and Alpine Microbial Collection (PAMC): A culture collection dedicated to polar and alpine microorganisms. Polar Biol. 2012, 35, 1433–1438. [Google Scholar] [CrossRef]
  2. Nichols, D.; Bowman, J.; Sanderson, K.; Nichols, C.M.; Lewis, T.; McMeekin, T.; Nichols, P.D. Developments with Antarctic microorganisms: Culture collections, bioactivity screening, taxonomy, PUFA production and cold-adapted enzymes. Curr. Opin. Biotechnol. 1999, 10, 240–246. [Google Scholar] [CrossRef]
  3. Helmke, E.; Weyland, H. Psychrophilic versus psychrotolerant bacteria-occurrence and significance in polar and temperate marine habitats. Cell. Mol. Biol. 2004, 50, 553–561. [Google Scholar] [PubMed]
  4. Cowan, D.A.; Casanueva, A.; Stafford, W. Ecology and biodiversity of cold-adapted microorganisms. In Physiology and Biochemistry of Extremophiles; American Society of Microbiology: Washington, DC, USA, 2007. [Google Scholar]
  5. D’Amico, S.; Collins, T.; Marx, J.C.; Feller, G.; Gerday, C. Psychrophilic microorganisms: Challenges for life. EMBO Rep. 2006, 7, 385–389. [Google Scholar] [CrossRef] [PubMed]
  6. De Maayer, P.; Anderson, D.; Cary, C.; Cowan, D.A. Some like it cold: Understanding the survival strategies of psychrophiles. EMBO Rep. 2014, 15, 508–517. [Google Scholar] [CrossRef] [PubMed]
  7. Struvay, C.; Feller, G. Optimization to low temperature activity in psychrophilic enzymes. Int. J. Mol. Sci. 2012, 13, 11643–11665. [Google Scholar] [CrossRef] [PubMed]
  8. Gerday, C. Catalysis and Protein Folding in Psychrophiles. In Cold-Adapted Microorganisms; Yumoto, I., Ed.; Caister Academic Press: Norfolk, UK, 2013; pp. 137–160. [Google Scholar]
  9. Siddiqui, K.S.; Cavicchioli, R. Cold-adapted enzymes. Annu. Rev. Biochem. 2006, 75, 403–433. [Google Scholar] [CrossRef] [PubMed]
  10. Fields, P.A.; Somero, G.N. Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proc. Natl. Acad. Sci. USA 1998, 95, 11476–11481. [Google Scholar] [CrossRef] [PubMed]
  11. Tang, M.A.; Motoshima, H.; Watanabe, K. Fluorescence studies on the stability, flexibility and substrate-induced conformational changes of acetate kinases from psychrophilic and mesophilic bacteria. Protein J. 2012, 31, 337–344. [Google Scholar] [CrossRef] [PubMed]
  12. Siddiqui, K.S.; Feller, G.; D’Amico, S.; Gerday, C.; Giaquinto, L.; Cavicchioli, R. The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase. J. Bacteriol. 2005, 187, 6197–6205. [Google Scholar] [CrossRef] [PubMed]
  13. Papaleo, E.; Riccardi, L.; Villa, C.; Fantucci, P.; De Gioia, L. Flexibility and enzymatic cold-adaptation: A comparative molecular dynamics investigation of the elastase family. Biochim. Biophys. Acta 2006, 1764, 1397–1406. [Google Scholar] [CrossRef] [PubMed]
  14. Merlino, A.; Russo Krauss, I.; Castellano, I.; De Vendittis, E.; Rossi, B.; Conte, M.; Vergara, A.; Sica, F. Structure and flexibility in cold-adapted iron superoxide dismutases: The case of the enzyme isolated from Pseudoalteromonas haloplanktis. J. Struct. Biol. 2010, 172, 343–352. [Google Scholar] [CrossRef] [PubMed]
  15. Papaleo, E.; Pasi, M.; Riccardi, L.; Sambi, I.; Fantucci, P.; De Gioia, L. Protein flexibility in psychrophilic and mesophilic trypsins. Evidence of evolutionary conservation of protein dynamics in trypsin-like serine-proteases. FEBS Lett. 2008, 582, 1008–1018. [Google Scholar] [CrossRef] [PubMed]
  16. Lonhienne, T.; Gerday, C.; Feller, G. Psychrophilic enzymes: Revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim. Biophys. Acta 2000, 1543, 1–10. [Google Scholar] [CrossRef]
  17. Casanueva, A.; Tuffin, M.; Cary, C.; Cowan, D.A. Molecular adaptations to psychrophily: The impact of ‘omic’ technologies. Trends Microbiol. 2010, 18, 374–381. [Google Scholar] [CrossRef] [PubMed]
  18. Ayala-del-Rio, H.L.; Chain, P.S.; Grzymski, J.J.; Ponder, M.A.; Ivanova, N.; Bergholz, P.W.; Di Bartolo, G.; Hauser, L.; Land, M.; Bakermans, C.; et al. The genome sequence of Psychrobacter arcticus 273–274, a psychroactive Siberian permafrost bacterium, reveals mechanisms for adaptation to low-temperature growth. Appl. Environ. Microbiol. 2010, 76, 2304–2312. [Google Scholar] [CrossRef] [PubMed]
  19. Methe, B.A.; Nelson, K.E.; Deming, J.W.; Momen, B.; Melamud, E.; Zhang, X.; Moult, J.; Madupu, R.; Nelson, W.C.; Dodson, R.J.; et al. The psychrophilic lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses. Proc. Natl. Acad. Sci. USA 2005, 102, 10913–10918. [Google Scholar] [CrossRef] [PubMed]
  20. Mavromatis, K.; Tsigos, I.; Tzanodaskalaki, M.; Kokkinidis, M.; Bouriotis, V. Exploring the role of a glycine cluster in cold adaptation of an alkaline phosphatase. Eur. J. Biochem. 2002, 269, 2330–2335. [Google Scholar] [CrossRef] [PubMed]
  21. Kulakova, L.; Galkin, A.; Nakayama, T.; Nishino, T.; Esaki, N. Cold-active esterase from Psychrobacter sp. Ant300: Gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability. Biochim. Biophys. Acta 2004, 1696, 59–65. [Google Scholar] [CrossRef] [PubMed]
  22. Sakaguchi, M.; Matsuzaki, M.; Niimiya, K.; Seino, J.; Sugahara, Y.; Kawakita, M. Role of proline residues in conferring thermostability on aqualysin I. J. Biochem. 2007, 141, 213–220. [Google Scholar] [CrossRef] [PubMed]
  23. Vieille, C.; Zeikus, G.J. Hyperthermophilic enzymes: Sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 2001, 65, 1–43. [Google Scholar] [CrossRef] [PubMed]
  24. Zanphorlin, L.M.; de Giuseppe, P.O.; Honorato, R.V.; Tonoli, C.C.; Fattori, J.; Crespim, E.; de Oliveira, P.S.; Ruller, R.; Murakami, M.T. Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 beta-glucosidase from Exiguobacterium antarcticum B7. Sci. Rep. 2016, 6, 23776. [Google Scholar] [CrossRef] [PubMed]
  25. Feller, G.; Gerday, C. Psychrophilic enzymes: Hot topics in cold adaptation. Nat. Rev. Microbiol. 2003, 1, 200–208. [Google Scholar] [CrossRef] [PubMed]
  26. Georlette, D.; Damien, B.; Blaise, V.; Depiereux, E.; Uversky, V.N.; Gerday, C.; Feller, G. Structural and functional adaptations to extreme temperatures in psychrophilic, mesophilic, and thermophilic DNA ligases. J. Biol. Chem. 2003, 278, 37015–37023. [Google Scholar] [CrossRef] [PubMed]
  27. D’Amico, S.; Marx, J.C.; Gerday, C.; Feller, G. Activity-stability relationships in extremophilic enzymes. J. Biol. Chem. 2003, 278, 7891–7896. [Google Scholar] [CrossRef] [PubMed]
  28. Siddiqui, K.S. Defying the activity-stability trade-off in enzymes: Taking advantage of entropy to enhance activity and thermostability. Crit. Rev. Biotechnol. 2017, 37, 309–322. [Google Scholar] [CrossRef] [PubMed]
  29. Truongvan, N.; Jang, S.H.; Lee, C. Flexibility and stability trade-off in active site of cold-adapted Pseudomonas mandelii esterase EstK. Biochemistry 2016, 55, 3542–3549. [Google Scholar] [CrossRef] [PubMed]
  30. Santiago, M.; Ramírez-Sarmiento, C.A.; Zamora, R.A.; Parra, L.P. Discovery, molecular mechanisms, and industrial applications of cold-active enzymes. Front. Microbiol. 2016, 7, 1408. [Google Scholar] [CrossRef] [PubMed]
  31. Kirk, O.; Christensen, M.W. Lipases from Candida antarctica: Unique biocatalysts from a unique origin. Org. Process Res. Dev. 2002, 6, 446–451. [Google Scholar] [CrossRef]
  32. Uppenberg, J.; Hansen, M.T.; Patkar, S.; Jones, T.A. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 1994, 2, 293–308. [Google Scholar] [CrossRef]
  33. Anderson, E.M.; Larsson, K.M.; Kirk, O. One biocatalyst–many applications: The use of Candida antarctica B-lipase in organic yynthesis. Biocatal. Biotransform. 1998, 16, 181–204. [Google Scholar] [CrossRef]
  34. Cavicchioli, R.; Charlton, T.; Ertan, H.; Mohd Omar, S.; Siddiqui, K.S.; Williams, T.J. Biotechnological uses of enzymes from psychrophiles. Microb. Biotechnol. 2011, 4, 449–460. [Google Scholar] [CrossRef] [PubMed]
  35. Joseph, B.; Ramteke, P.W.; Thomas, G. Cold active microbial lipases: Some hot issues and recent developments. Biotechnol. Adv. 2008, 26, 457–470. [Google Scholar] [CrossRef] [PubMed]
  36. Margesin, R.; Feller, G. Biotechnological applications of psychrophiles. Environ. Technol. 2010, 31, 835–844. [Google Scholar] [CrossRef] [PubMed]
  37. Sarmiento, F.; Peralta, R.; Blamey, J.M. Cold and hot extremozymes: Industrial relevance and current trends. Front. Bioeng. Biotechnol. 2015, 3, 148. [Google Scholar] [CrossRef] [PubMed]
  38. Siddiqui, K.S. Some like it hot, some like it cold: Temperature dependent biotechnological applications and improvements in extremophilic enzymes. Biotechnol. Adv. 2015, 33, 1912–1922. [Google Scholar] [CrossRef] [PubMed]
  39. Lane, M.D.; Seelig, B. Advances in the directed evolution of proteins. Curr. Opin. Chem. Biol. 2014, 22, 129–136. [Google Scholar] [CrossRef] [PubMed]
  40. Porter, J.L.; Rusli, R.A.; Ollis, D.L. Directed evolution of enzymes for industrial biocatalysis. Chembiochem 2016, 17, 197–203. [Google Scholar] [CrossRef] [PubMed]
  41. Kim, H.S.; Le, Q.A.T.; Kim, Y.H. Development of thermostable lipase B from Candida antarctica (CalB) through in silico design employing B-factor and RosettaDesign. Enzyme Microb. Technol. 2010, 47, 1–5. [Google Scholar] [CrossRef]
  42. Narasimhan, D.; Nance, M.R.; Gao, D.; Ko, M.C.; Macdonald, J.; Tamburi, P.; Yoon, D.; Landry, D.M.; Woods, J.H.; Zhan, C.G.; et al. Structural analysis of thermostabilizing mutations of cocaine esterase. Protein Eng. Des. Sel. 2010, 23, 537–547. [Google Scholar] [CrossRef] [PubMed]
  43. Fang, L.; Chow, K.M.; Hou, S.; Xue, L.; Chen, X.; Rodgers, D.W.; Zheng, F.; Zhan, C.G. Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification. ACS Chem. Biol. 2014, 9, 1764–1772. [Google Scholar] [CrossRef] [PubMed]
  44. Eijsink, V.G.H.; Gåseidnes, S.; Borchert, T.V.; van den Burg, B. Directed evolution of enzyme stability. Biomol. Eng. 2005, 22, 21–30. [Google Scholar] [CrossRef] [PubMed]
  45. Miyazaki, K.; Wintrode, P.L.; Grayling, R.A.; Rubingh, D.N.; Arnold, F.H. Directed evolution study of temperature adaptation in a psychrophilic enzyme. J. Mol. Biol. 2000, 297, 1015–1026. [Google Scholar] [CrossRef] [PubMed]
  46. Lehmann, M.; Wyss, M. Engineering proteins for thermostability: The use of sequence alignments versus rational design and directed evolution. Curr. Opin. Biotechnol. 2001, 12, 371–375. [Google Scholar] [CrossRef]
  47. Boyineni, J.; Kim, J.; Kang, B.S.; Lee, C.; Jang, S.H. Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation. Biochim. Biophys. Acta 2014, 1844, 1076–1082. [Google Scholar] [CrossRef] [PubMed]
  48. Perl, D.; Mueller, U.; Heinemann, U.; Schmid, F.X. Two exposed amino acid residues confer thermostability on a cold shock protein. Nat. Struct. Biol. 2000, 7, 380–383. [Google Scholar] [PubMed]
  49. Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan, J.M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451–1463. [Google Scholar] [CrossRef]
  50. Homaei, A.A.; Sariri, R.; Vianello, F.; Stevanato, R. Enzyme immobilization: An update. J. Chem. Biol. 2013, 6, 185–205. [Google Scholar] [CrossRef] [PubMed]
  51. Cao, L. Carrier-bound Immobilized Enzymes: Principles, Applications and Design; Wiley-VCH: Weinheim, Germany, 2005. [Google Scholar]
  52. Guisan, J.M. Immobilization of Enzymes and Cells, 3rd ed.; Humana Press: New York, NY, USA, 2013. [Google Scholar]
  53. Hermanson, G.T. Bioconjugate Techniques, 3rd ed.; Elsevier/AP: London, UK, 2013. [Google Scholar]
  54. Cowan, D.A.; Fernandez-Lafuente, R. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme Microb. Technol. 2011, 49, 326–346. [Google Scholar] [CrossRef] [PubMed]
  55. Sheldon, R.A.; van Pelt, S. Enzyme immobilisation in biocatalysis: Why, what and how. Chem. Soc. Rev. 2013, 42, 6223–6235. [Google Scholar] [CrossRef] [PubMed]
  56. Garcia-Galan, C.; Berenguer-Murcia, Á.; Fernandez-Lafuente, R.; Rodrigues, R.C. Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synth. Catal. 2011, 353, 2885–2904. [Google Scholar] [CrossRef]
  57. Szczesna-Antczak, M.; Kaminska, J.; Florczak, T.; Turkiewicz, M. Cold-active yaeast lipases: Recent issues and future prospects. In Cold-Adapted Yeasts; Buzzini, P., Margesin, R., Eds.; Springer: Heidelberg, Germany, 2014; pp. 353–375. [Google Scholar]
  58. Brígida, A.I.S.; Amaral, P.F.F.; Coelho, M.A.Z.; Gonçalves, L.R.B. Lipase from Yarrowia lipolytica: Production, characterization and application as an industrial biocatalyst. J. Mol. Catal. B Enzym. 2014, 101, 148–158. [Google Scholar] [CrossRef]
  59. Idris, A.; Bukhari, A. Immobilized Candida antarctica lipase B: Hydration, stripping off and application in ring opening polyester synthesis. Biotechnol. Adv. 2012, 30, 550–563. [Google Scholar] [CrossRef] [PubMed]
  60. Chen, B.; Hu, J.; Miller, E.M.; Xie, W.; Cai, M.; Gross, R.A. Candida antarctica lipase B chemically immobilized on epoxy-activated micro- and nanobeads: Catalysts for polyester synthesis. Biomacromolecules 2008, 9, 463–471. [Google Scholar] [CrossRef] [PubMed]
  61. Rahman, M.A.; Culsum, U.; Kumar, A.; Gao, H.; Hu, N. Immobilization of a novel cold active esterase onto Fe3O4~cellulose nano-composite enhances catalytic properties. Int. J. Biol. Macromol. 2016, 87, 488–497. [Google Scholar] [CrossRef] [PubMed]
  62. Fernandes, S.; Geueke, B.; Delgado, O.; Coleman, J.; Hatti-Kaul, R. Beta-galactosidase from a cold-adapted bacterium: Purification, characterization and application for lactose hydrolysis. Appl. Microbiol. Biotechnol. 2002, 58, 313–321. [Google Scholar] [CrossRef] [PubMed]
  63. Fresco-Taboada, A.; Serra, I.; Fernandez-Lucas, J.; Acebal, C.; Arroyo, M.; Terreni, M.; de la Mata, I. Nucleoside 2′-deoxyribosyltransferase from psychrophilic bacterium Bacillus psychrosaccharolyticus-preparation of an immobilized biocatalyst for the enzymatic synthesis of therapeutic nucleosides. Molecules 2014, 19, 11231–11249. [Google Scholar] [CrossRef] [PubMed]
  64. Mukhopadhyay, A.; Bhattacharyya, T.; Dasgupta, A.K.; Chakrabarti, K. Nanotechnology based activation-immobilization of psychrophilic pectate lyase: A novel approach towards enzyme stabilization and enhanced activity. J. Mol. Catal. B Enzym. 2015, 119, 54–63. [Google Scholar] [CrossRef]
  65. Mukhopadhyay, A.; Dasgupta, A.K.; Chakrabarti, K. Enhanced functionality and stabilization of a cold active laccase using nanotechnology based activation-immobilization. Bioresour. Technol. 2015, 179, 573–584. [Google Scholar] [CrossRef] [PubMed]
  66. Rajaei, S.; Noghabi, K.A.; Sadeghizadeh, M.; Zahiri, H.S. Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp. SH3. Extremophiles 2015, 19, 1145–1155. [Google Scholar] [CrossRef] [PubMed]
  67. Vaghari, H.; Jafarizadeh-Malmiri, H.; Mohammadlou, M.; Berenjian, A.; Anarjan, N.; Jafari, N.; Nasiri, S. Application of magnetic nanoparticles in smart enzyme immobilization. Biotechnol. Lett. 2016, 38, 223–233. [Google Scholar] [CrossRef] [PubMed]
  68. Pavlidis, I.V.; Vorhaben, T.; Tsoufis, T.; Rudolf, P.; Bornscheuer, U.T.; Gournis, D.; Stamatis, H. Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials. Bioresour. Technol. 2012, 115, 164–171. [Google Scholar] [CrossRef] [PubMed]
  69. Zhang, J.; Zhang, F.; Yang, H.; Huang, X.; Liu, H.; Guo, S. Graphene oxide as a matrix for enzyme immobilization. Langmuir 2010, 26, 6083–6085. [Google Scholar] [CrossRef] [PubMed]
  70. Zhang, Y.; Wu, C.; Guo, S.; Zhang, J. Interactions of graphene and graphene oxide with proteins and peptides. Nanotechnol. Rev. 2013, 2, 27–45. [Google Scholar] [CrossRef]
  71. Zhang, Y.; Zhang, J.; Huang, X.; Wu, H.; Guo, S. Assembly of graphene oxide-enzyme conjugates through hydrophobic interaction. Small 2012, 8, 154–159. [Google Scholar] [CrossRef] [PubMed]
  72. Lee, H.; Jeong, H.K.; Han, J.; Chung, H.S.; Jang, S.H.; Lee, C. Increased thermal stability of cold-adapted esterase at ambient temperatures by immobilization on graphene oxide. Bioresour. Technol. 2013, 148, 620–623. [Google Scholar] [CrossRef] [PubMed]
  73. Makowski, K.; Bialkowska, A.; Szczesna-Antczak, M.; Kalinowska, H.; Kur, J.; Cieslinski, H.; Turkiewicz, M. Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis. FEMS Microbiol. Ecol. 2007, 59, 535–542. [Google Scholar] [CrossRef] [PubMed]
  74. Wang, Y.B.; Gao, C.; Zheng, Z.; Liu, F.M.; Zang, J.Y.; Miao, J.L. Immobilization of cold-active cellulase from Antarctic bacterium and its use for kelp cellulose thanol fermentation. BioResources 2015, 10, 1757–1772. [Google Scholar]
  75. Fernandez-Lafuente, R.; Armisén, P.; Sabuquillo, P.; Fernández-Lorente, G.; Guisán, J.M. Immobilization of lipases by selective adsorption on hydrophobic supports. Chem. Phys. Lipids 1998, 93, 185–197. [Google Scholar] [CrossRef]
  76. Issa, B.; Obaidat, I.M.; Albiss, B.A.; Haik, Y. Magnetic nanoparticles: Surface effects and properties related to biomedicine applications. Int. J. Mol. Sci. 2013, 14, 21266–21305. [Google Scholar] [CrossRef] [PubMed]
  77. Fresco-Taboada, A.; Serra, I.; Arroyo, M.; Fernández-Lucas, J.; de la Mata, I.; Terreni, M. Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine. Catal. Today 2016, 259, 197–204. [Google Scholar] [CrossRef]
  78. Fernandez-Lafuente, R. Stabilization of multimeric enzymes: Strategies to prevent subunit dissociation. Enzyme Microb. Technol. 2009, 45, 405–418. [Google Scholar] [CrossRef]
  79. Sheldon, R.A. Cross-linked enzyme aggregates (CLEAs): Stable and recyclable biocatalysts. Biochem. Soc. Trans. 2007, 35, 1583–1587. [Google Scholar] [CrossRef] [PubMed]
  80. Sheldon, R.A. Cross-linked enzyme aggregates as industrial biocatalysts. Org. Process Res. Dev. 2011, 15, 213–223. [Google Scholar] [CrossRef]
  81. Stepankova, V.; Bidmanova, S.; Koudelakova, T.; Prokop, Z.; Chaloupkova, R.; Damborsky, J. Strategies for stabilization of enzymes in organic solvents. ACS Catal. 2013, 3, 2823–2836. [Google Scholar] [CrossRef]
  82. Adlercreutz, P. Immobilisation and application of lipases in organic media. Chem. Soc. Rev. 2013, 42, 6406–6436. [Google Scholar] [CrossRef] [PubMed]
  83. Gorman, L.A.; Dordick, J.S. Organic solvents strip water off enzymes. Biotechnol. Bioeng. 1992, 39, 392–397. [Google Scholar] [CrossRef] [PubMed]
  84. Zaks, A.; Klibanov, A.M. Enzyme-catalyzed processes in organic solvents. Proc. Natl. Acad. Sci. USA 1985, 82, 3192–3196. [Google Scholar] [CrossRef] [PubMed]
  85. Sellek, G.A.; Chaudhuri, J.B. Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb. Technol. 1999, 25, 471–482. [Google Scholar] [CrossRef]
  86. Ogino, H.; Miyamoto, K.; Ishikawa, H. Organic-solvent-tolerant bacterium which secretes organic-solvent-stable lipolytic enzyme. Appl. Environ. Microbiol. 1994, 60, 3884–3886. [Google Scholar] [PubMed]
  87. Gupta, A.; Khare, S.K. Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology. Crit. Rev. Biotechnol. 2009, 29, 44–54. [Google Scholar] [CrossRef] [PubMed]
  88. Doukyu, N.; Ogino, H. Organic solvent-tolerant enzymes. Biochem. Eng. J. 2010, 48, 270–282. [Google Scholar] [CrossRef]
  89. Song, J.K.; Rhee, J.S. Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution. Biochim. Biophys. Acta 2001, 1547, 370–378. [Google Scholar] [CrossRef]
  90. Reetz, M.T. Changing the enantioselectivity of enzymes by directed evolution. Methods Enzymol. 2004, 388, 238–256. [Google Scholar] [PubMed]
  91. Dror, A.; Shemesh, E.; Dayan, N.; Fishman, A. Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in methanol. Appl. Environ. Microbiol. 2014, 80, 1515–1527. [Google Scholar] [CrossRef] [PubMed]
  92. Kawata, T.; Ogino, H. Enhancement of the organic solvent-stability of the LST-03 lipase by directed evolution. Biotechnol. Prog. 2009, 25, 1605–1611. [Google Scholar] [CrossRef] [PubMed]
  93. Guo, H.; Zhang, Y.; Shao, Y.; Chen, W.; Chen, F.; Li, M. Cloning, expression and characterization of a novel cold-active and organic solvent-tolerant esterase from Monascus ruber M7. Extremophiles 2016, 20, 451–459. [Google Scholar] [CrossRef] [PubMed]
  94. Wu, G.; Zhang, X.; Wei, L.; Wu, G.; Kumar, A.; Mao, T.; Liu, Z. A cold-adapted, solvent and salt tolerant esterase from marine bacterium Psychrobacter pacificensis. Int. J. Biol. Macromol. 2015, 81, 180–187. [Google Scholar] [CrossRef] [PubMed]
  95. Kim, J.; Jang, S.H.; Lee, C. An organic solvent-tolerant alkaline lipase from cold-adapted Pseudomonas mandelii: Cloning, expression, and characterization. Biosci. Biotechnol. Biochem. 2013, 77, 320–323. [Google Scholar] [CrossRef] [PubMed]
  96. Ganasen, M.; Yaacob, N.; Rahman, R.N.Z.R.A.; Leow, A.T.C.; Basri, M.; Salleh, A.B.; Ali, M.S.M. Cold-adapted organic solvent tolerant alkalophilic family I.3 lipase from an Antarctic Pseudomonas. Int. J. Biol. Macromolec. 2016, 92, 1266–1276. [Google Scholar] [CrossRef] [PubMed]
  97. Li, M.; Yang, L.-R.; Xu, G.; Wu, J.-P. Screening, purification and characterization of a novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC 4254. Bioresour. Technol. 2013, 148, 114–120. [Google Scholar] [CrossRef] [PubMed]
  98. Dachuri, V.; Boyineni, J.; Choi, S.; Chung, H.-S.; Jang, S.-H.; Lee, C. Organic solvent-tolerant, cold-adapted lipases PML and LipS exhibit increased conformational flexibility in polar organic solvents. J. Mol. Catal. B Enzym. 2016, 131, 73–78. [Google Scholar] [CrossRef]
  99. Kamal, M.Z.; Yedavalli, P.; Deshmukh, M.V.; Rao, N.M. Lipase in aqueous-polar organic solvents: Activity, structure, and stability. Protein Sci. 2013, 22, 904–915. [Google Scholar] [CrossRef] [PubMed]
  100. Fitzpatrick, P.A.; Ringe, D.; Klibanov, A.M. X-ray crystal structure of cross-linked subtilisin Carlsberg in water vs. acetonitrile. Biochem. Biophys. Res. Commun. 1994, 198, 675–681. [Google Scholar] [CrossRef] [PubMed]
  101. Allen, K.N.; Bellamacina, C.R.; Ding, X.; Jeffery, C.J.; Mattos, C.; Petsko, G.A.; Ringe, D. An experimental approach to mapping the binding surfaces of crystalline proteins. J. Phys. Chem. 1996, 100, 2605–2611. [Google Scholar] [CrossRef]
Catalysts 07 00112 g001 550
Figure 1. Immobilization of cold-adapted enzymes. (a) Adsorption; (b) covalent binding; (c) cross-linking; (d) entrapment.
Figure 1. Immobilization of cold-adapted enzymes. (a) Adsorption; (b) covalent binding; (c) cross-linking; (d) entrapment.
Catalysts 07 00112 g001
Table
Table 1. Immobilization of cold-adapted enzymes from bacteria.
Table 1. Immobilization of cold-adapted enzymes from bacteria.
Enzyme NameSpeciesSupportChemistryCommentsReference
AdsorptionEsterase (EstH)Zunongwangia sp.Fe3O4-celluloseHydrogen bonding48% activity after 30 min at 50 °C[61]
β-GalactosidasePseudoalteromonas sp.DEAE-SepharoseIonic interaction87%–89% storage stability after 1 week at 4 °C[62]
Nucleoside 2′-deoxyribosyltransferaseBacillus psychrosaccharolyticusPEI-coated agaroseIonic interactionUnstable; lost activity within 2 h[63]
Covalent binding PullulanaseExiguobacterium sp.Epoxy-functionalized silicaEpoxyl groupMaintained thermal stability at 50 °C[66]
Esterase (EstK)Pseudomonas mandeliiGraphene oxideSulfo-NHS and EDCEnhanced thermal stability at 40 °C; catalytic efficiency reduced to 40% of free enzyme[72]
β-GalactosidasePseudoalteromonas sp.Epoxy-activated SepharoseEpoxyl group87%–89% storage stability after 1 week at 4 °C[62]
β-GalactosidasePseudoalteromonas sp.PEI-coated SepharoseGlutaraldehyde98% storage stability after 1 week at 4 °C[62]
β-GalactosidasePseudoalteromonas sp.Glutaraldehyde-treated chitosanGlutaraldehydeEnhanced themal stability at 50 °C; longer shelf life over 12 months[73]
Nucleoside 2′-deoxyribosyltransferaseBacillus psychrosaccharolyticusPEI-coated agaroseAldehyde-dextranOperational stability at 37 °C with 75% activity after 30 cycles[63]
Entrapment CellulasePseudoalteromonas sp.Sodium alginate beadsGlutaraldehyde cross-linking entrapment58% activity after seven cycles[74]
Pectate lyaseBacillus subtilisLipid-functionalized SWCNT-Thermal stability at 4–80 °C[64]
LaccasePseudomonas putidaLipid-functionalized SWCNT-Thermal stability at 4–80 °C[65]
DEAE: diethylaminoethyl; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; PEI: polyethylenimine; Sulfo-NHS: hydroxy-2,5-dioxopyrrolidine-3-sulfonicacid sodium salt; SWCNT: single-walled carbon nanotube.

Share and Cite

MDPI and ACS Style

Lee, C.; Jang, S.-H.; Chung, H.-S. Improving the Stability of Cold-Adapted Enzymes by Immobilization. Catalysts 2017, 7, 112. https://doi.org/10.3390/catal7040112

AMA Style

Lee C, Jang S-H, Chung H-S. Improving the Stability of Cold-Adapted Enzymes by Immobilization. Catalysts. 2017; 7(4):112. https://doi.org/10.3390/catal7040112

Chicago/Turabian Style

Lee, ChangWoo, Sei-Heon Jang, and Hye-Shin Chung. 2017. "Improving the Stability of Cold-Adapted Enzymes by Immobilization" Catalysts 7, no. 4: 112. https://doi.org/10.3390/catal7040112

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop