WO2009042090A2

WO2009042090A2 - A novel superoxide dismutase gene and uses thereof

Info

Publication number: WO2009042090A2
Application number: PCT/US2008/010964
Authority: WO
Inventors: Manuel Gidekel; Ana Gutierrez; Jaime Sanchez; Gustavo Cabrera; Claudia Rabert; Ivan Mihovilovic
Original assignee: Manuel Gidekel; Ana Gutierrez; Jaime Sanchez; Gustavo Cabrera; Claudia Rabert; Ivan Mihovilovic
Priority date: 2007-09-21
Filing date: 2008-09-22
Publication date: 2009-04-02

Description

TITLE: A Novel Superoxide dismutase gene and uses thereof

INVENTORS: GIDEKEL, Manuel; GUTIERREZ₅ Ana; SANCHEZ, Jaime; CABRERA, Gustavo; RABERT, Claudia and Ivan MIHOVILOVIC

FIELD OF THE INVENTION

The present invention relates generally to the field of plant molecular biology. More specifically the invention relates to nucleic acid fragment encoding plant superoxide dismutase and uses thereof.

BACKGROUND OF THE INVENTION

Superoxide dismutase (SOD) is a widely spread enzyme in the nature; it has been found in almost all organism that are living in the presence of oxygen. SOD is known to have a major role in preventing oxidative stress in the cells. In the plant kingdom superoxide dismutase activities are higher in plants that have grown in low temperatures than in higher temperatures. Superoxide dismutase has been recently researched a lot in relation to its antioxidant characteristics, for example in relation to cancer research.

Accordingly, identification and characterization of genes coding for superoxide dismutase like proteins in plant species tolerant to low temperatures can provide novel enzymes for various applications. A plant species extremely tolerant to low temperatures is Deschampsia antarctica Desv. (Poacea). Accordingly, we have studied the gene expression of this vascular plant naturally colonizing Maritime Antarctic Peninsula. SUMMARY OF THE INVENTION

Due to the increased interest in superoxide dismutases in various fields of industry, there is a clear need for novel superoxide dismutase enzymes. Especially, there is a need for novel enzymes functional in wide range of temperatures. Moreover, there is a need for a method for economic production of superoxide dismutase enzymes.

Accordingly, an object of the current invention is to provide a novel recombinant superoxide dismutase, which is psycrophilic and can therefore stand low temperatures.

Another object of the current invention is to provide a simple and rapid method of producing superoxide dismutase enzyme in host cell and subsequent purification of recombinant superoxide dismutase.

According to a preferred embodiment the superoxide dismutase enzyme is encoded by a nucleotide sequence essentially according to SEQ ID NO: 1 isolated from Deschampsia antarctica.

Yet another embodiment of the present invention is a chimeric gene comprising an isolated nucleotide sequence encoding superoxide dismutase like protein of Deschampsia antarctica.

An even further embodiment of the present invention is isolated host cells comprising the chimeric gene. The host cell may be eukaryotic, such as yeast or a plant cell, or it may be prokaryotic such as a bacterial cell. According to one embodiment the host cell comprising the chimeric gene is a Pichia cell.

An even further embodiment of the instant invention is to provide a process for cultivating host cells comprising the chimeric gene and isolating the recombinant enzyme produced in the cells.

Yet another embodiment of the instant invention is to use the novel SOD enzyme in preservation of pharmaceuticals, nutritional compositions and foods. Still another embodiment of the instant invention is to protect fruits and vegetables from oxidative stress.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 DNA sequence (SEQ ID NO:1) coding for superoxide dismutase of De schampsia antarctica.

Fig. 2. BLAST analysis of clone Cl 3 with a putative Oryza sativa mRNA for copper/zinc- superoxide dismutase, complete cds, clone :RSODB.

Fig.3. Comparison of Cu/Zn-SOD Deschampsia Antarctica (SEQ ID NO:2) and Cu/Zn-SOD Oryza sativa (SEQ ID NO:3). Superoxide dismutase protein-encoding gene showing the consensus region.

Fig. 4. Phylogenetic tree displaying the relationships between superoxide dismutase Cl 3 of Deschampsia antarctica (Da) with putative Cu/Zn-superoxide dismutase of Oyiza sativa (Os) and Zea mays (Zm). In case of the superoxide dismutase of Ananas comosus (Ac) and Malus Xiaojinensis (Mx) it showed a remote relatioa

Fig. 5. Multiple alignment of Deschampsia antartica superoxide dismutase Cl 3 with previously cloned superoxide dismutases using the Vector NTI 7 software. Deschampsia antarctica superoxide dismutase Cl 3 (Da) (SEQ ID NO:2) , putative Cu/Zn-superoxide dismutase of Oryza sativa (Os) (SEQ ID NO:3) , superoxide dismutase-Sod4A of Zea mays (Zm) (SEQ ID NO:4) , Cu/Zn-superoxide dismutase-Sodl of Ananas comosus (Ac) (SEQ ID NO:5) and superoxide dismutase-Sodl of Malus xiaojinensis (Mx) (SEQ ID NO: 6) and consensus region.

Fig. 6. Expression vector pGAPZalphaA-superoxide dismutase Cl 3 Clon7 of Deschampsia antarctica. The vector shows correct ORF with the alpha factor (secretor signal), myc epitope tag to recognize the excreted protein and the 6xHis tag to facilitate the protein purification. Fig. 7. Kinetics of the expression in the time of P. pastoris SDMI 168H (transformed with pGAPZaA-superoxide dismutase Cl 3) in YPD medium. (A) SDS-PAGE 18%. Lane Mw, protein molecular mass markers; Lane 1, Positope (100 ng, 53 kD) positive as antibody control protein; lane 2-5, culture supernatants of P. pastoris SDMI 168H transfected with pGAPZαA-SOD in the times 24, 48, 72 and 96 hours; Lane 6, culture supernatants of P. pastoris SDMI 168H transfected with the control plasmid pGAPZαA as negative control. (B) Western blot of the samples resolved in SDS-PAGE 18 %.

Fig. 8. Superoxide dismutase activity at different expression times (24, 48, 72 and 96 hours).

Fig. 9. Activity samples of concentrated protein at 96h. (M-SOD) activity of the sample P pastoris SMDl 168H transformed with pGAPZαA-SOD (69.4 LVL). (M-SI) activity of the sample P. pastoris SMDl 168H transformed with pGAPZα-A as control without D. antarctica SOD protein (35. I LVL).

Fig. 10. Determination of the expression of concentrated samples at 96h. (A) SDS-PAGE 15%. Lane Mw, protein molecular mass markers; Lane 1, expression of P. pastoris SMDl 168H (pGAPZαA-SOD). Lane 2, expression de P. pastoris (pGAPZαA) as control without superoxide dismutase. (B) Western blot of the samples resolved in SDS-PAGE 15 %.

Fig. 11. Activity of P. pastoris SMDl 168H expression for electrophoresis. (A) Native-page 12%. Lane 1, SOD 3μl (3.5 U/μl) as positive control; Lane 2, expression with pGAPZαA (at 96 h) as negative control. Lane 3-6, expression with pGAPZαA-SOD in the times 24, 48, 72 and 96 hours. Lane 7, supernatant concentrated (1/6) of the expression with pGAPZαA (at 96 h). Lane 8, supernatant concentrate (1/6) of the expression with pGAPZαA-SOD (at 96 h). (B) Western blot of a native gel realized in the same conditions of the Native-page 12 %.

Fig. 12. SOD protein purification analysis. (A) SDS-page 15%. Line 1, positive control (10 μl, crude harvest of P. pastori). Line 2, 4, 6 and 8 showed the elutions number El, E2, E3, and E4 (20 μl each) from P. pastoris SMD1168H::pGAPZαA-SOD purification. Line 3, 5, 7 and 9 showed elutions number El, E2, E3 and E4 (20 μl each) from P. pastoris SMD1168H::pGAPZαA. (B) Western blot of the gel transferred to a nitrocelulose membrane and inmunodetected using anti-myc antibody.

DESCRIPTION OF THE INVENTION

We started working with cDNAs expression library of samples of Deschampsia antarctica in which we identified one gene that codified for a type enzyme like superoxide dismutase {SOD: EC 1.15.1.1). The DNA sequence that codified the superoxide dismutase of Deschampsia antarctica shown in Figure 1 and is identified as SEQ ID NO: 1.

This gene was compared with other genes available in a public GeneBank (BLAST). The clone Cl 3 (superoxide dismutase Cl 3) showed homology (86% positive) with a putative Oryza sativa mRNA for copper/zinc-superoxide dismutase, complete cds, clone:RSODB having the sequence identified as DO 1000.1 GI:218225. Deschampsia antarctica gene sequence was found to be full length (Fig. 2).

The alignment of superoxide dismutase C13 with the putative Oryza sativa mRNA for copper/zinc-superoxide dismutase, complete cds, clone:RSODB gene was conducted by using the Vector NTI 7 software. The amino acid sequence for both superoxide dismutases showed a 84.9% identity and 90.1% homology (Fig. 3). From this it was concluded that we have a different plant Cu/Zn-superoxide dismutase gene.

A phylogenetic analysis of several plant superoxide dismutases was carried out in order to establish the presumed evolutionary relationships among them and to infer their evolutionary history. For this purpose, the superoxide dismutase Cl 3 of Deschampsia antarctica (Da) (SEQ ID NO:2), putative Cu/Zn-superoxide dismutase of Oryza sativa (Os) (SEQ ID NO:3), superoxide dismutase-Sod4A of Zea mays (Zm)(SEQ ID NO:4), Cu/Zn-superoxide dismutase- Sodl of Ananas comosus (Ac) (SEQ ID NO:5) and superoxide dismutase-Sodl of Malus xiaojinensis (Mx) (SEQ ID NO: 6) were compared. Finally, a phylogenetic tree was built and it is shown in Fig. 4. The phylogenetic tree revealed that there was no phylogenetic relation between Deschampsia superoxide dismutase C13 with superoxide dismutase-Sodl of Ananas comosus and superoxide dismutase-Sodl of Malus xiaojinensis. The phylogenetically related proteins are the Oryza sativa Cu/Zn-superoxide dismutase and Zea mays superoxide dismutase-Sod4A, which have 90.1 % and 88.2% of similarity, respectively.

In order to ascertain that Deschampsia antarctica superoxide dismutase Cl 3 protein was not a previously reported protein, a further comparison with functional related superoxide dismutase was conducted by using a GenBank information. The simultaneous alignment of Deschampsia superoxide dismutase Cl 3, putative Cu/Zn-superoside dismutase of Oryza sativa, superoxide dismutase-Sod4A of Zea mays, Cu/Zn-superoxido dismutase-Sodl of Ananas comosus and superoxide dismutase-Sodl of Malus xiaojinensis are shown in figure 5. The results from the comparison and the biochemical analysis are summarized in Tables 1 and 2.

Table 1. Comparison of the amino acid sequence of superoxide dismutase C13 of D. antarctica with other superoxide dismutases.

Table 2. Biochemical comparison of superoxide dismutase properties isolated from different sources

The results prove again that Deschampsia antarctica superoxide dismutase Cl 3 is different from other proteins with similar function.

Example 1. Cloning and expression of superoxide dismutase

Strains, vectors and culture media

Escherichia coli strain TOP 10 F' (Invitrogen) was selected for vector construction and Pichia pastoris strain SMDl 168H (Invitrogen) was used to express the Deschampsia antarctica superoxide dismutase C 13. E. coli was grown in low salt LB-Zeocin medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl and 25 μg/ml of zeocin).

Pichia pastoris was grown in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) for general growth and constitutive expression. For selection of transformants, zeocin 100 μg/ml agar plates were used (1% yeast extract, 2% peptone, 2% dextrose, 2% agar, 100 μg/ml zeocin). The P. pastoris SMDl 168H and pGAPZαA, used as fungal host and expression vector were purchased from Invitrogen Corporation.

Vector construction and transformation

The Deschampsia antarctica superoxide dismutase C13 gene was isolated by polymerase chain reaction (PCR) amplification using primers SODGapFw

(5'- AAGAATTCATGGTGAAGGCTGTAGCTGTGC-S' ; SEQ ID NO:7) and SODGapRev

(5'-ATATTCTAGACCCTGGAGCCCGATGATCC-S ' ;SEQ ID NO:8).

These primers introduced restriction site £coRI and Xbal respectively at the gene. PCR fragments were purified and DNA fragments were recovered from agarose gels using Ultra

Clean 15 DNA purification Kit (Carlsbad, CA, USA). DNA was purified and manipulated essentially as described by Sambrook et al., 1989. The PCR product was cloned into pGEM-T Easy (Promega) and liberated with the enzymes EcoRl and Xbal and ligated to the vector pGAPZαA digested with the same restriction enzymes. This resulted in the expression vector pGAPZαA-superoxide dismutase Cl 3 Clon7 (Fig. 6). All amplifications by PCR were performed by using Taq DNA Polymerase recombinant LC (Fermentas, Hanover, USA).

The resulting plasmid construct were transformed into E. coli TOP 10 F' and transformants were selected on low salt LB-Zeocin. The plasmid recombinant DNA was sequenced with α-Factor sequencing primer (5'-TACTATTGCCAGCATTGCTGC-S '; SEQ ID NO: 9) and 3'AOXl primer (5^'- GCAAATGGCATTCTGACATCC-3'; SEQ ID NO: 10) which annealed with the pGAPZαA sequence. Sequence alignment was performed by BLAST.

Pichia transformation and superoxide dismutase expression

Electrocompetent cells of P. pastoris SMDl 168H were prepared according to the supplier's instruction (Invitrogen). Ten micrograms recombinant plasmid linearized with Avr II was mixed with 80 μl electrocompetent cells, and electroporated by means of two pulse discharge (1500 V, 25 F, Bio-Rad Gene Pulser). After pulsing, 1 ml ice-cold IM sorbitol was immediately added to the cuvette. Then, 200 μl aliquots were spread on YPDS + zeocin agar (1% yeast extract, 2% peptone, 2% dextrose, IM sorbitol, 2% agar, 100 μg/ml zeocin) plates, and the plates were incubated at 30°C to screen for zeocin resistant transformants according to their capacity to grow in the presence of zeocin. Resistant zeocin clones were grown on 10 ml of YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30°C in shaking incubator (250-300 rpm) over night. 0.1 ml of the overnight cultivate was used to inoculate 50 ml of YPD in a 250 ml flask. The samples grew in a shaking incubator in the same conditions for 4 days and aliquots of 1 ml were taken every 24 hours. The samples were centrifuged at maximum speed for 2-3 minutes and the supernatant and the cell pellet were stored at -8O⁰C until ready to assay. The detection of superoxide dismutase protein in Pichia was realized using technique of SDS-PAGE stained with Coomassie blue R-250 and western blot. The analysis with anti-myc antibody and for method alkaline phosphatase showed two bands with sizes are of 25 and 29 kD (Fig 7). Anti-myc antibody (Invitrogen) was used for the analysis of the blotting and goat anti-mouse IgG alkaline phosphatase conjugate (Upstate) was used as second antibody.

Example 2. Determination of superoxide dismutase protein activity

SOD protein activity expression in time

Modified P. pastori was grown in YPD media for 96 h. The samples were collected every 24 hours and the supernatant was separated from cells by centrifugation. One ml aliquot of the sample was taken at selected time. Superoxide dismutase activity was determined according to McCord and Fridovich (1969) with modifications by Schδner and Krause (1990) by following the rate of ferricytochrome C reduction at 550 nm in a thermoregulated spectrophotometer. One unit of superoxide dismutase is defined as the amount of enzyme that inhibits in 50% the reduction of ferricytochrome C at pH 7.8 and 25°C. The maximum superoxide dismutase activity was observed a 72 and the activity remained high for 96 hours (Fig. 8).

SOD activity of the concentrated protein

The harvest of the protein expressed at 96 h was concentrated by filtration across an anisotropic cellulose membrane arranged in centrifugal filter devices (Centriplus, cut of 10 KD, Millipore).

The volume of the concentrate was about 1/6 of the initial volume. The same procedure was followed for P. pastoris transformed with pGAPZαA-SOD and pGAPZαA vectors, respectively.

The latter sample was used as negative control. The SOD activity was measured as described above and the results are presented in Figure 9. Results showed that in the harvest of pGAPZαA- SOD transformed P. pastori the SOD protein activity was 69.4 U/L which is almost twice the activity obtained in the negative control (35.1 U/L). This indicates the expression of a new protein in the microorganism.

Western blot of the concentrated samples expression Detection of superoxide dismutase protein from samples concentrated to 1/6 was realized by using the SDS-PAGE 15% technique stained with Coomassie blue R-250 (Fig.10 A). Two bands are observed between 20 and 25 KD in the sample transformed with pGAPZαA-SOD. The western blotting of the gel SDS-PAGE was detected by using anti Cu/Zn-SOD and Cu/Zn-SOD. The gels were developed by chemiluminescense (Fig. 10 B). The film shows four bands (15, 18, 20 and 25 kD) in the sample which overexpresses the superoxide dismutase enzyme.

Superoxide dismutase activity assay for electrophoresis (zymogram)

The superoxide dismutase was determined according to Rao et al (1996). Equal amounts of superoxide dismutase (1 Unit) were subjected to native PAGE (12%) at 80 mV for 6 hours at 4°C. Gels were stained to visualize the bands of superoxide dismutase by incubating in a solution containing 2.5 niM nitrozolium for 25 min, followed by incubation in 50 mM sodium phosphate buffer (pH 7.8) containing 28 μM riboflavin and 28 mM tetramethyl ethylene diamine for 20 min in the dark. The gel was placed in distilled water and exposed on a light at room temperature up to visualizing the clear bands that indicate enzyme activity (Fig. 1 IA). Another gel was run in the same conditions of native-page to transfer it to membrane of nitrocellulose. The western blot of the membrane was carried on by using anti-myc (Invitrogen) as the first antibody and goat anti-mouse IgG alkaline phosphatase conjugate (Upstate) as the second antibody to corroborate the location of the bands of the native gel (Fig. 1 IB).

PURIFICATION AND THERMOSTABILITY OF SOD ENZYME

Purification of the SOD enzyme expressed by P. pastoris SMDl 168H (pGAPZαA-SOD)

The superoxide dismutase protein secreted by P. pastoris SMDl 168H (pGAPZαA-SOD) to the culture media was purified by using "Probond Purification system" kit (Invitrogen Life Technologies). This system was designed for recombinant protein purification and it contains polyhystidines (6xHis-tagged). The purification results were showed in the SDS-PAGE gel at 15% and by inmunodetection with a western blot analysis using the anti-myc antibody. The P. pastoris SMD1168H (pGAPZαA) expression was used as control and also purified and treated under the same conditions (Fig. 12).

These results showed a progresive protein purification form El to E4 which is demonstrated by the thickness of the band as showed in line 8 in Fig. 12. Furthermore, it was demonstrated that control P. pastori SOD enzyme activity is absent in this culture filtrate.

Example 3. Purified SOD enzyme thermo stability

The thermo stability of the novel SOD enzyme was determined by incubation of this protein during 30 minutes at different temperatures ranging from: -20, 0, 20, 40, 60, 80 and 100°C. For subzero temperatures (-2O⁰C) glycerol was added to the mixture at a final concentration of 50% (v/v). The SOD protein was also autoclaved (121° C) at 1.1 kg.cm^"2 for 20 min for thermo stability determination. The enzyme activity results were expressed as activity percentage of the control (Table 3). The control activity was determined at 25⁰C without incubation for 30min.

Table 3. Thermo stability analysis of the purified SOD protein from D. antarctica.

Temperature Enzyme Activity

(⁰C) (U/μl)

25 (Control ) 26.3 (100%)

- 20 22 (86.6%)

0 19.8 (75.3%)

20 19.2 (73.0%)

40 16.0 (60.8%)

60 1.3 (4.9%)

80 0 (0%)

121 (Autoclaved) 0 (0%)

* Parenthesis values are referred as retained activity expressed as percentage of the control treatment. The SOD enzyme from D. antarctica cloned in P. pastori showed a psycrophile behavior by retaining an 86% of this activity at subzero temperatures (-20⁰C), which is unusual in this type of enzyme. It is worth to note that it retained more than 70% of the activity in the temperature range of zero to 20⁰C. This characteristic makes it useful in applications where low temperatures are required to scavenge superoxide radicals. The SOD enzyme lost its activity at 60⁰C and was denatured by autoclaving it for 20min.

Example 4. Use of the novel SOD enzyme in pharmacy

The SOD enzyme, due to its antioxidant activity, could be used for preservation of a wide number of pharmaceutical products during cold storage, because it can catalyze dismutation of O^2" at lower temperature (-20⁰C to 4°C). In addition, it could be used in several formulations (emulsions, creams, ointments, gels, tablets) that can be stored from low (-20⁰C) to room temperatures (25⁰C).

Such enzyme could be mixed with natural and synthetic polymers to form microcapsules, nanocapsules, as well as film preparations, in order to build a controlled release system of any drug that does not interact with the SOD protein.

One of the possible applications of the novel enzyme is in treating skin diseases such as burning, psoriasis and erythema.

Example 5. Use of the novel SOD enzyme in as nutraceutical

Conjugated linoleic acid (CLA; 9c, 1 It-18:2) and CLA isomers have been reported, in animals ad humans, to exhibit a variety of health-related benefits. Most commercially available samples of CLA, prepared by base-catalyzed isomerization of linoleic acid (9c, 12c- 18 : 2), are composed of mixtures of four major isomers. The four cis/trans isomers (8t, 10c- 18 : 2, 9c, 1 It- 18 : 2, 1Ot, 12c-18 : 2 and l ie, 13t-18 :2) were present in a ratio of approximately 1 :2:2:1. If indeed certain daily levels of CLA intake are required to produce suggested health benefits in humans, changes in concentrations of specific CLA isomers could significantly impact these effects. With this purpose, an addition of a certain amount of SOD enzyme (ranging from 0.001% to 20% (w/v)) could be added to nutritional formulations to preserve it during storage and human consuming.

There is a mayor quantity of commercial essential oils with different values and applications. It is known these products are formed by a mixture of compounds with different chemical structure and oxydation stability. That is why the SOD enzyme could be used to preserve them from oxygen action during storage and use.

Example 6. Use of the novel SOD enzyme in agricultural applications

Antioxidant UVB protecting composition

An emulsion mixture containing 0.001 to 25% (w/v) SOD enzyme dissolved in water and a surfactant compound (0.01 to 10% v/v) could be sprayed to different fruit trees such as apples, pear, peaches, avocado, mango, banana, guava, blueberry and other berries and so on. This is in order to protect fruits from damage due to the combination of oxygen-high temperatures typical of the tropical weathers or oxygen- sunlight radiation (UVA and UVB) of the rest of the world.

Example 7. Use of the novel SOD enzyme in food preservation.

One of the possible uses of this enzyme is in food preservation, mainly those one which are able to oxydized easily such as oil containing food. Among then butterflie, pate, mayonnaise, oil containing sauses, cheeses, juices, soft drinks and so on. The amount of SOD enzyme to be added to food products are in the range of 0.001% to 20% (w/v or w/w).

References.

McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprin (hemocuprin). The Journal of biological Chemistry 224, 6049-6055. Rao MV, Gopinadhan P, Ormrod DP (1996) Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiology 110, 125-136. Schόner S, Krause GH (1990) Protective systems against active oxygen species in spinach: response to cold acclimation in excess light. Planta 180, 383-389.

Claims

CLAIMSWhat is claimed is:

1. An isolated nucleic acid molecule encoding plant superoxide dismutase enzyme comprising an amino aoid sequence of SEQ ID NO: 2.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid comprises SEQ ID NO: 1.

3. A nucleic acid sequence according to SEQ ID NO: 1 encoding a plant superoxide dismutase enzyme having an amino acid sequence of SEQ ID NO:2.

4. An expression vector comprising the nucleic acid of claim 1.

5. A host cell comprising the expression vector of claim 4.

6. The host cell according to claim 5 wherein the cell is a Pichia cell.

7. The host cell according to claim 5, wherein the cell is a plant cell.

8. A superoxide dismutase enzyme comprising SEQ ID NO:2.

9. The superoxide dismutase enzyme of claim 8, wherein the enzyme is obtained from Deschampsia Antarctica.

10. The superoxide dismutase enzyme of claim 9, wherein the enzyme is thermostable at subzero temperatures.

11. The superoxide dismutase enzyme of claim 10, wherein the enzyme retains at least 70% of its activity at temperatures between -20°C to 20°C.

12. A process for producing recombinant superoxide dismutase enzyme encoded by

SEQ ID NO: 1 by cultivating Pichia cells of claim 6 and recovering the superoxide dismutase enzyme from the culture medium.

13. A method to preserve pharmaceutical products at subzero temperatures, said method comprising a step of mixing the superoxide dismutase enzyme of claim 8 into pharmaceutical preparations.

14. A method to preserve nutritional formulations, said method comprising a step of mixing 0.001% to 20% (w/v) of superoxide dismutase of claim 8 into a nutritional formulation.

15. A method to protect fruit trees and berries from UV damage, said method comprising the steps of : a) providing a mixture of super oxide dismutase enzyme of claim 7 , water and a surfactant compound; and b) spraying the fruit trees or berries with the mixture.

16. The method of claim 15 wherein the mixture contains 0.001 to 25% w/v of super oxidedismutase enzyme and 0.01 to 10% v/v of surfactant.