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Research Article
 

Purification and Characterization of D-psicose 3 Epimerase (DPEase) From Escherichia coli BL21 (DE3) pET21b dpe



Deby EdyLiani, Yurnaliza Yurnaliza and Budi Saksono
 
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ABSTRACT

Background and Objective: The DPEase enzyme from Agrobacterium tumefaciens is more efficient and has a high activity in D-fructose. The dpe gene has been successfully cloned to Escherichia coli BL21 (DE3) pET-21b dpe but the enzyme has not been purified and its character is unknown. The intent of this study was to purify and assign of DPEase enzyme by recombinant E. coli. Materials and Methods: The enzyme was clarified by affinity chromatography and then characterized by following pH, temperature, co-factor parameters. Analysis of molecular weight proteins was done by SDS-PAGE. Results: Through purification, the purified DPEase activity was increased 1,01 times than crude and with 84.2% of yield. The DPEase had an the maximum temperature is 40°C and pH was 8.5. The presence of Mg2+, Mo2+, Cu2+, Ca2+ and Zn2+ inhibited the activity of the enzyme while of Co2+, Mn2+, Fe2+, Ni2+ enhanced the activity. Estimation of molecular weight through SDS-PAGE revealed that weight of DPEase was 32 kDa. Conclusion: Purified DPease enzymes shows clear bands that demonstrate successful purification using affinity chromatography. It is expected that after pure enzymes are obtained the character of the enzymes working will be maximized.

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Deby EdyLiani, Yurnaliza Yurnaliza and Budi Saksono, 2020. Purification and Characterization of D-psicose 3 Epimerase (DPEase) From Escherichia coli BL21 (DE3) pET21b dpe. Pakistan Journal of Biological Sciences, 23: 561-566.

DOI: 10.3923/pjbs.2020.561.566

URL: https://scialert.net/abstract/?doi=pjbs.2020.561.566
 
Copyright: © 2020. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

D-Psicose (C-3 epimerase of D-Fructose) is a limited amount of monosaccharide with sweetness level of 30% lower than sucrose. D-Psicose is not only synthesized by microorganisms but also present as a free sugar in wheat1. A side from being food sweetener, D-psicose also plays a role in increasing insulin levels2, hepatic lipid accumulation3, securing pancreatic beta cells4. The D-psicose has low calorie5 and increases blood sugar levels6. These characteristics indicate that D-psicose prevent diabetes and other diseases. The D-psicose is used as a sweetener with calories that is safe for consumption by the drug and food regulatory agency in America (In August 2011 GRN No. 400) and has been commonly used as sweetener and food additives7. The D-psicose can be applied in the food attentiveness8.

The D-psicose converted from fructose enzymatically by DPEase encoded by dpe gene. This DPEase enzyme is included in the metalloprotein enzyme group because it requires metal ions to increase its enzyme activity9 especially in Co2+ and Mn2+ metal ions10. This characteristic distinguishes DPEase from other epimerases. The DPEase enzyme was originally announced as D-tagatose 3-epimerase (DTEase), but because the specificity of the product produced was D-psicose, so this enzyme then appeared as the DPEase enzyme11.

Several studies reported that dpe gene were isolated from various microorganisms8, i.e. Clostridium cellulolyticum H10, C. bolteae ATCC BAA-6139, C. scindens ATCC 3570412, Ruminococcus sp., 5139 BFAA13 and A.tumefaciens14. Among recombinant DPEases, DPEase from A. tumafaciens is more effective in the comparison of D-fructose to D-psicose and it is proven that the activity of the enzyme produced is greater with D-fructose than D-tagatose as a substrate11.

The ability of bacteria in producing D-psicose is low in nature. Genetic engineering is one way to improve bacterial ability to produce D-psicose by isolating dpe gene which is responsible for DPEase synthesis. Isolation of dpe gene from A. tumefaciens and cloning to E. coli BL21 (DE3) has been reported from previous study15. However, the expression and characterization of the DPEase has not been studied yet. Therefore, the characterization of pure enzyme should be done prior its application.

MATERIALS AND METHODS

This study was conducted in October, 2017 to March, 2018 at the Bioengineering Carbohydrate Research Laboratory (CBRG) LIPI Cibinong Biotechnology Laboratory, Bogor Regency, West Java.

Bacterial strain and culture condition: Different things done in this study were bacterial conditions and culture of E. coli BL21 (DE3) pET-21b containing dpe gene (recombinant E. coli strain) from A. tumefaciens local15. The recombinant bacteria was cultivated in Luria-Bertani (LB) containing Ampicillin (20 mg mL1) at 37°C under agitation in rotary shaker (160 rpm) for 24 h.

Production of DPEase: The DPEase enzyme from recombinant E. coli strain are made in a specific medium that has been designed by researchers. Overnight raising was vaccinate into 1 L specific medium and incubated at 37°C under agitation. After stretching an OD600 of 0.5-1, the inducer, 1 mM IPTG was mixed into culture and maintained at 18°C for 16 h. Induced culture were then centrifuged to obtain biomass. Biomass was redrooping into 10 mM Tris buffer pH 8.0, sonicated and centrifuged to obtain supernatant. The supernatant was applianced as crude enzyme.

Enzyme assay: Determination of the activity of the enzyme DPEase was measured based on the formation of D-psicose with D-fructose as a substrate. The confession mixtures implied 50 μL crude enzyme and 500 μL fructose solution (100 g mL1) in 50 μL Tris-HCl buffer 1 M pH 8.0 and 400 μL aquadest for 10 min at 40°C stop reaction with heat up at 100°C for 10 min in boiling water. Remaining sucrose was digested by adding culture of Saccharomyces cerevisiae into mixture with ratio of 1:1 (v/v) and maintained at 37°C under agitation at 160 rpm for 18 h. To eliminate culture of S. cerevisiae using centrifugation at 5.000 rpm for 5 min. The D-psicose is checked by Cysteine-carbazole method16. One unit of enzyme activity is an amount enzyme that catalyzes 1 μmol D-psicose per minute. Protein levels were checked by the Lowry analysis17, with serum albumin protein as standards.

Enzyme purification: The purification of the recombinant DPEase enzyme was carried out by affinity chromatography using the His-Tag resin in HisTrap affinity column (HisTrap HP). The DPEase enzyme was prepared before loading to chromatography column. Crude enzyme was recovered and filtered through 0.22 μm sterile syringe filter. Filtrate enzyme was ready to insert to column after added with 10 mM of CoCl2+ solution. The His-Tag resin column was rinsed with distilled water and then equilibrated with 10 mL of buffer A (20 mM imidazole, 50 mM Tris-HCl, 250 mM NaCl, pH 8.0). Two milliliters of crude enzyme was loaded to the list. The list was washed with 20 mL buffer A and with 20 mL buffer B (250 mM imidazole, 50 mM Tris HCl, 250 mM NaCl, pH 8.0), respectively. The fractions were was collected every 1,5 mL. This study was carried out under temperature 20oC. The purity of the enzyme was checked its activity and the purity. The profile protein of purified DPEase was observed using SDS-PAGE.

Temperature and pH parameters to the DPEase enzyme: To see the effect of temperature on DPEase activity was conducted by reacting enzymes at various temperature of 30, 35, 40, 45 and 50°C. The reaction mixtures containing 0.5 mL of D-fructose (50 g L1), 0.1 mL of 1 M Tris-HCl buffer pH 8.5 and 0.1 mL were reacted at various temperature parameters for 10 min.

To see the effect of pH on DPEase activity was conducted by reacting enzymes at various pH. Sodium phosphate buffer (1 M) was used pH 6.0-7.0 meanwhile pH 8.0, 8.5, 9.0 was used Tris-HCl buffer (1M) and Glycine buffer (1M) was utilized for pH values 10.0. The enzyme was reacted at 40°C for 10 min.

Metal ions parameters to the DPEase enzyme: To see the effect of metals on the activity of the DPEase enzyme and then reacted by adding nine metal ions i.e., Mn2+, Mg2+, Mo2+, Cu2+, Co2+, Ca2+, Fe2+, Zn2+, Ni2+ into the reactions. The crude enzymes was dialyzed prior characterization. The enzyme dialysis was performed at 16°C for 24 h using the 1 M Hepes buffer (pH 8.0). Hepes buffer was replaced to new solution at each 30, 60 and 120 min. After 24 h of dialysis, enzymes are ready to be characterized.

Enzyme assay conducted as above. The reaction mixture contain the metal ions were added each as a solution for 0.1 mL metal solution (MnCl2, MgCl2, MoCl2, CuSO4, CoCl2, CaCO3, FeSO4, ZnSO4, NiCl2), this was followed by incubating an enzyme of 0.1 mL at 40°C for 1 hr. The effect of metal ion as co-factor if increase enzyme activity while inhibitor reduce.

Statistical analysis: Descriptive statistics, data are presented in the mean and standard deviation of the test. Relative activity, the highest value becomes 100% and other values are divided into the highest value.

RESULTS

Enzyme purification and molecular mass determination: Crude DPEase had a specific activity of 5.02 U mg1 and total protein of 9.55 mg from 2 mL of crude enzyme volume. The use of affinity chromatography increased enzyme purity 1.01 times than crude with specific activity of 5.09 U mg1 and yield 84.2% (Table 1).

The purity of pure enzymes is proven through SDS-PAGE, which can be seen at Fig. 1. In addition, to knowing the molecular mass of the protein obtained from the purification results, it was checked using SDS-PAGE. Crude enzyme from supernatant of lysate cell contains many soluble protein including DPEase (Fig. 1, lane 5). In Line 6 was unbound protein fraction which did not interact with His-Tag resins. The washing fraction (lane 7) was the proteins that weak interacting with the resin and will come out. Mean while in lane 8 was eluate, a fraction of target protein which removed from the column (Fig. 1). The existence DPEase protein was shown as a single band at 32 kDa of molecular weight in the eluation fraction. Thus, pure DPEase enzyme was obtained successfully only by single step chromatography using His-Tag affinity chromatography.

Temperature parameters to the DPEase enzyme: Based on the results obtained by testing the various temperatures, the DPEase enzyme activity is obtained in Fig. 2. The DPEase activity increased significantly and reached the optimum activity when the temperature was 40°C. Increasing of temperature more than 40oC will decrease enzyme activity significantly. The DPEase activity was in moderate temperature. The DPEase was categorized in the mesophilic enzyme group. The enzyme belong to this group had maximum activity at the moderate temperatures.

Fig. 1: Molecular mass of purified DPEase from E. coli BL21 (DE3) pET-21b contain gene dpe by affinity chromatography
  Lane 1-3: Bovine serum albumin (BSA) at concentration 2, 1 and 0.5 μg mL1, Lane 4: Molecular weight marker, Lane 5-8: Crude enzyme, unbound proteins, washing protein, eluent protein of DPEase

Table 1: Profile of crude and purified enzymes of DPEase

Fig. 2: Testing the temperature parameters for DPEase enzyme activity with temperatures varying at pH 8.5 for 10 min

Fig. 3: Testing the pH parameters for DPEase enzyme activity with pH varying at temperature 40°C for 10 min

Table 2: Enzyme activity assay of metal ions

Table 3: Optimum character of various DPEase enzymes produced from strains A. tumefaciens

pH parameters to the DPEase enzyme: Based on the results obtained by testing the various pH, the DPEase enzyme activity is obtained in Fig. 3. The greatest activity of the DPEase enzyme is pH 8.5. The DPEase activity is at its lowest point at pH 6.0 and increases slowly with increasing pH and optimum at pH 8.5. An increase above of pH 8.5 resulted a significant decrease in activity.

Metal ions parameters to the DPEase enzyme: Based on the results obtained by testing nine type of metal ions, the DPEase enzyme activity was obtained in Table 2. The DPEase activity increased in the presence of co-factors Co2+, Mn2+, Fe2+ and Ni2+, but decreased in Mg2+, Mo2+, Cu2+Ca2+, Zn2+. The best co-factor to improved the DPEase activity was Co2+, followed by Mn2+, Fe2+ and Ni2+. The potential metal inhibitor for decreased of DPEase activity was Cu2+, followed by Mo2+, Ca2+, Zn2+, Mg2+ (Table 2).

DISCUSSION

The DPEase was successfully purified in active state, by using single step of affinity chromatography with His-Tag resin column and confirmed through SDS-PAGE electrophoresis and enzyme activity of evaluation foil. The DPEase activity in this study was lower than DPEase from Kim et al.11 research that the dpe gene also isolated from A. tumefaciens by using three purification step with 3 different column (HisTrap HP, HiPrep and Sephacryl S-300 HR). The pure DPEase had lower maximal temperature than crude. Crude DPEase had an optimum temperature at 45°C meanwhile pure DPEase at 40°C. The crude enzyme was more stable at high temperatures than a pure state. Its may be caused by effect of the co-factor. Co-factor will improve enzyme stability to temperature. The pure enzyme was loss its stability because during the purification step, all co-factor was removed from the enzyme18. Purified DPEase enzymes have optimum activity at temperature of 40°C and pH 8.5 . This characters similar to DPEase character isolated from Shinorizobium sp.19. Compared to previous report Kim et al.11, theirs local DPEase possess differences of enzyme activities in pH and temperature. DPEase character11 have optimum at pH 8.5 and 8.0. The differences amino acids of DPEase constructing may yield a different result of activity of same enzyme20.

The DPEase activity was increased in the present of co-factors, Co2+, Mn2+, Fe2+, Ni2+and inhibited by Mg2+, Mo2+, Cu2+, Ca2+, Zn2+. Among co-factors, addition of Fe2+ was recommended in food industry, considering its benefits to human bodies and safe for body consumption. Overall, from isolation DPEase enzyme of Agrobacterium sp., theirs DPEase has different character has been identified. Low optimum temperature is useful to minimize velocity of miliard reaction on base pH and absolutely useful to minimize dependancy on metal ions of Cu2+ and Mn2+ danger in body health. And from industry application aspect especially in small industry absolutely has the uniqueness to explore. Fe2+ usage as co-factor and very important, because Fe2+ is needed to our body. At this time, we are developing functional food based on DPEase enzyme usage with the hope that this research can provide information for readers and other useful research that wants to conduct research on the enzymes DPEase and D-psicose.

The interesting thing is all of the genetic source from A. tumafaciens (Table 3). Theirs DPEase has the similarity with DPEase reported by Tseng et al.23 that needs Co2+ ion, but it is different from DPEase reported by Kim et al.11 which requires more Mn2+ ions. If seen from the character of the optimal temperature of DPEase in this study only about 40°C, this value is relatively lower than the other DPEase 50°C11,10,21, 55°C23 and 60oC22.

What is different in this study is the strain of Agrobacterium tumafaciens used is a local strain other than that the media for the production of enzymes uses a specific media that has been designed itself that comes from waste. Means that this research has succeeded in achieving Indonesia's natural wealth and using unused waste is beneficial again.

CONCLUSION

The DPEase enzyme was purified using a single step column His-Tag in the active state. Confirmed through SDS-PAGE electrophoresis and enzyme activity assay. Based on electrophoresis testing, the resulting enzyme appears in a single band with an atomic mass of 32 kDa which shows the success of purification with enzyme specific activity of 5.09 U mg1. With the acquisition of pure enzymes in this study indicates that the purification conducted by researchers has been successful. Pure enzyme DPEase shows optimum activity at pH 8.5 and temperature of 40oC. the DPEase activity was improved by co-factors, Co2+, Mn2+, Fe2+, Ni2+and inhibited by Mg2+, Mo2+, Cu2+, Ca2+, Zn2+. From various metal ion tests, the use of Fe2+metal ions as cofactors in the DPEase enzyme has potential in the food industry.

SIGNIFICANCE STATEMENT

This research succeeded in purifying the cloned DPEase enzyme from a local strain of Agrobacterium tumafaciens which is useful in producing D-Psicose as a low-calorie natural sugar, increases insulin levels which is good for diabetes and protects the pancreas.

ACKNOWLEDGEMENT

To the Biotechnology Research Center, Ministry of Research and Higher Education for the 2017 INSINAS Project the authors say many thanks.

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