Global Advanced Research Journal of Microbiology (ISSN: 2315-5116) Vol. 1(7) pp. 108-119, August, 2012 Available online http://garj.org/garjm/index.htm Copyright 2012 Global Advanced Research Journals Full Length Research Paper Production and Optimal Performance Studies of Glucose Isomerase from Agricultural Raw Materials *Lawal AK, Banjoko AM, Osikoya AF, Olatope SO, Kayode OF, Etoamaihe M, Emoleila I, Alebiosu FA, Majolagbe YL, Shittu KA, Buhari F, and Dike EN. Department of Biotechnology, Enzymology and Genetics Division. Federal Institute of Industrial Research Oshodi Lagos State Nigeria. Accepted 06 August, 2012 An Investigation was carried out to study the production and Optimal activity of glucose Isomerase enzyme from agricultural raw materials. A total of 81 Bacilli Isolates were isolated and thereafter screened for their ability to produce glucose isomearase by using the plate assay technique on Nutrient agar fortified with 4% (w/v) of glucose. Bacilli promising Isolates for Isomerase production were identified as Bacillus coagulans (B 1 8, C 2 1, C 2 6, B 2 13, C 3 5 and C 2 4) and Bacillus megaterium (A 3 1,C 3 9, B 3 1, B 1 1 and B 1 7). Studies on the optimal conditions for the performance for temperature and ph of the produced Isomerase enzyme was in a range of 30 0 C - 70 0 C and 5.0-9.0 respectively. Comparative Studies of the enzyme for its activity as produced by Isolate B 1 8 and C 3 9 was 0.488µ/ml and 0.762µ/ml to 0.647µ/ml and 0.732µ/ml respectively. These values compares favourably with the standard (Sweetzyme) with 0.893µ/ml. Keywords: Bacilli, agricultural raw materials, glucose isomerase, optimal condition. INTRODUCTION Enzymes are valuable tools in industrial processes as the reactions they catalyse generally proceed under mild conditions, highly selective and are alternative in solving environmental problems (Fabiana et al.,1998). Biologically active enzymes may be extracted from living organisms. A very wide range of sources are used for the commercial enzyme production Actinoplanes and Zymonas, from spinach to snake venom. A very much larger number of *Corresponding author E-mail: kolakuns@yahoo.com; Tel. +2348023164214 enzymes are used in chemical analyses and clinical diagnosis. Non- microbial sources provide a larger proportion of these at present time. According to Fabiana et al., (1998), of the hundred or so enzymes being used industrially, over a half is from fungi and yeast and over a third are from bacteria with the remainder divided between animal (8%), and plant (4%) sources. However, microbes are preferred to plants and animals as sources of enzymes because they are generally cheaper to produce and their enzyme contents are more predictable and controllable. Reliable supplies of raw materials of constant composition are more easily arranged. Plant and animal tissues contain
109 Glo. Adv. Res. J. Microbiol. more potentially harmful materials than microbes, including phenolic compounds (from plants), endogenous enzyme inhibitors. Glucose isomerase, otherwise referred to as xylose isomerase (EC.5.3.1.5) is now a common object of commerce used to produce some three million tonnes of High Fructose Syrup per annum. It is an intracellular enzymes produced from a range of genera, principally Streptomyces, Bacillus, Corynebacterium and Arthrobacter spp. The enzyme is induced in organisms grown on media containing xylose as the free sugar or as xylans. High Fructose Syrup produced by the activity of glucose isomerase is useful sweetener; principally in carbonated beverages such as coca- cola and Pepsi- cola. High Fructose Syrup has found wide market in the United States in her soft drink industries. In Nigeria, Glucose Isomerase along with other Industrial enzymes are being imported up to a tune of about 200million. Also, this enzyme is scarce because they are not locally produced and the ones produced are often patented. Glucose Isomerase despite its diverse uses as sweetner in the carbonated beverage industries has never really received much attention in terms of local production and utilization. It is however on the basis of adding value to the production of glucose isomerase from locally available raw material that the research was focused at achieving the following objectives: To carryout local production of glucose isomerase using screened identified Bacilli Isolates. To partially characterize the enzyme inorder to determine their optimal performance. To utilize the enzyme for the production of fructose syrup. MATERIALS AND METHODS Collection of Soil Samples: Soil samples were collected from fruit market along Cappa Oshodi, along Agege motor road,lagos state,nigeria in sterile disposable Petri dishes and taken to the laboratory for immediate use. Isolation of Bacillus Spp. From Soil Samples: This was carried out by accurately weighing 10 grammes of the soil samples in to 90mls of sterile distilled water in a boiling tube. The tubes were placed in water bath at 90 C and maintained at this temperature for 1hr. Serial dilutions were performed and from an appropriate dilutions of 10 1 and 10 ⁴. 1ml was plated out into sterile Petri dishes and molten, cooled nutrient agar and tryptone soy- agar were poured on each plate by pour plate method. The plates were later incubated at 37 C for 24h- 48h. Morphological/Biochemical Test on Bacillus Isolates: Morphological test which include Gram- staining, spore test were carried out on the Bacillus isolates. Biochemical characterization for catalase test, gelatin test, MR, VP test, urease test and fermentation of sugars following standard methods as described by Cowan and Steel, (1974) and Buchanan and Gibson (1974). Screening for Bacillus Producing Glucose Isomerase: This was carried out by growing the Bacillus isolates on solid nutrient agar fortified with 1-4 % (w/v) of glucose. The plates were thereafter incubated at 37 C for 24h. The incubated plates were then flooded with Dinitrosalicylic acid (DNS) reagent, and later transferred into the oven and incubated at 45 C for 30 mins. Haloes produced around the zone of inoculation of the isolates were measured using graduated ruler. Production of Corn Steep Liquor: Maize grains were bought from local market at Mushin Metropolis,Lagos State Nigeria. Five (5) kilograms of the maize grains were weighed accurately and five litres of distilled water was added to it in a 10 litre plastic container. Steeping was allowed to proceed at 350C at 121 rpm for 72h at room temperature (28 ± 2 C). After 72h, steeped was carefully decanted from the maize into plastic container and kept in the refrigerator (4 C). Analysis of The Corn Steep Liquor: The corn steep liquor was analysed for the following: glucose as a reducing sugar; using the method of Pearson, (1991), ph, acidity and some minerals which include phosphorus, magnesium, calcium, iron and manganese using the method of AOAC,(1990). Production of Glucose Isomerase: Promising B. coagulans (B₁8) and B. megaterium (C₃9) were used for the production of glucose isomerase using submerged fermentation, though with little modifications as described by Kelly et al, 1977 as follows: Medium composition: Lablemco 2.5g, NaCl 2.5g, MgSO₄.7H₂O 2.5g, CoCl ₂ 0.025g, D-Xylose 0.25g, yeast extract 0.25g, 2.5% (v/v) corn steep liquor, 1 litre distilled water. The ph of the medium was adjusted to 7.2; and thereafter dispensed as 200mls into 250mls conical flasks. The flasks were later autoclaved at 121 c for 15 mins, cooled and later inoculated with 5mls of a 24h old culture of B. coagulans (B₁8) and B.megaterium (C₃9) in different flasks. The flasks were later placed in a shaker incubator at 35 c at 121rmp for 72h. Parameters Monitored During Production: The following parameters were monitored during the production of glucose isomerase by the Bacillus isolates at 24 hours intervals. Determination of ph: This was monitored using ph meter (Unicam 9450 model). Determination of Cell Growth: This was done using spectrophotometer (T70 UV/VIS) at 540nm.
Lawal et at. 110 Table 1. Spore forming Bacilli isolated from soil samples Lab code Gram reaction Cell morphology A 31 Positive reaction Presence of spores C 39 Positive reaction Presence of spores B 18 Positive reaction Presence of spores C 21 Positive reaction Presence of spores C 26 Positive reaction Presence of spores B 31 Positive reaction Presence of spores B 213 Positive reaction Presence of spores C 35 Positive reaction Presence of spores B 11 Positive reaction Presence of spores B 17 Positive reaction Presence of spores C 24 Positive reaction Presence of spores Determination of Protein: The method of Lowry et al (1951) was used at 750nm using spectrophotometer (T70 UV/VIS) and the concentrations of protein in the enzyme samples were determined with reference to standard Bovine Serum Albumin, (BSA). Recovery of Glucose Isomerase Enzyme: The extracellular enzyme solution was obtained by centrifugation at 4000rpm for 15 mins. Also, we obtained the intracellular enzyme by lysing the bacteria cell wall via freezing, thawing, sonication and centrifugation. The supernatant obtained was collected and also used as enzyme source. Determination of Glucose Isomerase Activity: This was carried out using the method of Cole, a modification of Bacon and Bell, 1948. The enzyme reaction mixture contained 0.5ml of 0.2M phosphate buffer (ph 7), 0.2ml of 1M D-glucose, 0.1ml of MgSO₄.7H₂O, 0.1ml of 0.01M CoCl₂.6H₂O and 0.2ml of enzyme solution. The mixture was incubated at 70 c for 1h; and the reaction was stopped by adding 2mls of 0.5M perchloric acid. Fructose Produced By the Enzyme Was Determined Reagents Consisting Of Two Solutions; A: resorcinol 0.05 % (w/v) in absolute ethanol and B: HCl (sp. gr.1.18) containing FeNH₄(SO₄)₂.12H₂O, 0.216g/l. 1ml of sample + 1.5ml of solution B. The contents were mixed thoroughly in tubes covered with foil and immersed in water bath at (80 ± 3 c) for 40 mins. The optical density of the resulting red coloured solution was read at 480nm using spectrophotometer. The concentration of fructose in the sample was determined with reference to fructose standard curve. A blank reagent containing 1ml of distilled water was used. One unit of glucose isomerase activity was defined as the amount of the enzyme that produced 1µmol of D- fructose per minute under the assay conditions described. Determination of the Optimum Conditions of Glucose Isomerase Temperature: The reaction mixture of 72h containing extracellular and intracellular enzymes from Bacillus isolates (B₁8 and C₃9) were prepared as earlier described and incubated at varying temperature ranges from 30 c - 100 c. The enzyme activity for each temperature was determined using Cole s method as described earlier. ph Studies: The reaction mixture of 72h containing extracellular and intracellular from Bacillus isolates (B₁8 and C₃9) were prepared as earlier described and therefore adjusted to varying ph ranges from 4.0 to 10.0. The enzyme activity for each ph was determined using Cole s method as described earlier. RESULTS AND DISCUSSION Isolation of Bacillus isolates at temperature of 90 c was to ensure that other forms of vegetative cells would be killed except Bacillus spps. that are known to be very resistant to high temperature probably as a result of their spore forming abilities. Gram reaction study revealed a total of 81 Bacillus isolates, since they were all positive (+ve). However, literature search indicated that Bacillus isolates that will probably produce glucose isomerase usually have spores at the centre or at the terminals of the cells. Based on this, only 11 Bacillus isolates were selected and subjected to some series of biochemical characterization (table 1). Biochemical characteristics of 11 Bacilli isolates are as shown in table 2. The result revealed that 6 were B.coagulans (B 1 8, C 2 1, C 2 6, B 2 13, C 3 5 and C 2 4) and 5 were B.megaterium (A 3 1,C 3 9, B 3 1, B 1 1 and B 1 7). All the 11 isolates are the probable glucose isomerase producers.
111 Glo. Adv. Res. J. Microbiol. Table 2. Biochemical characteristics of the Bacilli isolates Biochemical Tests. Lab Catalase Urease MR VP Gelatin Glucose Mannitol Probable identity code A 31 + + _ ± _ + _ B.megaterium C 39 + + _ ± + + _ B.megaterium B 18 + _ - - ± + _ B.coagulans C 21 + _ - + _ ± _ B.coagulans C 26 + - _ ± _ B.coagulans B 31 + + + ± + + _ B.megaterium B 213 + - ± ± + + - B.coagulans C 35 + - ± + - + - B.coagulans B 11 + + ± ± + + - B.megaterium B 17 + - - - + - - B.megaterium C 24 + - - + - + - B.coagulans Table 3. Zone of Clearance (MM) for Bacillus Isolates Grown on Glucose Medium Zone of Clearance/ Glucose Concentration Isolates 1% 2% 3% 4% A 31 N.D N.D 1.5 N.D C 39 N.D N.D 3.5 1.0 B 18 N.D N.D 4.0 1.5 C 21 N.D N.D 1.0 N.D C 26 N.D N.D 0.4 0.4 B 31 N.D N.D 3.2 N.D B 213 N.D N.D N.D 0.5 C 35 1.5 1.6 N.D 0.5 B 11 N.D N.D N.D N.D B 17 N.D N.D 2.0 N.D C 24 0.5 0.5 4.0 0.5 N.D No Obvious Detection The 11 Bacilli isolates were screened for their abilities to produce high concentration of glucose isomerase enzyme on nutrient agar fortified with glucose (1%- 4% w/v). From the result (table 3), isolates C 3 9, B 1 8 and C 2 4 showed very good clearance on the plates at 3% (w/v) concentration with 3.5mm, 4.0mm and 4.0mm respectively. Hence, they were used for further works. Chemical analyses of the corn steep liquor are shown in table 4. The result showed the absence of glucose, the ph and the total acidity was 5.32 and 140ppm respectively. Minerals were present in the sample though in varying concentration as 32.7ppm, 6.22ppm, 5.80ppm and 2.63ppm for phosphorus, magnesium, calcium and iron respectively. Manganese was completely absent from the sample. These analyses showed that corn steep liquor could be used as substrate for glucose isomerase production. This agrees with the observations of Chen et al., (1979). Changes in ph, growth and protein content of the isolates in the production medium. Growth studies (table 5, figure 1) shown an enhanced growth by the isolates throughout the period of production up to 72h. The growth as observed may be probably due to the utilization of the substrate by the
Lawal et at. 112 Table 4. Chemical Analyses of Corn Steep Liquor. Parameter Glucose Results N.D ph 5.32 Total acidity Phosphorus Magnesium 140ppm 32.7ppm 6.22ppm Calcium Iron Manganese 5.80ppm 2.63ppm N.D Figure 1. Changes in growth of B 18 (B.coagulans) and C 39 (B.megaterium)
113 Glo. Adv. Res. J. Microbiol. Table 5. Changes in growth of B. coagulans (B 18) and B. megaterium (C 39) Time(h) Growth B 18 C 39 0 0.070 0.050 24 0.087 0.056 48 0.104 0.778 72 0.445 1.143 Table 6. Changes in protein content (mg/ml) of isolates B 18 (B.coagulans) and C 39 (B.megaterium) Time (h) Protein content (mg/ml) B 18 C 39 0 0.360 0.230 24 0.700 0.380 48 1.300 0.980 72 1.840 2.000 Figure 2. Changes in Protein content (mg/ml) of B. coagulans (B 18) and B. megaterium (C 39). Bacillus isolates. The amount of protein (table 6, figure 2) in the production medium also rose steadily from 0h with 0.23mg/ml to 2.00mg/ml by 72h in C 3 9 isolate and 0.36mg/ml at 0h to 1.84mg/ml by 72h in B 1 8. Studies of the
Lawal et at. 114 Table 7. Changes in ph of isolates B 18 (B. coagulans) and C 39 (B. megaterium) Time (h) ph B 18 C 39 0 6.27 6.45 24 6.18 6.24 48 6.28 6.29 72 6.26 6.27 Figure 3. Changes in ph of isolates B 18 (B. coagulans) and C 39 (B. megaterium) ph (table 7, figure 3) of isolates in the production medium showed a fall in ph by 24h, then slight increase by 48h and were maintained at 72h. However, the ph during production was adjusted to neutrality. The studies of the enzyme activity as obtained from isolate B 1 8 from extracellular and intracellular samples during production (table 8, figure 4); the result revealed that the isolate produce glucose isomerase enzyme intracellularly and extracellularly. The isolate B 1 8, produced glucose isomerase enzyme intracellularly and extracellularly which have highest activities at 48h (0.50U/ml and 0.55U/ml respectively). Similar observation was reported by Chen et al (1979), during the studies of enzyme activity of glucose isomerase obtained from Streptomyces flavogriseus. The effect of temperature on extracellular glucose isomerase activity obtained from isolates B 1 8 and C 3 9 as shown in table 9, figure 5; showed that the enzyme activity of extracellular glucose isomerase of B 1 8 and C 3 9 increase with increasing temperature (30 C - 70 C). Enzyme
115 Glo. Adv. Res. J. Microbiol. Table 8. Enzyme Activities (U/ml) of Glucose Isomerase Produced by isolate B 18 (B.coagulans). Time (h) B 18 Intracellular Extracellular 24 0.450 0.339 48 0.500 0.555 72 0.444 0.460 Table 8c. Effect of temperature on the intracellular activity of glucose isomerase from isolates B 18 and C 39 Temperature( c) Enzyme activity (U/ml) B 18 C 39 30 0.47 0.45 40 0.44 0.58 50 0.34 0.43 60 0.50 0.41 70 0.44 0.36 80 0.33 0.56 90 0.64 0.37 100 0.41 0.63 Figure 4a. Enzyme Activity of Glucose Isomerase Produced by Isolate B 18 (B.coagulans).
Lawal et at. 116 Figure 4b. Enzyme activity of glucose isomerase produced by isolate B 18 (B.coagulans). Table 9. Effect of ph on the activity of extracellular glucose isomerase from isolates B 18 and C 39 ph Enzyme activity (U/ml) B 18 C 39 4 0.342 0.466 5 0.454 0.539 6 0.472 0.433 7 0.682 0.546 8 0.676 0.708 9 0.715 0.821 10 1.411 1.085 Figure 5. Effect of temperature on the extracellular activity of glucose isomerase from isolates B 18 and C 39
117 Glo. Adv. Res. J. Microbiol. Figure 5b. Effect of temperature on the intracellular activity of glucose isomerase from isolates B 18 and C 39 Figure 6a. Effect of ph on the activity of extracellular glucose isomerase from isolates B 18 and C 39 Figure 6b. Effect of ph on the activity of intracellular glucose isomerase from isolates B 18 and C 39
Lawal et at. 118 Table 10. Effect of ph on the activity of intracellular glucose isomerase from isolates B 18 and C 39 ph Enzyme activity (U/ml) B 18 C 39 4 0.403 0.354 5 0.318 0.482 6 0.505 0.363 7 0.444 0.363 8 0.560 0.666 9 0.622 0.687 10 1.220 1.140 Table 11. Comparison of enzyme activity from isolates B 18 and C 39 with sweetzyme (Novozyme). SAMPLE ENZYME ACTIVITY (U/ml) B 18(I) 0.488 B 18(E) 0.762 C 39(I) 0.647 C 39(E) 0.732 Standard (Sweetzyme) 0.893 Key: I Intracellular, E Extracellular activity was highest at 70 C with 0.682U/ml and 0.546U/ml respectively. The activity of the enzyme started decreasing at 80 c - 100 c. This is in line with the report of Kelly et al (1997) that during isomerization, temperature is preferably maintained within the range 20 c - 90 c and the best activity is obtained within 50 C 75 C. Studies carried out on the effects of ph of intracellular and extracellular enzyme showed that it affects enzyme activity and stability (tables 9,10 figure 6). Maximum activity of enzymes produced from both isolates (B 1 8 and C 3 9) was at a ph 10. This agrees with the information from novozyme and Kelly et al (1977). The former reported that maximum activity was obtained at a ph above 8.0; whereas maximum stability was found between ph 7.2 and 7.5. The latter reported that the ph of glucose containing medium undergoing isomerization is preferably maintained within the range 5 to 9, particularly 7.0-8.5. Comparative studies of glucose isomerase enzyme produced using intracellular and extracellular extracts from isolates B 1 8 and C 3 9 when salted out with ammonium sulphate (50% w/v saturation) have activities that compete favorably with the standard glucose isomerase obtained from Novozymes, Demark. The result is shown in table 11.
119 Glo. Adv. Res. J. Microbiol. CONCLUSION From the studies carried out so far, the followings have been established: Promising strains of Bacillus spp. of B.megaterium (C 3 9) and B.coagulans (B 1 8) are good producers of extracellular and intracellular glucose isomerase enzyme. Glucose isomerase produced was further used to produce fructose syrup. The stability of the enzyme for optimum temperature and ph were also been established. The enzyme produced competes favorably with the standard obtained from Novozyme. REFERENCES AOAC (1990). Official methods of analysis, 13 th edition. Washington D.C. Association of official analytical chemist. Bacon JSD and Bell DJ (1948). Biochem. J. 42. 379. Buchanan RE and Gibbson ME (1974). Bergey s manual of determinative bacteriology. Baltimore. The Williams and Wilkings Co. U.S.A Pp510-593. Chen WP, Anderson AW, Han YW (1979).Production of Glucose Isomerase by Streptomyces flavogriseus. Applied and Environmental Microbiology, Feb. 1979 Pp 324-331 Cowan ST, Steel KJC (1974). Manual of identification of medical bacteria. 2 nd edition. Cambridge University Press. Inc. (London) limited Pp 452. Effective conversion of glucose to fructose for sweetners. Novozyme starch application sheet. Fabiana G, Stanislav MM, Francesco P (1998). Genetic Eng. and Biotechnol. 3 and 4, 1998, 14-16. Forbes AL, Bowman BA, Filer LJ, Glinsman WH, White JS (1993). Health effects of dietary fructose. AJCN, 1993; 58(5S). Glinsman W.H, Bowman B.A. The public health significance of crystalline fructose. AJCN, 1993; 58(5S):820S. Kelly JM and John L (1977). Process for the preparation of glucose isomerase using Curtobacterium. US Patent Documents: 3821086; 3956066. Pearson D (1991). The Chemical Analysis of foods. 7 th edition. Churchell,Edinburgh.