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5280 Afr. J. Microbiol. Res. Table 1. Purification of α-L-rhamnosidase. Purification step Figure 3. SDS-PAGE of the purified α-L-rhamnosidase. Lane 1 is the broth supernatant, lane 2 is the ammonium sulphate precipitation fraction, lane 3 is the dialysate of the ammonium sulphate precipitation, lane 4 is the fraction from DEAE-sephrose column, and lane 5 is the fraction from Sephacryl-S200 column. Total protein (mg) Total activity (U) Specific activity (U/mg) Yield (%) Purified fold Broth supernatant 922.96 91243.8 97.2 100 1 Ammonium sulphate precipitation 161.5 54442.8 334.8 59.67 3.41 Dialysis 32.7 44857.8 1371.6 49.17 13.88 DEAE-Sepharose chromatography 5.64 40294.8 7144.2 44.16 72.27 Sephacryl S-200 chromatography 4.34 33328.8 7678.8 36.53 77.68 Figure 4. Effect of pH on α-L-rhamnosidase activity: pH optimum (■); pH stability(□).
prunin so far. By using the HPLC method to facilitate detection of the enzymatic activity (Figure 7A), the α-L-rhamnosidase from A. niger has been purified to homogeneity (Figures 2 and 3) by ammonium sulphate precipitation and chromatographies on DEAE-sepharose and Sephacryl S-200 HR columns. The MW of the purified α-L-rhamnosidase was measured to be around 87 kDa, similar to a previously reported α-L-rhamnosidase from Ni et al. 5281 A. niger (Manzanares et al., 1997). Although our purified α-L-rhamnosidase had similar pH optimal value in range of pH 4.0 to 5.0 as the previously reported α-L-rhamnosidases determined by the pNPR method (Manzanares et al., 1997; Spagna et al., 2000; Yoshikazu et al., 1973), their stable pH values are significantly different. In addition, our α-L-rhamnosidase showed an optimal temperature within 50 to 60°C, which was also different from other reported α-L-rhamnosidases from A. niger (Yoshikazu et al., 1973; Manzanares et al., 1997). Yet, its optimal pH, stable pH, optimal temperature and stable temperature were all in agreement with those of the naringinase from A. niger (Puri and Kalra, 2005). Moreover, the molecular weight of our α-L-rhamnosidase which was similar to the SDS-PAGE analysis of the naringinase from A. niger, but less than the result of gel filtration analysis of the same naringinase (168 kDa) (Puri and Kalra, 2005), suggesting it was a subunit from naringinase. This enzyme was easy to be purified. Based on the chromatogram, remaining enzymatic activity and calculated purified fold (Figure 2B, Table 1), it is obvious that the ion exchange chromatography was so efficient to purify the α-L-rhamnosidases that further purification procedures, for example, gel filtration chromatography and affinity chromatography, were not necessary. Its Km value (0.27 mM) indicating that our purified α-L-rhamnosidase has a high affiliation to naringin. Furthermore, the transforming rate and the residual naringin (Figure 7) were above 97% and less than 4%, respectively revealing the high efficiency and validity of the new purified α-L-rhamnosidase. These properties suggest our α-L-rhamnosidase was in high purity that can be used in food and pharmaceutical industries. The purified enzyme and its process possessed the following three advantages: the enzyme that displayed a strong enzymatic activity and good stability in a wide pH and temperature range (Figures 3 and 4) would facilitate the bioconversion of naringin to prunin, It also facilitated the purification of prunin because the transforming rate was very high and the remaining naringin was left in small amount (Figure 7) and the enzymatic reaction can be promoted by K + ion and some other chemicals (Tables 2 and 3 ). Moreover, this α-L-rhamnosidase can be applied to eliminate the bitter taste of citrus juice, which is expected to having the following four advantages: the Figure 5. Effect of temperature on α-L-rhamnosidase activity. (A) Temperature profiles; (B) temperature Fig.5 stability at 4(■), 25(▲), 37°C(○); (C) temperature stability at 50(□), 55°C(◆); (D) thermal process is safe; it will not cause other nutritional loss, for ature profiles; stability (B) temperature at 60°C. stability at 4(■), 25(▲), 37 ºC(○); (C) temperature the enzyme only specifically hydrolyzes naringin; it can at 50(□), 55 ºC(◆); (D) thermal stability at 60 ºC. hydrolyze naringin at natural and weak acidic pH value of the juice and unlike naringinase, it would not convert naringin to non-soluble naringenin which has to be removed by centrifuge and filtration during the juice processing. Conclusion The α-L-rhamnosidase from a fermented broth of A. niger was purified and characterized in light of its MW, optimal
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African Journal of Microbiology Res
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Editors Prof. Dr. Stefan Schmidt Ap
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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nces Table of Contents: Volume 6 Nu
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Table of Contents: Volume 6 Number
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Table 1. Overview of the Soil prope
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exactly. MICROBIAL BIOMASS IN ORGAN
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Table 3. Advantages and disadvantag
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analysis of polymerase chain reacti
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Enzyme assay Triplicate samples of
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Figure 2. Effect of a) incubation t
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Table 2. Plasmid profile. Emerenini
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Table 1. Properties and identity of
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more understandable by the fact tha
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fertilizers considered the most imp
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Table 3. Effect of NPK levels and s
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Table 4. Effect of NPK levels and s
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Table 5. Effect of NPK levels and f
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with mineral NPK on wheat plant. Eg
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to synthetic antibiotics. In spite
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Among these, the K. pneumonia and V
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Figure 5. Pseudomonas aeruginosa An
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pathogens of their environment (She
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cultivable indigenous fishes of Ind
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Table 1. Oxidative stress parameter
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of tularemia. Ann N Y Acad. Sci., 1
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al., 1998; Heubuelt 1929) have alre
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Table 2. Influence of organic compo
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NO2 NO2 Zare et al. 5131 Figure 4.
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Sundermeyer-Klinger H, Meyer W, War
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Table 1. Composition of Mueller-Hin
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dose level, and is effective antibi
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wearing a mask is useful during the
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Novel Swine-Origin Influenza A H1N1
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that it produces lactic acid, bacte
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Figure 2. DMRT Graph, effect of var
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Bacillus colony Fig 1. Colony morph
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thuringiensis isolate S1 (Figure 6)
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Figure 1. The Kinetic of P. aerugin
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Figure 3. DNA-dosimeter determined
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Relative Fluorescence Unit Figure 4
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Conclusion The public health risk i
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general are members of the normal i
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Table 2. Distribution of Nosocomial
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and also to assess the influence of
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Table 1. List of decamers used in R
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Figure 2. RAPD profile of Fusarium
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Table 1. Biosurfactant production p
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Figure 3. The correlation of reduct
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To estimate the water content, stra
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Kraiem et al. 5183 Table 2. Effect
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log CFU/g log CFU/g log CFU/g 8.5 7
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delays to cooling and wrapping on s
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pilus (tcp) that is a subtle of pol
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protein. DISCUSSION 70 kda 60 kda 5
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Percentage repellency (%) Percentag
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Fumigant toxicity of essential oils
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Table 1. Primers used for PCR and s
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Table 3. Amino acid substitutions i
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composition in qnrB alleles. Althou
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Figure 1. Overview the Yongxing isl
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standard, XSLJ1, XSLJ2, XSLJ5, XSLJ
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Table 1. primer information: sequen
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conventional method. The reasons fo
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Figure 1. Comparison of amplificati
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Table 3. Fruiting-body formation an
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Yokoyama E, Yamagishi K, Hara A (20
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Table 1. Primer oligonucleotide seq
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Figure 2. Nucleotide and putative a
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Figure 6. Ghrelin expression induce
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Page 167 and 168: Table 1. The SNPs related to the pr
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Page 181 and 182: Swimming time (s) 1000 800 600 400
Page 183 and 184: Hepatic glycogen (mg/g) Hemoglobin
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Page 213 and 214: Figure 1. Mechanism of enzymatic co
Page 215: Enzymatic activity (U/mL) Enzymatic
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