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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.144, #3)

Biotransformation of Valdecoxib by Plant Cell Cultures by V. Molmoori; K. Srisailam; V. Ciddi (pp. 201-212).

Valdecoxib is a new anti-inflammatory drug that is highly selective for inhibition of the inducible form of cyclooxygenase (COX-2). In the present study, biotransformation of valdecoxib was investigated in cell cultures of five medicinal plants, viz., Catharanthus roseus, Azadirachta indica, Capsicum annuum, Ervatamia heyneana, and Nicotiana tabacum. Identification of the biotransformed products was carried out by using high-performance liquid chromatography coupled with diode array detection and liquid chromatography–tandem mass spectrometry analysis. All the cultures transformed valdecoxib into more polar compounds, and C. roseus also produced one unknown compound that is less polar than the substrate. The reactions performed by these plant cell cultures include hydroxylation, methylation, and demethylation. Optimization studies were performed to investigate the effect of the day of extraction and substrate concentration on biotransformation.

Keywords: Valdecoxib; Biotransformation; Plant cell cultures; Catharanthus roseus ; Azadirachta indica ; Capsicum annuum ; Ervatamia heyneana ; Nicotiana tabacum

Effect of Chain Length on Enzymatic Hydrolysis of p-Nitrophenyl Esters in Supercritical Carbon Dioxide by Mahesh N. Varma; Giridhar Madras (pp. 213-223).

The effect of chain length on the enzymatic hydrolysis of various p-nitrophenyl esters was investigated. Specifically, the hydrolysis of various esters p-nitrophenyl butyrate (PNPB), p-nitrophenyl caprylate (PNPC), p-nitrophenyl laurate (PNPL), p-nitrophenyl myristate (PNPM) and p-nitrophenyl palmitate (PNPP) was studied in supercritical carbon dioxide (ScCO₂) with lipase (Novozym 435). This indicates that the conversion of nitrophenyl esters decreases with increasing chain length. The effect of various parameters such as amount of water added, temperature, and enzyme loading was studied. The optimum temperature for the hydrolysis of PNPB and PNPC was 50°C but was 55°C for PNPL, PNPM, and PNPP in ScCO₂. The reactions were also conducted in acetonitrile as the solvent, and it was found that the reactions reach equilibrium much faster in ScCO₂ than in acetonitrile. The kinetics of the hydrolysis reactions were modeled using a Ping Pong Bi Bi model.

Keywords: Hydrolysis; PNP; PNPL; Supercritical carbon dioxide; Equilibrium

Artificial Neural Network-Genetic Algorithm Approach to Optimize Media Constituents for Enhancing Lipase Production by a Soil Microorganism by M. A. Haider; K. Pakshirajan; A. Singh; S. Chaudhry (pp. 225-235).

Results of lipase production by a soil microorganism, expressed in terms of lipolytic activities of the culture were modeled and optimized using artificial neural network (ANN) and genetic algorithm (GA) techniques, respectively. ANN model, developed based on back propagation algorithm, were highly accurate in predicting the system with coefficient of determination (R ²) value being close to 0.99. Optimization using GA, based on the ANN model developed, resulted in the following values of the media constituents: 9.991 ml/l oil, 0.100 g/l MgSO₄ and 0.009 g/l FeSO₄. And a maximum value of 7.69 U/ml of lipolytic activity at 72 h of culture was obtained using the ANN-GA method, which was found to be 8.8% higher than the maximum values predicted by a statistical regression-based optimization technique-response surface methodology.

Keywords: Artificial neural networks; Genetic algorithms; Response surface methodology; Optimization; Lipase production; Soil microorganism

The Flexibility of the Non-Conservative Region at the C Terminus of d-Hydantoinase from Pseudomonas putida YZ-26 is Extremely Limited by Xue-Yao Zhang; Li-Xi Niu; Ya-Wei Shi; Jing-Ming Yuan (pp. 237-247).

We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of d-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

Keywords: d-Hydantoinase; C-terminal deletion and substitution; Subunit dissociation; Structural modeling; Properties

Expression of Recombinant Human Epidermal Growth Factor in Escherichia coli and Characterization of its Biological Activity by Ahmad Faizal Abdull Razis; Elysha Nur Ismail; Zarida Hambali; Muhammad Nazrul Hakim Abdullah; Abdul Manaf Ali; Mohd Azmi Mohd Lila (pp. 249-261).

Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.

Keywords: Human epidermal growth factor; Fusion protein; Signal sequence; Cell proliferation; Percentage of growth

Characterization of Cyclodextrin Glucanotransferase Produced by Bacillus megaterium by Ivan Pishtiyski; Viara Popova; Boriana Zhekova (pp. 263-272).

Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of soluble starch, with a K _m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr²⁺, Mg²⁺, Co²⁺, Mn²⁺, and Cu²⁺, and it was inhibited by Zn²⁺and Ag⁺. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of cyclodextrins, but the predominant product was β-cyclodextrin.

Keywords: Cyclodextrin glucanotransferase; Cyclodextrins; Enzyme characterization; Bacillus megaterium ; Application

Importance of C-Terminal Region for Thermostability of GH11 Xylanase from Streptomyces lividans by Qin Wang; Tao Xia (pp. 273-282).

The amino acid sequences of xylanase B (XlnB) and xylanase C (XlnC) from Streptomyces lividans show significant homology. However, the temperature optima and stabilities of the two enzymes are quite different. XlnB exhibits an optimum temperature of 40 °C and retains 50% of its maximum activity at 43 °C, whereas the corresponding values for XlnC are 60 and 70 °C. To analyze these properties further, as well as to study the effect of the exchange of homologous segments in the C-terminal region, four chimeras designated as BSC, BFC, CSB, and CFB were constructed by substituting segments from the C-terminal homologous region of XlnB gene with that of XlnC and in turn substituting XlnC gene with that of XlnB. The purified chimeric enzymes were characterized with respect to pH/temperature activity, stability, and kinetic parameters. Most of enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45–55 °C and pH 7.0. Both K _m and k _cat values of chimeric enzymes for p-nitrophenyl-β-d-cellobioside were admixtures of both parental enzymes, except that the k _cat value of chimeric BFC (2.79 s⁻¹) was higher than that of parental XlnC (1.99 s⁻¹). Notably, thermal stability of chimeric BSC and BFC was increased by 25 and 13 °C separately, as compared to one of parental XlnB, whereas the thermal stability of chimeric CSB and CFB was decreased by 23 and 21 °C, respectively, as compared to another parental XlnC. These results suggest that homologous C-terminal region in S. lividans GH11 xylanase appears to play an important role in determining enzyme characteristics, and exchanging of different segments of gene in this region might significantly alter or improve the enzymatic properties such as thermal stability.

Keywords: Xylanase; Chimeric enzyme; Thermostability; Streptomyces lividans

An Inhibitive Determination Method for Heavy Metals Using Bromelain, A Cysteine Protease by M. Y. Shukor; N. Masdor; N. A. Baharom; J. A. Jamal; M. P. A. Abdullah; N. A. Shamaan; M. A. Syed (pp. 283-291).

A heavy-metal assay has been developed using bromelain, a protease. The enzyme is assayed using casein as a substrate with Coomassie dye to track completion of hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion, and the solution is brown. In the presence of metal ions such as Hg²⁺ and Cu²⁺, the hydrolysis of casein is inhibited, and the solution remains blue. Exclusion of sulfhydryl protective agent and ethylenediaminetetraacetic in the original assay improved sensitivity to heavy metals several fold. The assay is sensitive to Hg²⁺ and Cu²⁺, exhibiting a dose–response curve with an IC₅₀ of 0.15 mg l⁻¹ for Hg²⁺ and a one-phase binding curve with an IC₅₀ of 0.23 mg l⁻¹ for Cu²⁺. The IC₅₀ value for Hg²⁺ is found to be lower to several other assays such as immobilized urease and papain assay, whilst the IC₅₀ value for Cu²⁺ is lower than immobilized urease, 15-min Microtox™, and rainbow trout.

Keywords: Bromelain; Cysteine protease; Inhibitive determination method

An Improved Enzyme Assay for Molybdenum-Reducing Activity in Bacteria by M. Y. Shukor; M. F. A. Rahman; N. A. Shamaan; C. H. Lee; M. I. A. Karim; M. A. Syed (pp. 293-300).

Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, K _m for the laboratory-prepared 10:4-phosphomolybdate is 2.56 ± 0.25 mM (arbitrary concentration), whereas the apparent V _max is 99.4 ± 2.85 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent Michaelis constant or K _m for NADH as the electron donor is 1.38 ± 0.09 mM, whereas the apparent V _max is 102.6 ± 1.73 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent K _m and V _max for another electron donor, NADPH, is 1.43 ± 0.10 mM and 57.16 ± 1.01 nmol Mo-blue min⁻¹ mg⁻¹ protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V _max obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.

Keywords: Assay; Mo-reducing enzyme; Molybdate; Phosphomolybdate; Molybdenum blue

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.144, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.144, #3)