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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.150, #2)
Production and Characterization of Cellulase-Free Xylanase from Trichoderma inhamatum by Leonor Alves de Oliveira da Silva; Eleonora Cano Carmona (pp. 117-125).
The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25°C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50°C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T1/2 was 2.2 h at 40°C and 2 min at 50°C. The pH stability was high from 4.0 to 11.0. These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence.
Keywords: Xylanase; Trichoderma inhamatum ; Optimization; Enzyme characterization; Enzyme regulation
Properties of a Cationic Peroxidase from Citrus jambhiri cv. Adalia by Saleh A. Mohamed; Mohamed O. El-Badry; Ehab A. Drees; Afaf S. Fahmy (pp. 127-137).
The major pool of peroxidase activity is present in the peel of some Egyptian citrus species and cultivars compared to the juice and pulp. Citrus jambhiri cv. Adalia had the highest peroxidase activity among the examined species. Four anionic and one cationic peroxidase isoenzymes from C. jambhiri were detected using the purification procedure including ammonium sulfate precipitation, chromatography on diethylaminoethanol-cellulose, carboxymethyl-cellulose, and Sephacryl S-200 columns. Cationic peroxidase POII is proved to be pure, and its molecular weight was 56 kDa. A study of substrate specificity identified the physiological role of POII, which catalyzed the oxidation of some phenolic substrates in the order of o-phenylenediamine > guaiacol > o-dianisidine > pyrogallol > catechol. The kinetic parameters (K m, V max, and V max/K m) of POII for hydrolysis toward H2O2 and electron donor substrates were studied. The enzyme had pH and temperature optima at 5.5 and 40 °C, respectively. POII was stable at 10–40 °C and unstable above 50 °C. The thermal inactivation profile of POII is biphasic and characterized by a rapid decline in activity on exposure to heat. The most of POII activity (70–80%) was lost at 50, 60, and 70 °C after 15, 10, and 5 min of incubation, respectively. Most of the examined metal ions had a very slight effect on POII except of Li+, Zn2+, and Hg2+, which had partial inhibitory effects. In the present study, the instability of peroxidase above 50 °C makes the high temperature short time treatment very efficient for the inactivation of peel peroxidase contaminated in orange juice to avoid the formation of off-flavors.
Keywords: Citrus jambhiri cv. Adalia; Peroxidase; Properties; Thermal inactivation
Properties of a Cationic Peroxidase from Citrus jambhiri cv. Adalia by Saleh A. Mohamed; Mohamed O. El-Badry; Ehab A. Drees; Afaf S. Fahmy (pp. 127-137).
The major pool of peroxidase activity is present in the peel of some Egyptian citrus species and cultivars compared to the juice and pulp. Citrus jambhiri cv. Adalia had the highest peroxidase activity among the examined species. Four anionic and one cationic peroxidase isoenzymes from C. jambhiri were detected using the purification procedure including ammonium sulfate precipitation, chromatography on diethylaminoethanol-cellulose, carboxymethyl-cellulose, and Sephacryl S-200 columns. Cationic peroxidase POII is proved to be pure, and its molecular weight was 56 kDa. A study of substrate specificity identified the physiological role of POII, which catalyzed the oxidation of some phenolic substrates in the order of o-phenylenediamine > guaiacol > o-dianisidine > pyrogallol > catechol. The kinetic parameters (K m, V max, and V max/K m) of POII for hydrolysis toward H2O2 and electron donor substrates were studied. The enzyme had pH and temperature optima at 5.5 and 40 °C, respectively. POII was stable at 10–40 °C and unstable above 50 °C. The thermal inactivation profile of POII is biphasic and characterized by a rapid decline in activity on exposure to heat. The most of POII activity (70–80%) was lost at 50, 60, and 70 °C after 15, 10, and 5 min of incubation, respectively. Most of the examined metal ions had a very slight effect on POII except of Li+, Zn2+, and Hg2+, which had partial inhibitory effects. In the present study, the instability of peroxidase above 50 °C makes the high temperature short time treatment very efficient for the inactivation of peel peroxidase contaminated in orange juice to avoid the formation of off-flavors.
Keywords: Citrus jambhiri cv. Adalia; Peroxidase; Properties; Thermal inactivation
Properties of a Cationic Peroxidase from Citrus jambhiri cv. Adalia by Saleh A. Mohamed; Mohamed O. El-Badry; Ehab A. Drees; Afaf S. Fahmy (pp. 127-137).
The major pool of peroxidase activity is present in the peel of some Egyptian citrus species and cultivars compared to the juice and pulp. Citrus jambhiri cv. Adalia had the highest peroxidase activity among the examined species. Four anionic and one cationic peroxidase isoenzymes from C. jambhiri were detected using the purification procedure including ammonium sulfate precipitation, chromatography on diethylaminoethanol-cellulose, carboxymethyl-cellulose, and Sephacryl S-200 columns. Cationic peroxidase POII is proved to be pure, and its molecular weight was 56 kDa. A study of substrate specificity identified the physiological role of POII, which catalyzed the oxidation of some phenolic substrates in the order of o-phenylenediamine > guaiacol > o-dianisidine > pyrogallol > catechol. The kinetic parameters (K m, V max, and V max/K m) of POII for hydrolysis toward H2O2 and electron donor substrates were studied. The enzyme had pH and temperature optima at 5.5 and 40 °C, respectively. POII was stable at 10–40 °C and unstable above 50 °C. The thermal inactivation profile of POII is biphasic and characterized by a rapid decline in activity on exposure to heat. The most of POII activity (70–80%) was lost at 50, 60, and 70 °C after 15, 10, and 5 min of incubation, respectively. Most of the examined metal ions had a very slight effect on POII except of Li+, Zn2+, and Hg2+, which had partial inhibitory effects. In the present study, the instability of peroxidase above 50 °C makes the high temperature short time treatment very efficient for the inactivation of peel peroxidase contaminated in orange juice to avoid the formation of off-flavors.
Keywords: Citrus jambhiri cv. Adalia; Peroxidase; Properties; Thermal inactivation
Two Step Purification of Acinetobacter sp. Lipase and Its Evaluation as a Detergent Additive at Low Temperatures by N. Saisubramanian; S. Sivasubramanian; N. Nandakumar; B. Indirakumar; N. Amaranath Chaudhary; R. Puvanakrishnan (pp. 139-156).
Acinetobacter sp. lipase was purified to homogeneity by a two-step process. The crude enzyme (along with biomass) was subjected to partial purification by aqueous two phase system (ATPS), avoiding centrifugation and filtration steps. Conditions for lipase partitioning by ATPS were optimized by response surface methodology (RSM) and a combination of 29.45% polyethylene glycol 8000, 15.5% phosphate, and a pH of 7.0 resulted in an optimal partition coefficient. Partially pure lipase was further purified by a modified batch process using Octyl Sepharose CL-4B in a vacuum filtration apparatus. This two-step process resulted in a purified lipase with a yield of 74.6% having a specific activity of 88.8 U/mg of protein and a purification fold of 14.92. The homogeneity of the lipase preparation obtained by the purification process was confirmed by reversed phase high performance liquid chromatography profile. The molecular weight of the purified lipase was found to be around 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified lipase exhibited pH and temperature optima of 8.5 and 37°C, respectively. The lipase was active at low temperatures and it retained 86.8% activity at 10°C. It also displayed other features such as stability over a broad range of pH (3.0–9.0) as well as stability in the presence of hydrogen peroxide and commercial detergents. Based on these characteristics, the potential of this lipase as an additive in laundry detergent formulation was evaluated under low temperature wash conditions. The results indicated that Acinetobacter sp. lipase increased the washing efficiency of the detergent Nirma by 21–24% at 15°C–20°C, respectively.
Keywords: ATPS; Lipase purification; Response surface methodology; Oxidant stable lipase; Detergent additive
Immobilization of Escherichia coli novablue γ-glutamyltranspeptidase in Ca-alginate-k-carrageenan beads by Chih-Peng Hung; Huei-Fen Lo; Wen-Hwei Hsu; Shih-Chun Chen; Long-Liu Lin (pp. 157-170).
The recombinant Escherichia coli γ-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-k-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-k-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of l-theanine was performed in 50 mM Tris–HCl buffer (pH 10) containing 25 mM l-glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40°C for 12 h, and a conversion rate of 27% was achieved.
Keywords: Escherichia coli ; γ-glutamyltranspeptidase; Alginate; Immobilization; Bioconversion; l-theanine
Immobilization of Escherichia coli novablue γ-glutamyltranspeptidase in Ca-alginate-k-carrageenan beads by Chih-Peng Hung; Huei-Fen Lo; Wen-Hwei Hsu; Shih-Chun Chen; Long-Liu Lin (pp. 157-170).
The recombinant Escherichia coli γ-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-k-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-k-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of l-theanine was performed in 50 mM Tris–HCl buffer (pH 10) containing 25 mM l-glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40°C for 12 h, and a conversion rate of 27% was achieved.
Keywords: Escherichia coli ; γ-glutamyltranspeptidase; Alginate; Immobilization; Bioconversion; l-theanine
Synthesis of a Novel pH-Sensitive Methacrylate Amphiphilic Polymer and Its Primary Application in Aqueous Two-phase Systems by Wei QIN; Xue-Jun CAO (pp. 171-183).
In this study, a novel pH-sensitive and reversible water-soluble polymer(PABC) forming aqueous two-phase systems(ATPS) was synthesized by using 2-(dimethylamino)ethyl methacrylate, t-butyl methacrylate, and methyl methacrylate as monomers and 2,2′-azo-bis-isobutyronitrile as initiator. The PABC could be recovered by adjusting isoelectric point (PI) to 8.4, and recovery at PI could reach 95%. ATPS was formed by 5% (w/w) PABC and 10% (w/w) PEG20000. The partition coefficient K of lysozyme was 6.8, and the partition coefficient K of bovine serum albumin could reach 12.5 in the ATPS.
Keywords: Aqueous two-phase systems; PH-sensitive; Amphiphilic polymer; Methacrylate; Polymer recycling
Construction of a Recombinant S. cerevisiae Expressing a Fusion Protein and Study on the Effect of Converting Xylose and Glucose to Ethanol by Jinxin Zhang; Shen Tian; Yazhen Zhang; Xiushan Yang (pp. 185-192).
Gene XYL1 from Candida shehatae and gene XYL2 from Pichia stipitis were amplified by polymerase chain reaction (PCR), and the two genes were both placed under the strong promoter of alcohol dehydrogenase (ADH) of plasmid pAD2 to produce the recombinant expression vector pAD2-P12. Because the amplified XYL1 fragment lacks the stop codon UAA, the polypeptide expressed in yeast cells should be a fusion protein, which is a fusion of xylose reductase and xylitol dehydrogenase. Subsequently, the pAD2-P12 vector was transformed into Saccharomyces cerevisiae YS58 to produce a recombinant S. cerevisiae YS58-12. It was indicated that S. cerevisiae YS58-12 has the ability of metabolizing xylose to produce ethanol by fermentation experiment. The result of cofermentation of glucose and xylose by using this recombinant S. cerevisiae YS58-12 showed a relatively satisfactory result. The highest percentage of xylose consumption rate reached 81.3% and the ethanol yield was equal to 67.14% of the ideal value.
Keywords: Recombinant Saccaromyces cerevisiae ; XYL1 ; XYL2 ; Fusion protein; Xylose; Ethanol
Effect of Agitation and Aeration Rates on Chitinase Production Using Trichoderma virens UKM1 in 2-l Stirred Tank Reactor by Suraini Abd-Aziz; Christine Cheryl Fernandez; Madihah Md. Salleh; Rosli Md. Illias; Mohd. Ali Hassan (pp. 193-204).
Shrimps have been a popular raw material for the burgeoning marine and food industry contributing to increasing marine waste. Shrimp waste, which is rich in organic compounds is an abundant source of chitin, a natural polymer of N-acetyl-d-glucosamine (GluNac), a reducing sugar. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma virens UKM1 was used in this study. The effect of agitation and aeration rates using colloidal chitin as control substrate in a 2-l stirred tank reactor gave the best agitation and aeration rates at 200 rpm and 0.33 vvm with 4.1 U/l per hour and 5.97 U/l per hour of maximum volumetric chitinase activity obtained, respectively. Microscopic observations showed shear sensitivity at higher agitation rate of the above system. The oxygen uptake rate during the highest chitinase productivity obtained using sun-dried ground shrimp waste of 1.74 mg of dissolved oxygen per gram of fungal biomass per hour at the k L a of 8.34 per hour.
Keywords: Agitation; Aeration; Chitinase; Trichoderma virens UKM1; Shrimp waste; Colloidal chitin
Biophysical and Biochemical Characterization of a Hyperthermostable and Ca2+-independent α-Amylase of an Extreme Thermophile Geobacillus thermoleovorans by J. L. Uma Maheswar Rao; T. Satyanarayana (pp. 205-219).
α-Amylases reported from various microbial sources have been shown to be moderately thermostable and Ca2+ dependent. The bacterial strain used in this investigation is an extremely thermophilic bacterium Geobacillus thermoleovorans that produces a novel α-amylase (26 kDa; α-amylase gt), which is hyperthermostable (T opt 100 °C) and does not require Ca2+ for its activity/stability. These special features of α-amylase gt make it applicable in starch saccharification process. The structural aspects of α-amylase gt are, therefore, of significant interest to understand its structure–function relationship. The circular dichroism spectroscopic data revealed the native α-amylase gt to contain 25% α-helix, 21% β-sheet, and 54% random coils. The addition of urea, at high concentration (8 M), appeared to expose the buried Trp residues of the native α-amylase gt to the aqueous environment and thus showed low fluorophore. Fluorescence-quenching experiments using KI, CsCl, N-bromosuccinimide, and acrylamide revealed interesting features of the tryptophan microenvironment. Analysis of K sv and f a values of KI, CsCl, and acrylamide suggested the overall Trp microenvironment in α-amylase to be slightly electropositive. Fluorescence-quenching studies with acrylamide revealed the occurrence of both collisional as well as static quenching processes. There was no change in the α-helix content or the enzyme activity with an increase in temperature (60–100 °C) that suggested a critical role of the α-helix content in maintaining the catalytic activity.
Keywords: Ca2+-independent α-amylase; Quenchers; Tryptophan; Fluorescence; Circular dichroism
Enhanced Production of High-Quality Biomass, δ-Aminolevulinic Acid, Bilipigments, and Antioxidant Capacity of a Food Alga Nostochopsis lobatus by Usha Pandey; J. Pandey (pp. 221-231).
The growing interest in natural food has raised the global demand for nutraceuticals. We studied enhanced production of biomass, delta-aminolevulinic acid (δ-ALA), bili pigments and antioxidant capacity of a food alga Nostochopsis lobatus in a full-factorial (three level) design with supplemental Zn, glutamine, and Zn + glutamine in batch culture. Production of biomass, pigments, and antioxidant capacity all were higher under immobilized cell cultures in comparison to free cell cultures. Maximum biomass (2,390 mg dry wt l−1), δ-ALA (2.715 μg mg−1 dry wt h−1), phycocyanin (98.50 mg g−1 dry wt), phycoerythrin (158.0 mg g−1 dry wt), and antioxidant capacity (140.50 μmoles ascorbic acid equivalent capacity g−1 fresh wt) were recorded when Zn and glutamine were supplemented together in the growth medium at pH 7.8. These effects were found to be significantly related to the activities of glutamine synthetase (GSmax: 490.2 nmoles mg protein−1 min−1), glutamate synthase (GOGATmax: 27.0 nmoles mg protein−1 min−1), and glutamate dehydrogenase (GDHmax: 159.9 nmoles mg protein−1 min−1). This study shows that N. lobatus could be a promising bioresource for the production of nutritionally rich biomass, δ-ALA, bili pigments, and antioxidants. Use of immobilized cells in batch culture supplemented with Zn and glutamine could be an effective approach for scaling up production for commercial use.
Keywords: Nostochopsis lobatus ; Cyanobacteria; δ-Aminolevulinic acid; Bilipigments; Phycocyanin; Phycoerythrin; Biomass