Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #3)

Analysis of Relationship Between Microbial and Methanogenic Biomass in Methane Fermentation by Kenzo Kubota; Yuya Ozaki; Yoshiki Matsumiya; Motoki Kubo (pp. 493-501).

To analyze the relationship between biomass of microorganisms and methane production, the total biomass of bacteria and archaea (BA) during methane fermentation was analyzed by the environmental DNA analysis method. In the case of using methanogenic sludge as a seed which is generally used for methane fermentation, the total BA biomass reached to 1.5 × 10⁸ to 3.6 × 10⁸ cells/ml when methane was produced. On the other hand, soil suspension was used as a seed; methane was not produced for 14-day cultivation. However, the total BA biomass reached to above 1.5 × 10⁸ cells/ml. The methanogen biomass was counted by using a fluorescence microscope (coenzyme F₄₂₀), and the methanogen biomass and the ratio of methanogens in the total of BA were analyzed during methane fermentation. At the methane-producing phase, the methanogen biomass reached to 1.3 × 10⁸ cells/ml, and the ratio of methanogens was above 70% of the total BA. When the ratio of methanogens in a seed was changed, the methane-producing phase was moved. However, the relationship between methanogens and other microorganisms at the methane-producing phase was almost similar.

Keywords: Methane fermentation; Microbial biomass; Methanogen; Environmental DNA (eDNA); Coenzyme F₄₂₀

Production of Metallothionein Polyclonal Antibodies Using Chickens as Model by Angélica María Ortiz-Bueno; Bertha Alicia León-Chávez; Alejandro Ruiz-Tagle; Patricia Lozano-Zarain; Leopoldo Castro-Caballero; William E. Achanzar; Eduardo Brambila (pp. 502-511).

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.

Keywords: Chicken antibodies; Metallothionein; Metallothionein isoforms; IgY production

Application of Calcium Alginate–Starch Entrapped Bitter Gourd (Momordica charantia) Peroxidase for the Removal of Colored Compounds from a Textile Effluent in Batch as well as in Continuous Reactor by Mahreen Matto; Rukhsana Satar; Qayyum Husain (pp. 512-523).

Calcium alginate–starch entrapped bitter gourd peroxidase has been employed for the treatment of a textile industrial effluent in batch as well as in continuous reactor. The textile effluent was recalcitrant to decolorization by bitter gourd peroxidase; thus, its decolorization was examined in the presence of a redox mediator, 1.0 mM 1-hydroxybenzotriazole. Immobilized enzyme exhibited same pH and temperature optima for effluent decolorization as attained by soluble enzyme. Immobilized enzyme could effectively remove more than 70% of effluent color in a stirred batch process after 3 h of incubation. Entrapped bitter gourd peroxidase retained 59% effluent decolorization reusability even after its tenth repeated use. The two-reactor system containing calcium alginate–starch entrapped enzyme retained more than 50% textile effluent decolorization efficiency even after 2 months of its operation. The absorption spectra of the treated effluent exhibited a marked difference in the absorption at various wavelengths as compared to untreated effluent. The use of a two-reactor system containing immobilized enzyme and an adsorbent will be significantly successful for treating industrial effluents at large scale, and it will help in getting water free from aromatic pollutants.

Keywords: Alginate; Bitter gourd peroxidase; Concanavalin A; Entrapment; Immobilization; Starch

Biosorption of Cr(VI) from Water Using Biomass of Aeromonas hydrophila: Central Composite Design for Optimization of Process Variables by D. Ranjan; P. Srivastava; M. Talat; S. H. Hasan (pp. 524-539).

The potential use of biomass of Aeromonas hydrophila for biosorption of chromium from aqueous solution was investigated. The variables (pH, initial Cr(VI) concentration, biomass dose, and temperature) affecting process were optimized by performing minimum number of experimental runs with the help of central composite design. The results predicted by design were found to be in good agreement (R ² = 99.1%) with those obtained by performing experiments. Multiple regression analysis shows that uptake decreases with increase in pH and biomass dose, whereas it increases with increase in temperature and concentration. The maximum removal of Cr(VI) predicted by contour and optimization plots was 184.943 mg/g at pH 1.5, initial Cr(VI) concentration 311.97 mg/L, temperature 60 °C, and biomass dose 1.0 g. The removal of Cr(VI) was governed by adsorption of Cr(VI) as well as its reduction into Cr(III), which further gets adsorbed. The sorption capacity of biomass was calculated from experimental data using Langmuir sorption model and was found to be 151.50 mg/g at 40 °C and pH 1.5, which is comparable to other biosorbents. In addition to this, Dubinin–Radushkevich model was applied, and it was found that nature of sorption was chemisorption.

Keywords: Aeromonas hydrophila ; Hexavalent chromium; Biosorption; Optimization; Response surface methodology; Isotherm

Covalent Immobilization of α-Galactosidase from Penicillium griseoroseum and its Application in Oligosaccharides Hydrolysis by Daniel Luciano Falkoski; Valéria Monteze Guimarães; Marisa Vieira de Queiroz; Elza Fernandes de Araújo; Maíra Nicolau de Almeida; Everaldo Gonçalves de Barros; Sebastião Tavares de Rezende (pp. 540-551).

Partially purified α-Galactosidase from Penicillium griseoroseum was immobilized onto modified silica using glutaraldehyde linkages. The effective activity of immobilized enzyme was 33%. Free and immobilized α-galactosidase showed optimal activity at 45 °C and pH values of 5 and 4, respectively. Immobilized α-galactosidase was more stable at higher temperatures and pH values. Immobilized α-galactosidase from P. griseoroseum maintained 100% activity after 24 h of incubation at 40 °C, while free enzyme showed only 32% activity under the same incubation conditions. Defatted soybean flour was treated with free and immobilized α-galactosidase in batch reactors. After 8 h of incubation, stachyose was completely hydrolyzed in both treatments. After 8 h of incubation, 39% and 70% of raffinose was hydrolyzed with free and immobilized α-galactosidase respectively. Immobilized α-galactosidase was reutilized eight times without any decrease in its activity.

Keywords: Penicillium griseoroseum ; α-Galactosidase; Enzyme immobilization; Modified silica; Raffinose oligosaccharides; Soybean products

Water Hyacinth as Carbon Source for the Production of Cellulase by Trichoderma reesei by Pradnya Deshpande; Sajitha Nair; Shubhangi Khedkar (pp. 552-560).

Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation, and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend.

Keywords: Water hyacinth; Trichoderma reesei ; Cellulase; Saccharification

Ethanol Production from Starch Hydrolyzates using Zymomonas mobilis and Glucoamylase Entrapped in Polyvinylalcohol Hydrogel by Martin Rebroš; Michal Rosenberg; Zuzana Grosová; L’udmila Krištofíková; Miroslav Paluch; Matej Šipöcz (pp. 561-570).

The glucoamylase from Aspergillus niger, immobilized into poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats®, was used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis in free form. This system was stable in both the repeated batch and continuous mode of SSF. The microorganism was found to adsorb on the capsules with immobilized enzyme. This increased the ethanol productivity of the repeated batch system with 5% w/v of immobilized glucoamylase almost 2.1 times (7.2 g l⁻¹ h⁻¹) compared to free enzyme–free microorganism system (3.5 g l⁻¹ h⁻¹). The continuous SSF with the immobilized glucoamylase (11.5% w/v) tested for 15 days had productivity 10 g l⁻¹ h⁻¹, which is comparable to continuous experiments on semi-defined glucose medium (10 g l⁻¹ h⁻¹). These two systems were stable in both glucoamylase activity and microorganism productivity.

Keywords: Ethanol; Glucoamylase; Immobilization; LentiKats; Poly(vinylalcohol); Zymomonas mobilis

Transfer of Ephedra Genomic DNA to Yeasts by Ion Implantation by Jie Lü; Xiang Jin; Pei-Hong Mao; Xiang-Dong Ma; Hai-Qiu Ling; Yong-Hong Fan; Long Yu; Bao-Shan Wu; Ping-Kai Ouyang (pp. 571-581).

The genomic DNA from Ephedra glauca was randomly transferred to Saccharomyces cerevisiae and Hansenula anomala by argon and nitrogen ion implantation. Through repeated subculturing and using reversed phase high-performance liquid chromatography analysis to quantify the concentrations of the secondary metabolites, l-ephedrine and d-pseudoephedrine, 12 recombinant strains of genetically stable yeast were obtained, each using glucose as a carbon source, NaNO₃ as a nitrogen source and producing l-ephedrine and/or d-pseudoephedrine. After culturing in liquid medium for 72 h, extracellular l-ephedrine and d-pseudoephedrine concentrations of 18.85 and 4.11 mg/L, respectively, were detected. Using l-ephedrine and d-pseudoephedrine as the target products, the transformation efficiencies of the genomic DNA from E. glauca transferred to S. cerevisiae and H. anomala were 1.15% (1/87) and 2.13% (8/376), respectively. The addition of the amino acid, L-Phe, to culture media substantially changed the amount of l-ephedrine and/or d-pseudoephedrine produced by the recombined yeasts. However, the change in metabolite production was not consistent among strains, rising in some, while dropping to nondetectable levels in others. After random amplification of polymorphic DNA (RAPD) analysis, four RAPD primers were obtained from the initial 100 RAPD primers, each amplifying different fragments with the recombined yeast Ar_Han0458 genome. Using one primer as polymerase chain reaction primer, the result showed that the recombined yeast Ar_Han0458 genome matched E. glauca genomic DNA at 150 bp, indicating a successful transfer of genetic information, facilitated by ion implantation.

Keywords: Ion implantation; Ephedra genomic DNA; Transformation; Recombined yeast; l-Ephedrine; d-Pseudoephedrine

Development of Magnetic Particle-based Chemiluminescence Enzyme Immunoassay for the Detection of 17β-estradiol in Environmental Water by Tian-Bing Xin; Xu Wang; Hui Jin; Shu-Xuan Liang; Jin-Ming Lin; Zhen-Jia Li (pp. 582-594).

In the present work, a simple, fast, and highly sensitive chemiluminescence enzyme immunoassay for 17β-estradiol (E2) in environmental water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix and the separation tools. The specific anti-E2 polyclonal antibody (PcAb) was produced against a conjugate of estradiol–bovine serum albumin. The specificity of the anti-E2 antibody was studied. The results showed that the antibody did not cross-react with the structurally related endocrine-disrupting compounds, including estrone, ethinyl E2, estriol, E2-17-glucuronide, E2-3-sulfate-17-glucuronide, androstenedione, and dihydrotestosterone. The water samples were pretreated with solid-phase extraction using C₁₈ cartridges for the removal of matrix effects. Several physicochemical parameters including the dilution ratios of E2-6–horseradish peroxidase conjugate and anti-E2 PcAb, immunoreaction time, volume of chemiluminescent substrate and MPs, chemiluminescence reaction time, and pH of assay solution were studied and optimized. At optimal experimental conditions, it was found that the proposed method exhibited high performance with detection limit of 2.0 pg/mL, linear range of 20–1,200 pg/mL, and total assay time of 45 min. Both inter- and intra-assay coefficient of variation were less than 10%. The average recoveries of three different spiked concentration samples ranged from 86.3% to 108%. The method was successfully applied to the determination of E2 in river, waste, and tap water, and showed a good correlation with the commercially available radioimmunoassay kit.

Keywords: Chemiluminescence enzyme immunoassay (CLEIA); 17β-Estradiol (E2); Magnetic particles (MPs); Water samples

Optimization of Fermentation Conditions for the Biosynthesis of l-Threonine by Escherichia coli by Ning Chen; Jin Huang; Zhi-bin Feng; Lei Yu; Qing-yang Xu; Ting-yi Wen (pp. 595-604).

In this study, the fed-batch fermentation technique was applied to improve the yield of l-threonine produced by Escherichia coli TRFC. Various fermentation substrates and conditions were investigated to identify the optimal carbon source, its concentration and C/N ratio in the production of l-threonine. Sucrose was found to be the optimal initial carbon source and its optimal concentration was determined to be 70 g/L based on the results of fermentations conducted in a 5-L jar fermentor using a series of fed-batch cultures of E. coli TRFC. The effects of glucose concentration and three different feeding methods on the production of l-threonine were also investigated in this work. Our results showed that the production of l-threonine by E. coli was enhanced when glucose concentration varied between 5 and 20 g/L with DO-control pulse fed-batch method. Furthermore, the C/N ratio was a more predominant factor than nitrogen concentration for l-threonine overproduction and the optimal ratio of ammonium sulfate to sucrose (g/g) was 30. Under the optimal conditions, a final l-threonine concentration of 118 g/L was achieved after 38 h with the productivity of 3.1 g/L/h (46% conversion ratio from glucose to threonine).

Keywords: Escherichia coli ; l-Threonine; Fed-batch fermentation; Condition optimization; Sucrose; C/N ratio

Molecular Cloning and Expression Analysis of an Mn-SOD Gene from Nelumbo nucifera by Chen Dong; Guolin Li; Zhuoqi Li; Honglin Zhu; Mingquan Zhou; Zhongli Hu (pp. 605-614).

A rapid amplification cDNA end (RACE) assay was established to achieve the complete sequence of mitochondrial manganese-superoxide dismutase (Mn-SOD) cDNA in Nelumbo nucifera. The obtained full-length cDNA of Mn-SOD was 926 bp and contained a 699-bp open reading frame encoding an Mn-SOD precursor of 233 amino acids. The recombinant of Mn-SOD expressed by PET-32a vector in Escherichia coli BL21 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting assays. A 3D structural model of the Mn-SOD was constructed by homology modeling. Real-time polymerase chain reaction analysis revealed that Mn-SOD mRNA was expressed in young leaves, blossom, stems, and terminal buds during reproductive stage but with the highest expression in young leaves. This significant difference demonstrated the differential expression of Mn-SOD in various organs of N. nucifera.

Keywords: Mn-SOD; RACE; Prokaryotic expression; Homology model; Real-time PCR

Transgenic Tobacco Expressing Zephyranthes grandiflora Agglutinin Confers Enhanced Resistance to Aphids by Song-hua Ye; Sheng Chen; Fan Zhang; Wei Wang; Qin Tian; Jin-zhi Liu; Fang Chen; Jin-ku Bao (pp. 615-630).

Plant lectins have been reported as transgenic resistance factors against a variety of insect pests. Herein, homologous analysis demonstrated that Zephyranthes grandiflora agglutinin (ZGA) exhibited high similarity with other monocot mannose-binding lectins (MBLs). Phylogenetic analysis revealed that it had taxonomical relationships with insecticidal MBLs. Subsequently, a plasmid expression vector pBI121 containing zga gene (pBIZGA) was constructed using the zga sequence, under the control of CaMV35S promoter and nos terminator. pBIZGA was then integrated into the genome of Nicotiana tabacum L. Polymerase chain reaction and Southern blot analysis demonstrated that this zga gene was integrated into the plant genome. Western blotting and agglutinating activity analysis also showed that transgenic tobacco plants expressed different levels of ZGA. Carbohydrate inhibition analysis indicated that recombinant ZGA and the native shared the same carbohydrate-binding specificity. Moreover, genetic analysis confirmed Mendelian segregation (3:1) of the transgenic in T1 progenies. In planta bioassays on T0 plants and their progenies indicated that expressed ZGA had an effect on reducing the survivability and fecundity of tobacco aphids (Myzus nicotianae). These findings demonstrate that the novel zga gene of ZGA can be expressed in crop plants susceptible to various sap-sucking insects.

Keywords: Zephyranthes grandiflora agglutinin; Tobacco aphid (Myzus nicotianae); Bioassay; Insect resistance; Transgenic tobacco

Characterization of the Interactions of Lysozyme with DNA by Surface Plasmon Resonance and Circular Dichroism Spectroscopy by Kuo-Chih Lin; Ming-Tsai Wey; Lou-Sing Kan; David Shiuan (pp. 631-641).

Association with nucleic acid has been recognized as a unique role of lysozyme and may explain why lysozyme was called a killer protein against HIV infection. In the present study, we characterized the interactions of lysozyme and its derived peptides with a biotin-labeled pUC19 plasmid DNA. Real-time detection of the macromolecular interaction was performed using the SPR (surface plasmon resonance) spectroscopy. The SPR sensorgrams were analyzed and the association and dissociation rate constants as well as the dissociation equilibrium constant K _D were, thus, estimated. The results reveal that other than the electrostatic interactions between the basic protein and the nucleotide sequences carrying negative charges, the specific DNA-binding motifs at the N- and C-termini of lysozyme were also involved in the interactions. The nonapeptide RAWVAWRNR (aa 107–115 of lysozyme) reported previously to block HIV-1 viral entrance and replication was also able to bind DNA with its K _D value comparable to that of histones. The possibilities of ligand-binding-induced conformational changes were investigated using the circular dichroism spectroscopy. The CD spectra (200–320 nm) reveal that the conformational changes indeed occur as the spectra of lysozyme–DNA interactions are much less at the major trough region than the sum of individual spectra. The interaction of lysozyme with DNA molecules may interfere with DNA replication, modulate gene expression, and block bacterial and viral infections. These all suggest that human lysozyme may represent part of the innate immune system with a very broad protective spectrum.

Keywords: Circular dichroism spectroscopy; Dissociation equilibrium constant; DNA; Lysozyme; Molecular interaction; Surface plasmon resonance

Validation and Comparative Studies of Four Sulfonamide Immunoassays Based on the Same Generic Polyclonal Antibody by Hongyan Zhang; Yan Zhang; Shuo Wang; Liguo Zang (pp. 642-652).

Four different immunoassays based on the same generic polyclonal antibody were validated by high performance liquid chromatography, respectively. They also were compared with each other in terms of sensitivity, precision, and accuracy for the quantification or screening of sulfonamide residues in food samples. Correlation studies showed that there was a good correlation between the immunoassays and liquid chromatography data. The conventional plate assay has better precision and the plate-enhanced chemiluminescent assay has higher sensitivity. These two methods all could be used as quantification methods for large numbers of samples and complements of the conventional analytical methods in laboratory. The flow-through strip assay has higher sensitivity and the dip-stick strip assay was less affected by matrix effect. These two methods all could be used as valuable tools for the rapid on-site screening of sulfonamide residues in animal-derived food samples.

Keywords: Sulfonamide; Immunoassay; Generic polyclonal antibody; High performance liquid chromatography; Animal-derived food

Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from Bacillus amyloliquefaciens by Dhanya Gangadharan; K. Madhavan Nampoothiri; Swetha Sivaramakrishnan; Ashok Pandey (pp. 653-662).

Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0–8.0 and temperature between 50 and 60°C. The enzyme did not show any obligate requirement of metal ions but Ca²⁺ and Cu²⁺ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca²⁺ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches. The K _m and V _max of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis.

Keywords: Bacillus amyloliquefaciens ; Alpha amylase; Raw starch digestion; Ion exchange chromatography; Submerged fermentation

Production, Characterization, and Properties of Sophorolipids from the Yeast Candida bombicola using a Low-cost Fermentative Medium by Achlesh Daverey; Kannan Pakshirajan (pp. 663-674).

The yeast Candida bombicola produces biosurfactant with properties akin to those of sophorolipid (SL) group of compounds. In the present work, the yeast was shown to produce 63.7 g l⁻¹ SL when grown on a cheap fermentative medium containing sugarcane molasses, yeast extract, urea, and soybean oil. The partially purified SL was characterized and confirmed by Fourier-transform infrared (FT-IR) spectroscopy, ¹H and ¹³C nuclear magnetic resonance (NMR) and liquid chromatography–mass spectroscopy (LC-MS) analysis. The critical micelle concentration (CMC) and minimum surface tension of the produced SL in aqueous solution were found to be 59.43 mg l⁻¹ and 34.15 m Nm⁻¹, respectively. The emulsification activity and stability with kerosene oil and organic solvents viz. xylene, benzene, and hexadecane were also tested using the produced SL, which yielded better results compared to those reported in the literature.

Keywords: Sophorolipids; Biosurfactant; Fermentation; Candida bombicola ; Sugarcane molasses

A Novel Endophytic Taxol-Producing Fungus Chaetomella raphigera Isolated From a Medicinal Plant, Terminalia arjuna by V. Gangadevi; J. Muthumary (pp. 675-684).

Taxol is the most important member of the clinically useful natural anticancer drug. An endophytic fungus Chaetomella raphigera (strain TAC-15) was isolated from a medicinal plant Terminalia arjuna and screened for its potential in Taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores. This fungus was grown in MID liquid medium and analyzed by chromatographically and spectrometrically for the presence of Taxol. The amount of Taxol produced by this endophytic fungus was quantified by HPLC which showed that it produced 79.6 μg/L, and further confirmative analyses were done by using UV, IR, FAB mass spectroscopy, and NMR spectroscopy. Thus, the fungus can serve as a potential material for fungus engineering to improve the production of Taxol.

Keywords: Endophytic fungus; Chaetomella raphigera ; Medicinal plant; Taxol; Anticancer drug.

Treatment of Wool with Laccase and Dyeing with Madder by Majid Montazer; Fatemeh Dadashian; Nahid Hemmatinejad; Kajal Farhoudi (pp. 685-693).

This research has explored the effect of laccase (Denilite ІІ S) on the physical properties of the wool fabric and confirms the anti-felting of wool. In the experiment, laccase was applied to a wool fabric and different characteristics including weight loss, strength, alkali solubility, felting shrinkage, water drop absorption, and dye ability with madder were studied. The surface morphology of the wool fabrics was also observed by scanning electron microscope. The results indicated that the wool fabric treated with laccase has a higher water drop absorption, lower felting shrinkage, and lower values of a ^* and b ^*. Treatment of a wool fabric with 10% or lower percentage of laccase reduced the fabric weight but increased the tensile strength. However, using higher concentration of laccase reduced fabric weight and tensile strength. The dyeing of laccase pre-treated wool fabric with madder indicated a lower lightness.

Keywords: Wool; Laccase; Tensile strength; Alkali solubility; Absorption

Analysis of Rhamnolipid Biosurfactants Produced Through Submerged Fermentation Using Orange Fruit Peelings as Sole Carbon Source by Seba George; K. Jayachandran (pp. 694-705).

The fermentative production of rhamnolipid biosurfactant from Pseudomonas aeruginosa MTCC 2297 was carried out by submerged fermentation using various cost-effective waste materials such as orange peelings, carrot peel waste, lime peelings, coconut oil cake, and banana waste. The orange peel was found to be the best substrate generating 9.18 g/l of rhamnolipid biosurfactant with a surface tension reduction up to 31.3 mN/m. The production was growth independent, and optimum conditions were standardized. The emulsifying activity was highest against kerosene (73.3%). Rhamnolipid components were purified and separated by ethyl acetate extraction, preparative silica gel column chromatography, high-performance liquid chromatography and thin-layer chromatography. The major rhamnolipid components were characterized, by fast atom bombardment mass spectrometry, as a mixture of dirhamnolipids and monorhamnolipids.

Keywords: Biosurfactant; Fermentation; Orange peel; Pseudomonas aeruginosa ; Rhamnolipid

Lipase-catalyzed Reactions at Interfaces of Two-phase Systems and Microemulsions by P. Reis; R. Miller; M. Leser; H. Watzke (pp. 706-721).

This work describes the influence of two polar lipids, Sn-1/3 and Sn-2 monopalmitin, on the activity of lipase in biphasic systems and in microemulsions. In previous communications, we have shown that Sn-2 monoglycerides can replace Sn-1,3 regiospecific lipases at the oil–water interface, causing a drastically reduced rate of lipolysis. We here demonstrate that even if the lipase is expelled from the interface, it can catalyze esterification of the Sn-2 monoglyceride with fatty acids in both macroscopic oil–water systems and in microemulsions, leading to formation of di- and triglycerides.

Keywords: Lipase; Esterification; Synthesis; Interface

Lipase-catalyzed Reactions at Interfaces of Two-phase Systems and Microemulsions by P. Reis; R. Miller; M. Leser; H. Watzke (pp. 706-721).

Keywords: Lipase; Esterification; Synthesis; Interface

Application of Artificial Neural Network for Yield Prediction of Lipase-Catalyzed Synthesis of Dioctyl Adipate by Mohd Basyaruddin Abdul Rahman; Naz Chaibakhsh; Mahiran Basri; Abu Bakar Salleh; Raja Noor Zaliha Raja Abdul Rahman (pp. 722-735).

In this study, an artificial neural network (ANN) trained by backpropagation algorithm, Levenberg–Marquadart, was applied to predict the yield of enzymatic synthesis of dioctyl adipate. Immobilized Candida antarctica lipase B was used as a biocatalyst for the reaction. Temperature, time, amount of enzyme, and substrate molar ratio were the four input variables. After evaluating various ANN configurations, the best network was composed of seven hidden nodes using a hyperbolic tangent sigmoid transfer function. The correlation coefficient (R ²) and mean absolute error (MAE) values between the actual and predicted responses were determined as 0.9998 and 0.0966 for training set and 0.9241 and 1.9439 for validating dataset. A simulation test with a testing dataset showed that the MAE was low and R ² was close to 1. These results imply the good generalization of the developed model and its capability to predict the reaction yield. Comparison of the performance of radial basis network with the developed models showed that radial basis function was more accurate but its performance was poor when tested with unseen data. In further part of the study, the feedforward backpropagation model was used for prediction of the ester yield within the given range of the main parameters.

Keywords: Esterification; Lipase; Adipate ester; Neural network; Modeling; Prediction

Evaluation of Native and Chemically Modified Sargassum glaucescens for Continuous Biosorption of Co(II) by Mina Ebrahimi; Reza Panahi; Reza Dabbagh (pp. 736-746).

In the present study, biosorption of stable cobalt was studied in an up-flow fixed-bed column using the brown alga Sargassum glaucescens treated with formaldehyde (FA) or MgCl₂. Notable increase in cobalt removal was observed for FA-treated biosorbent with 2.7 and 1.4 times higher dynamic capacity (DC) and uptake capacity (UC) than native alga, respectively. Consequently, FA-treated S. glaucescens was selected for further investigations. In particle size experiments, the DCs of 0.5–1 and 1–2 mm particles were both equal to 27.6 mg/g, and corresponding UCs were 34 and 38 mg/g, respectively. The maximum DC was obtained at residence time of 2.5 min. Studying the effect of additional ions indicated partial effect of Na⁺ and K⁺ ions on DC and UC, Mg²⁺ reduced highly the DC and slightly the UC while heavy metal ions (Ni²⁺, Cd²⁺, Cu²⁺, Zn²⁺, Pb²⁺ and Cr³⁺) caused decrease in both DC and UC about 1.5–4.7 and 1.8–3.2 times, respectively. Moreover, the column regeneration studies were carried out for four sorption–desorption cycles. The DC and the UC highly decreased in the second cycle, partially decreased or remained constant in the third and in the fourth one.

Keywords: Biosorption; Chemical modification; Waste-water treatment; Fixed-bed column; Sargassum glaucescens ; Cobalt

A Novel Reversible pH-Triggered Release Immobilized Enzyme System by Lili Gai; Daocheng Wu (pp. 747-760).

A novel immobilized enzyme system supported by poly(acrylic acid/N,N’-methylene-bisacryl-amide) hydrogel microspheres was prepared. This system exhibited characteristics of reversible pH-triggered release. The morphology, size, and chemical structure were examined through optical microscopy, particle size analyzer, and Fourier transform infrared spectrometer. Immobilization and release features were further investigated under different conditions, including pH, time, and microsphere quantity. Results showed the microspheres were regularly spherical with 3.8 ~ 6.6 μm diameter. Loading efficiencies of bovine serum albumin immobilized by gel entrapment and adsorption methods were 93.9% and 56.2%, respectively. The pH-triggered protein release of the system occurred when medium pH was above 6.0, while it was hardly detected when medium pH was below 6.0. Release efficiencies of entrapped and adsorbed protein were 6.38% and 95.0%, respectively. Hence, adsorption method was used to immobilize trypsin. Loading efficiency of 77.2% was achieved at pH 4.0 in 1 h. Release efficiency of 91.6% was obtained under optimum pH catalysis condition set at 8.0 and trypsin was free in solutions with retention activity of 63.3%. And 51.5% of released trypsin could be reloaded in 10 min. The results indicate this kind of immobilized enzyme system offers a promising alternative for enzyme recovery in biotechnology.

Keywords: pH-dependent hydrogel; Reversible pH-triggered release; BSA; p(AAc/MBA) microspheres; Immobilized trypsin

Production of Milk-Clotting Protease from Bacillus subtilis by Kakoli Dutt; Pritesh Gupta; Saurabh Saran; Swati Misra; Rajendra Kumar Saxena (pp. 761-772).

An indigenous Bacillus subtilis strain isolated from soil was found to be a potent milk-clotting protease (mcp) producer. Production optimized using response surface methodology (RSM) yielded 1,190 U/ml of enzyme in medium containing 6% fructose, 1% casein, 0.3% NH₄NO₃, 10 mM CaCl₂, pH 6.0 and inoculated with 3% inoculum and incubated at 250 rpm for 72 h. Solid-state fermentation resulted in 1,080 and 952.3 U/gds of milk-clotting protease using soybean meal and rice bran, respectively, with higher proteolytic values of 18.97 and 9.1 IU/gds. Production in a biphasic system using an overlay of RSM-optimized medium on solid layer of 6% fructose and 1% casein with 1.5% agar resulted in significant enzyme production. Maximum mcp was obtained using a biphasic system where solid: liquid ratio of 3.0 resulted in a final yield of 1,276.65 U/ml with a yield index of 1.80 as compared to static liquid culture. However, significant increase or difference was noted as compared to yield obtained after RSM. This is the first report on the use of RSM for production of mcp from a bacterial species.

Keywords: Milk clotting protease; Response surface methodology; Solid-state fermentation; Biphasic system

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #3)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #3)