Applied Biochemistry and Biotechnology (v.150, #1)

The aims of this study were to understand the genotypic variability in cell-wall composition and cell-wall accessibility to enzymes in select switchgrass plants obtained from two different populations derived from a base population of octaploid cultivars. Population C+3 was developed by three breeding generations for high digestibility and population C−1 developed by one generation of breeding for low digestibility. Above-ground biomass from 12 selected genotypes, three each with high or low digestibility within each population, was analyzed for their cell-wall aromatics and polysaccharides. The ratio of p-coumaric acid/ferulic acid was greater (P ≤ 0.05) for the high-lignin C−1 population over the low-lignin C+3 population, although the amounts of these two phenolics did not differ between populations. Combined values of guaiacyl + syringyl-lignin were consistently higher in genotypes from the C−1 population as compared to the genotypes from the C+3 population. Overall, p-coumaric acid was released by enzymes in greater amounts than ferulic acid in all these genotypes. Genotypes in the C−1 population exhibited lower dry weight loss as compared to the genotypes in the C+3 population after enzymatic digestion, suggesting changes in cell-wall architecture. Overall, our data highlight the phenotypic plasticity coded by the switchgrass genome and suggest that combining dry matter digestibility with other more specific cell-wall traits could result in genotypes with greater utility as bioenergy feedstocks.
Keywords: Cell wall; Chemical analyses; Digestibility; Genotypes; Phenolic acids; Lignin; Sugars; Switchgrass; Enzymes

The insulin-like growth factor (IGF-I) gene (GenBank accession no. AY247412) of Qiantang River triangular bream (Megalobrama terminalis) was cloned for the first time from the liver by reverse transcriptase polymerase chain reaction. The gene was inserted into pMD 18-T vector to construct the recombinant plasmid pMD 18-T/IGF-I. Sequence analysis indicated that the IGF-I cDNA consisted of 486 nucleotides encoding 161 amino acids, which spanned the complete signal peptide, mature peptide (including B, C, A, and D domains), and E-domain. Analysis of the E domain indicated that triangular bream IGF-I gene belonged to the IGF-I Ea-2 subtype. To construct the expression plasmid, the IGF-I gene was subcloned into prokaryotic expressing vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/IGF-I was transformed into Escherichia coli BL21 (DE3), and the transgene expression was observed after being induced with isopropylthiogalactoside. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting indicated that the recombinant fusion protein had immune activity, and the molecular weight was about 47 kDa. The results of SDS-PAGE and thin-layer scanning showed that the yield of fusion protein had been enlarged with prolonging time. When the time of induced expression was 1, 2, 3, 4, 5, and 6 h, the expression amount was approximately 1.4, 4.3, 8.1, 11.3, 16.3, and 18.8% of total bacterial protein, respectively.
Keywords: Qiantang River triangular bream (Megalobrama terminalis); Insulin-like growth factor-I (IGF-I); Cloning; Prokaryotic expression; Antigenicity

Sequential Production of Amylolytic and Lipolytic Enzymes by Bacterium Strain Isolated from Petroleum Contaminated Soil by Nayara Bezerra Carvalho; Ranyere Lucena de Souza; Heizir F. de Castro; Gisella M. Zanin; Álvaro Silva Lima; Cleide M. F. Soares (25-32).
Amylases and lipases are highly demanded industrial enzymes in various sectors such as food, pharmaceuticals, textiles, and detergents. Amylases are of ubiquitous occurrence and hold the maximum market share of enzyme sales. Lipases are the most versatile biocatalyst and bring about a range of bioconversion reactions such as hydrolysis, inter-esterification, esterification, alcoholysis, acidolysis, and aminolysis. The objective of this work was to study the feasibility for amylolitic and lipolytic production using a bacterium strain isolated from petroleum contaminated soil in the same submerged fermentation. This was a sequential process based on starch and vegetable oils feedstocks. Run were performed in batchwise using 2% starch supplemented with suitable nutrients and different vegetable oils as a lipase inducers. Fermentation conditions were pH 5.0; 30°C, and stirred speed (200 rpm). Maxima activities for amyloglucosidase and lipase were, respectively, 0.18 and 1,150 U/ml. These results showed a promising methodology to obtain both enzymes using industrial waste resources containing vegetable oils.
Keywords: Amylolytic enzymes; Lipolytic enzymes; Submerged fermentation; Petroleum

Processing Parameters Matching Effects upon Rhizobium tropici Biopolymers’ Rheological Properties by Flávia Duta Pimenta; Léa Maria de Almeida Lopes; Francisca Pessôa de França (33-49).
The combined effects of the processing parameters upon rheological properties of biopolymers produced by Rhizobium tropici were studied as a function of the Ca+2 ions’ concentration variation, yeast extract concentration added to the medium, aeration, and agitation, maintaining the mannitol concentration in 10 g/L. The experiments were carried out using a fermenter with 20-L capacity as a reactor. All processing parameters were monitored online. The temperature [(30 ± 1) °C] and pH values (7.0) were kept constant throughout the experimental time. As a statistical tool, a complete 23 factorial design with central point and response surface was used to investigate the interactions between relevant variables of the fermentation process: calcium carbonate concentration, yeast extract concentration, aeration, and agitation. The processing parameter setup for reaching the maximum response for rheological propriety production was obtained when applying mannitol concentration of 10.0 g/L, calcium carbonate concentration 1.0 g/L, yeast extract concentration 1.0 g/L, aeration 1.30 vvm, and agitation 800 rpm. The viscosimetric investigation of polysaccharide solutions exposed their shear-thinning behavior and polyelectrolytic feature.
Keywords: Rhizobium tropici ; Biopolymer; Exopolysaccharides; Experimental design; Rheological properties; Shear-thinning behavior

Comparison of Yarrowia lipolytica Lipase Immobilization Yield of Entrapment, Adsorption, and Covalent Bond Techniques by Wazé Aimée Mireille Alloue; Jacqueline Destain; Thami El Medjoub; Hakim Ghalfi; Philomène Kabran; Philippe Thonart (51-63).
The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic properties. Sodium alginate and chitosan were the polymers selected to immobilize lipase by inclusion. The beads of each polymer were dried by freeze drying and fluidization. The results show that chitosan was more adapted to the inclusion of lipase. Even though freeze dried, bead activity was low compared to that of fluidized beads. The freeze-drying process seems to produce suitable beads for storage at 4 and 20 °C. The immobilization by adsorption was carried out on both celite and silica gel. Maximum immobilization yield of 76% was obtained with celite followed by 43% in silica gel. The enzyme adsorbed on the two supports exhibited greater stability at a certain temperature (50 °C) and in no polar solvents (Isooctane, n-heptane, and n-hexane). In addition, the lipase immobilized by covalent bond retained residual activity equitable to 70%. It was demonstrated that the enzyme immobilized by covalent bond showed greater activity (80%) after 5 months of storage.
Keywords: Yarrowia lipolytica lipase; Enzymatic activity; Immobilization; Reusability

This study uses an overall evaluation criterion for improving the immobilized bead reusability and extracellular enzyme production by immobilized cells by assigning relative weightage to bead reusability, enzyme production, and cell leakage. Initially, alkaline protease production by alginate-immobilized Bacillus circulans (MTCC 6811) was analyzed using L18 orthogonal array (OA). The resultant optimized parameters were further fine-tuned with L9 OA experimentation. At L18-OA analysis, inoculum level and CaCl2 had least influence at individual level. At the interactive level, incubation time revealed maximum and minimum interaction with sodium alginate and glucose concentration, respectively. L9 experimentation indicated that glucose concentration contributed the major influence on protease production followed by matrix material and incubation time at the individual level, and at the interactive level, matrix concentration played a vital role by interacting with incubation time, inoculum, and CaCl2 concentration. All selected input parameters showed significance either at individual level or interactive in both OAs. Scanning electron microscopy analysis showed bacterial morphology variation with variation of matrix concentration. Overall, glucose concentration depicted a major influence at the individual level for the enzyme production. Significant improvement, approximately 147%, in enzyme yield was observed. Economic enzyme production by immobilized B. circulans is regulated by interactive influence of fermentation parameters, which influence the immobilized bead stability, reusability, and enzyme yield.
Keywords: Overall evaluation criteria (OEC); Taguchi methodology; Optimization; Protease production; Immobilization; Sodium alginate

Bioactive Benzopyrone Derivatives from New Recombinant Fusant of Marine Streptomyces by Mervat M. A. El-Gendy; M. Shaaban; A. M. EL-Bondkly; K. A. Shaaban (85-96).
In our searching program for bioactive secondary metabolites from marine Streptomycetes, three microbial benzopyrone derivatives (13), 7-methylcoumarin (1) and two flavonoides, rhamnazin (2) and cirsimaritin (3), were obtained during the working up of the ethyl acetate fraction of a marine Streptomyces fusant obtained from protoplast fusion between Streptomyces strains Merv 1996 and Merv 7409. The structures of the three compounds (13) were established by nuclear magnetic resonance, mass, UV spectra, and by comparison with literature data. Marine Streptomyces strains were identified based on their phenotypic and chemotypic characteristics as two different bioactive strains of the genus Streptomyces. We described here the fermentation, isolation, as well as the biological activity of these bioactive compounds. The isolated compounds (1–3) are reported here as microbial products for the first time.
Keywords: Marine Streptomyces ; New recombinant fusant; Benzopyrones; Antibacterial and antifungal agents

Purification of a PHA-Like Chitin-binding Protein from Acacia farnesiana Seeds: A Time-dependent Oligomerization Protein by T. Santi-Gadelha; B. A. M. Rocha; C. C. Oliveira; K. S. Aragão; E. S. Marinho; C. A. A. Gadelha; M. H. Toyama; V. P. T. Pinto; C. S. Nagano; P. Delatorre; J. L. Martins; F. R. Galvani; A. H. Sampaio; H. Debray; B. S. Cavada (97-111).
A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 ± 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.
Keywords: Acacia farnesiana ; Lectin-like protein; Purification; Oligomerization; Tandem mass spectrometry

Kinetic and Thermodynamic Study of a Chemically Modified Highly Active Xylanase from Scopulariopsis sp. by Ahmed Jawaad Afzal; Saleem Ahmed Bokhari; Khawar Sohail Siddiqui (113-113).

Enzymatic Microreactors for the Determination of Ethanol by an Automatic Sequential Injection Analysis System by Eliana M. Alhadeff; Andrea M. Salgado; Oriol Cos; Nei Pereira Jr.; Belkis Valdman; Francisco Valero (115-116).