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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.143, #3)

Linoleic Acid Isomerase from Propionibacterium acnes: Purification, Characterization, Molecular Cloning, and Heterologous Expression by Ming-De Deng; Alan D. Grund; Kenneth J. Schneider; Kim M. Langley; Sarah L. Wassink; Susan S. Peng; Reinhardt A. Rosson (pp. 199-211).

Propionibacterium acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR), a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased 376-fold by medium and process optimization in bench-scale fermentors.

Keywords: Biotransformation; Chromatography; Cloning; Conjugated linoleic acid; Expression; Fermentation; Linoleic acid isomerase

Developments in Directed Evolution for Improving Enzyme Functions by S. Sen; V. Venkata Dasu; B. Mandal (pp. 212-223).

The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature’s recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages.

Keywords: Directed evolution; DNA shuffling; In vitro recombination; Mutagenesis; Random priming

Optimization of Fermentation Parameters to Enhance the Production of Ethanol from Palmyra Jaggery Using Saccharomyces cerevisiae in a Batch Fermentor by Veera Venkata Ratnam Bandaru; Sita Ramalakshmi Bandaru; Subba Rao Somalanka; Damodara Rao Mendu; Sarat Babu Imandi; Srinivasa Rao Bejawada; Narasimha Rao Medicherla; Thangadurai Devarajan; Jayaraju Karothi; Ayyanna Chityala (pp. 224-235).

Application of statistical experimental designs for optimization of fermentation parameters to enhance ethanol production, which is an economical and renewable energy source using Saccharomyces cerevisiae NCIM 3090 from palmyra jaggery, was studied in a batch fermentor. Using Plackett–Burman design, impeller speed, concentrations of CoCl₂ and KH₂PO₄ were identified as significant variables, which highly influenced ethanol production, and these variables were further optimized using a central composite design (CCD). The ethanol production was adequately approximated with a full quadratic equation obtained from three factors and five levels of CCD. Maximum ethanol concentration of 132.56 g/l (16.8% [v/v]) was obtained for an impeller speed of 247.179 (≈250) rev/min, CoCl₂ of 0.263 g/l and KH₂PO₄ of 2.39 g/l. A second-order polynomial regression model was fitted and was found adequate with R ² of 0.8952. This combined statistical approach enables rapid identification and investigation of significant parameters for improving the ethanol production and could be very useful in optimizing processes.

Keywords: Saccharomyces cerevisiae ; Batch fermentor; Palmyra jaggery; Ethanol; Plackett–Burman design; Central composite design

An Organic Soluble Lipase for Water-Free Synthesis of Biodiesel by Xueyan Zhao; Bilal El-Zahab; Ryan Brosnahan; Justin Perry; Ping Wang (pp. 236-243).

Lipase AK was modified with short alkyl chains to form a highly organic soluble enzyme and was used to catalyze the synthesis of biodiesel from soybean oil in organic media. The effects of several key factors including water content, temperature, and solvent were examined for the solubilized enzyme in comparison with several other commercially available lipases. Whereas native lipases showed no activity in the absence of water, the organic soluble lipase demonstrated reaction rates of up to 33 g-product/g-enzyme h. The biocatalyst remains soluble in the biodiesel product, and therefore, there is no need to be removed because it is expected to be burned along with the diesel in combustion engines. This provides a promising one-pot mix-and-use strategy for biodiesel production.

Keywords: Biodiesel; Transesterification; Nonaqueous biocatalysis; Lipases; Enzymes

Comparison between Different Hydrolysis Processes of Vine-Trimming Waste to Obtain Hemicellulosic Sugars for Further Lactic Acid Conversion by A. B. Moldes; G. Bustos; A. Torrado; J. M. Domínguez (pp. 244-256).

Trimming vine shoot samples were treated with water under selected operational conditions (autohydrolysis reaction) to obtain a liquid phase containing hemicellulose-decomposition products. In a further acid-catalyzed step (posthydrolysis reaction), xylooligosaccharides were converted into single sugars for the biotechnological production of lactic acid using Lactobacillus pentosus. A wide range of temperatures, reaction times, and acid concentrations were tested during the autohydrolysis–posthydrolysis process to investigate their influence on hemicellulose solubilization and reaction products. The maximum concentration of hemicellulosic sugars was achieved using autohydrolysis at 210 °C followed by posthydrolysis with 1% H₂SO₄ during 2 h. Data from autohydrolysis–posthydrolysis were compared with the results obtained at the optima conditions assayed for prehydrolysis (3% H₂SO₄ at 130 °C during 15 min) based on previous works. Prehydrolysis extracted more hemicellulosic sugars from trimming vine shoots; however, the protein content in the hydrolysates from autohydrolysis–posthydrolysis was higher. The harsher conditions assayed during the autohydrolysis process and the higher content of protein after this treatment could induce Maillard reactions decreasing consequently the concentration of hemicellulosic sugars in the hydrolysates. Therefore, despite the several advantages of autohydrolysis (less equipment caused by the absence of mineral acid, less generation of neutralized sludges, and low cost of reagents) the poor results obtained in this work with no detoxified hydrolysates (Q _P = 0.36 g/L h, Q _S = 0.79 g/L h, Y _P/S = 0.45 g/g, Y _P/Sth = 61.5 %) or charcoal-treated hydrolysates (Q _P = 0.76 g/L h, Q _S = 1.47 g/L h, Y _P/S = 0.52 g/g, Y _P/Sth = 71.5 %) suggest that prehydrolysis of trimming vine shoots with diluted H₂SO₄ is more attractive than autohydrolysis-posthydrolysis for obtaining lactic acid through fermentation of hemicellulosic sugars with L. pentosus. Besides the higher hemicellulosic sugars concentration achieved when using the prehydrolysis technology, no detoxification steps are required to produce efficiently lactic acid (Q _P = 1.14 g/L h; Q _S = 1.64 g/L h; Y _P/S = 0.70 g/g; Y _P/Sth = 92.6 %), even when vinification lees are used as nutrients (Q _P = 0.89 g/L h; Q _S = 1.54 g/L h; Y _P/S = 0.58 g/g; Y _P/Sth = 76.1 %).

Keywords: Trimming vine shoots; Autohydrolysis; Prehydrolysis; L. pentosus ; Lactic acid

Whey Treatment by AnSBBR with Circulation: Effects of Organic Loading, Shock Loads, and Alkalinity Supplementation by Roberto A. Bezerra Jr.; José A. D. Rodrigues; Suzana M. Ratusznei; Marcelo Zaiat; Eugenio Foresti (pp. 257-275).

The main objective of this work was to investigate the effect of volumetric loading rate (VLR), shock load, and alkalinity supplementation on the efficiency and stability of an Anaerobic Sequencing Batch Biofilm Reactor (AnSBBR) containing polyurethane foam cubes. Mixing in the reactor, which was kept at 30 ± 1°C, occurred by recirculating the liquid phase. The reactor treated 2.5 l cheese whey in 8-h cycles, at concentrations of 1, 2, and 4 g COD l⁻¹, which corresponded to VLRs of 3, 6, and 12 g COD l⁻¹ day⁻¹, respectively. Application of single-cycle shock loads of 6, 12, and 24 g COD l⁻¹ day⁻¹ did not impair reactor performance. In addition, for VLRs of 3, 6, and 12 g COD l⁻¹ day⁻¹, alkalinity supplementation to the influent, at the end of each assay, could be reduced to 75, 50, and 50%, respectively, in relation to supplementation at the beginning of the assay. During reactor operation a viscous polymer-like material was formed between the polyurethane foam cubes, which increased at higher VLR. Finally, addition of salts to the influent improved reactor efficiency.

Keywords: AnSBBR; Volumetric loading rate; Shock load; Cheese whey; Alkalinity supplementation

Improvement and Modelling of Hexenal Transfer in Liquid-Gas Reactor by Najla Ben Akacha; Om Elkhir Guizani; Mohamed Gargouri (pp. 276-283).

The aim of the present work is to improve the extraction of an aromatic compound, 2E-hexenal, from a continuous liquid–gas reactor. Having an improved process to recover hexenal could be of interest to obtain this chemical if produced by any biotechnological process. The experimental program proposed on the basis of a full 2³ factorial design demonstrated that the conditions optima for the extraction are 40°C as a reactor temperature and −10°C as a temperature of trap for a nitrogen flow of 45 mL s⁻¹. These conditions allowed to recover more than 75% of hexenal, when this compound was stripping by a nitrogen stream from the liquid phase. The mass transfer was theoretically and experimentally studied. The model predictions were validated against experimental results obtained for a reactor, and good correlation was observed for a simulation.

Keywords: Extraction; Factorial design; Green note; Mass transfer coefficient; Mathematical modeling; Reactor

Comparison of Different Pretreatment Strategies for Enzymatic Hydrolysis of Wheat and Barley Straw by Lisa Rosgaard; Sven Pedersen; Anne S. Meyer (pp. 284-296).

In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L® from Trichoderma reesei, and a β-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, ∼48% (g g⁻¹), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of ∼39% (g g⁻¹). Addition of extra enzyme (Celluclast 1.5 L®+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32–39 g L⁻¹, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.

Keywords: Lignocellulose; Enzymatic hydrolysis; Glucose yield; Pretreatment

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.143, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.143, #3)