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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.79, #1-3)

Bioprocess evaluation and confirmation by Raphel Katzen; Nhuan P. Nghiem (pp. 583-584).

Two-phase methanization of food wastes in pilot scale by Joon Pyo Lee; Jin Suk Lee; Soon Chul Park (pp. 585-593).
A 5 ton/d pilot scale two-phase anaerobic digester was constructed and tested to treat Korean food wastes in Anyang city near Seoul.The easily degradable presorted food waste was efficiently treated in the two-phase anaerobic digestion process. The waste contained in plastic bags was shredded and then screened for the removal of inert materials such as fabrics and plastics, and subsequently put into the two-stage reactors. Heavy and light inerts such as bones, shells, spoons, and plastic pieces were again removed by gravity differences. The residual organic component was effectively hydrolyzed and acidified in the first reactor with 5 d space time at pH of about 6.5. The second, methanization reactor converted the acids into methane with pH between 7.4 and 7.8. The space time for the second reactor was 15 d. The effluent from the second reactor was recycled to the first reactor to provide alkalinities.The process showed stable steady-state operation with the maximum organic loading rate of 7.9 kg volatile solid (VS)/m3/d and the volatile solid reduction efficiency of about 70%. The total of 3.6 tons presorted MSW containing 2.9 tons of food organic was treated to produce about 230 m3 of biogas with 70% (v/v) of methane and 80 kg of humus.This process is extended to full-scale treating 15 tons of food waste a day in Euiwang city and the produced biogas is utilized for the heating/cooling of adjacent buildings.

A process economic approach to develop a dilute-acid cellulose hydrolysis process to produce ethanol from biomass by Nick Nagle; Kelly Ibsen; Edward Jennings (pp. 595-607).
Successful deployment of a bioethanol process depends on the integration of technologies that can be economically commercialized. Pretreatment and fermentation operations of the traditional enzymatic bioethanol-production process constitute the largest portion of the capital and operating costs. Cost reduction in these areas, through improved reactions and reduced capital, will improve the economic feasibility of a large-scale plant.A technoeconomic model was developed using the ASPEN PlusTN modeling software package. This model in cluded a two-stage pretreatment operation with a co-current first stage and countercurrent second stage, a lignin adsorption unit, and a cofermentation unit. Data from kinetic modeling of the pretreatment reactions, verified by bench-scale experiments, were used to create the ASPEN Plus base model. Results from the initial pretreatment and fermentation yields of the two-stage system correlated well to the performance targets established by the model. The ASPEN Plus model determined mass and energy-balance information, which was supplied, to an economic module to determine the required selling price of the ethanol. Several pretreatment process variables such as glucose yield, liquid: solid ratio, additional pretreatment stages, and lignin adsorption were varied to determine which parameters had the greatest effect on the process economics. Optimized values for these key variables became target values for the bench-scale research, either to achieve oridentify as potential obstacles in the future commercialization process. Results from this modeling and experimentation sequence have led to the design of an advanced two-stage engineering-scale reactor for a dilute-acid hydrolysis process.

Keywords: ASPEN Plus; pretreatment; bioethanol production; economic feasibility; rZ. mobilis

Biomass conversion to mixed alcohol fuels using the MixAlco process by Mark T. Holtzapple; Richard R. Davison; M. Kyle Ross; Salvador Aldrett-Lee; Murlidhar Nagwani; Chang-Ming Lee; Champion Lee; Seth Adelson; William Kaar; David Gaskin; Hiroshi Shirage; Nan-Sheng Chang; Vincent S. Chang; Mitchell E. Loescher (pp. 609-631).
The MixAlco process is a patented technology that converts any biodegradable material (e.g., sorted municipal solid waste, sewage sludge, industrial biosludge, manure, agricultural residues, energy crops) into mixed alcohol fuels containing predominantly 2-propanol, but also higher alcohols up to 7-tridecanol. The feed stock is treated with lime to increase its digestibility. then, it is fed to a fermentor in which a mixed culture of acid-forming microorganisms produces carboxylic acids. Calcium carbonate is added to the fermentor to neutralize the acids to their corresponding carboxylate salt. The dilute (−3%) carboxylate salts are concentrated to 19% using an amine solvent that selectively extracts water. Drying is completed using multi-effect evaporators. Finally, the dry salts are thermally converted to ketones which subsequently are hydrogenated to alcohols. All the steps in the MixAlco process have been proven at the laboratory scale. A techno-economic model of the process indicates that with the tipping fees available in New York ($126/dry tonne), mixed alcohol fuels may be sold for $0.04/L ($0.16/gal) with a 60% return on investment (ROI). With the average tipping fee in the United States rates ($63/dry tonne), mixed alcohol fuels may be sold for $0.18/L ($0.69/gal) with a 15% ROI. In the case of sugarcane bagasse, which may be obtained for about $26/dry ton, mixed alcohol fuels may be sold for $0.29/L ($1.09/gal) with a 15% ROI.

Keywords: MixAlco process; municipal solid waste; sewage sludge; alcohol fuels; biomass

Economic analysis of selected lignocellulose-to-ethanol conversion technologies by K. S. So; R. C. Brown (pp. 633-640).
The objective of this case study was to examine the economics of three lignocellulose-to-ethanol conversion technologies: fast pyrolysis integrated with a fermentation step, simultaneous saccharification and fermentation (SSF), and dilute sulfuric acid hydrolysis and fermentation. All technologies were assumed to have an annual production rate of 25 million gallons of ethanol. The three technologies were compared in terms of capital costs, operating costs, and ethanol production costs. Sensitivity analyses were carried out to study the uncertainties of wood costs and ethanol production rates on ethanol production costs. Final economic analysis showed that fast pyrolysis integrated with a fermentation step is comparable with the other two processes and suggests that it should be considered for further development.

Keywords: Biomass-to-ethanol; economic analysis; fast pyrolysis; fermentation; acid hydrolysis

Financing biotechnology projects by J. B. Keller; P. B. Plath (pp. 641-648).
An increasing number of biotechnology projects are being brought to commercialization using conventional structured finance sources, which have traditionally only been available to proven technologies and primary industries. Attracting and securing competitive cost financing from main-stream lenders, however, will require the sponsor of a new technology or process to undergo a greater level of due diligence. The specific areas and intensity of investigation, which are typically required by lenders in order to secure long-term financing for biotechnology-based manufacturing systems, is reviewed. The processes for evaluating the adequacy of prior laboratory testing and pilot plant demonstrations is discussed. Particular emphasis is given to scale-up considerations and the ability of the proposed facility design to accommodate significant modifications, in the event that scale-up problems are encountered.

Keywords: Biotechnology; commercialization; scale-up; risk assessment; project finance; due diligence

Simultaneous saccharification and cofermentation of dilute-acid pretreated yellow poplar hardwood to ethanol using xylose-fermenting Zymomonas mobilis by James D. McMillan; Mildred M. Newman; David W. Templeton; Ali Mohagheghi (pp. 649-665).
Simultaneous saccharification and cofermentation (SSCF) was carried out at approximately 15% total solids using conditioned dilute-acid pretreated yellow poplar feedstock, an adapted variant of National Renewable Energy Laboratory (NREL) xylose-fermenting Zymomonas mobilis and either commercial or NREL-produced cellulase enzyme preparations. In 7 d, at a cellulase loading of 12 filter paper units pergram cellulose (FPU/g), the integrated system produced more than 3% w/v ethanol and achieved 54% conversion of all potentially available biomass sugars (total sugars) entering SSCF. A control SSCF employing Sigmacell cellulose and a commercial cellulase at an enzyme loading of 14 FPU/gachieved 65% conversion of total sugars to ethanol.

Keywords: Hydrolyzate conditioning; Zymomonas mobifis ; enzymatic saccharification; ethanol fermentation; SSCF; integrated testing

Enzymatic processes and enzyme production by Karl Sanford; Mike Himmel (pp. 669-670).

Bioconversion of acrylonitruke to acrylamide using a thermostable nitrile hydratase by Rugmini Padmakumar; Patrick Oriel (pp. 671-679).
Although providing an attractive route for production of crrylamide from acrylonitrile, utilization of nitrile hydratase en zymes has been limited by the requirement for low temperatu rebioconversion conditions. Thisrreportsummarizes a search for thermostable nitrile hydratases from aerobic moderate thermophiles screened for ability to grow on acrylonitrileatt concentrations to 1% at elevated temperatures. A new isolate Bacillus sp. BR449 constitutively expresses a thermostble nitrile hydratase with properties iccluding low substrate inhibition and broad temperature range with optimal activity at 55°C. With prolonged exposure, BR449 nitrile hydratase exhibited temperature-dependent inactivation by acrylonitrile, which is attributed to alkylation of nucleophilic sites on the enzymes/

Keywords: Nitrile hydratase; thermophile; acrylamide

Lignin peroxidase production by Streptomyces viridosporus T7A by José E. Zerbini; Edna M. M. Oliveira; Elba P. S. Bon (pp. 681-688).
The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in submerged batch fermentations using growth media containing 6.5 g/L yeast extract and 2.5–10.0 g/L glucose, corresponding to carbon to nitrogen (C/N) ratios from 7.1–12.4. The kinetics for biomass and enzyme accumulation and glucose consumption were followed allowing definition of optimized conditions for enzyme production. Considering the physiological response of the microorganism in relation to enzyme production, a sharp increase on enzyme activity was consistently observed upon glucose depletion, indicating glucose regulation. In accordance to that the plot of maximal enzyme vs maximal enzyme per gram of glucose consumption showed a linear inversely proportional relationship, indicating that the characteristics of the metabolic pool at the studied C/N ratios affected enzyme biosynthesis even after glucose depletion.

Keywords: Strep to myces viridosporus ; lignin peroxidase; medium C/N ratio; nitrogen nutrition; gluccse regulation

Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli by Ziyu Dai; Brian S. Hooker; Ryan D. Quesenberry; Jianwei Gao (pp. 689-699).
Expression of Trichoderma reesei exo-cellobiohydrolase I (CBHI) gene in transgenic tobacco was under the control of CaMV 35S promoter. In transgenic leaf tissues, CBHI activity up to 66.1 μmol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 μmol h/g total protein. Protein immunoblot analysis confirms the presence of CBHI enzyme in both transgenic calli and leaf tissues. CBHI expression levels accounted for about 0.11% and 0.082% of total protein in transgenic leaf tissues and calli, respectively, Furthermore, expression of CBHI gene did not affect normal growth and development of transgenic plants.

Keywords: Bioreactor; cellulase; exo-cellobiohydrolase I; transgenic tobacco; Trichoderma reesei

Preserving the activity of cellulase in a batch foam fractionation process by Veara Loha; Aleš Prokop; Liping Du; Robert D. Tanner (pp. 701-712).
Foam fractionation isone of the low operating-cost techniques for removing proteins from a dilute solution. The initial bulk solution pH and air superficial velocity play an importantrole in the foam-fractionation process. Denaturation of proteins (enzymes) can occur, however, during the foamfractionation process from the shear forces resulting from bursting air bubbles. At the extreme bulk solution pHs (lower than 3.0 and higher than 10.0), the en zymatic activity of cellulase in the foamate phase drops significantly. Within these two pH boundsan increase in the air superficial velocity, Vo, and a decrease in the bulk solution pH leads to a decrease in the separation ratio (SR), defined as theratio of the protein concentration in the foamate to the protein concentration in the residue. On the other hand, an increase in Vo provides a higher foamate-protein recovery. The process efficiency is defined as the product of foamate-protein recovery times the SR times the cellulase activity. The optimal operating condition of the cellulase foamfractionation process is taken into account at the maximum value of the processefficiency. In this study, that optimal condition is atan air superficial velocity of 32 cm/min and a bulk-solution pH of 10.0. At this condition, the recovered foamate is about 80% of the original protein mass, the SR is about 12, and the en zymatic activity is about 60% of the original cellulase activity.

Keywords: Cellulase; cellulase activity; foam fractionation; batch foam fractionation; protein separation

Bleaching of kraft pulp with commercial xylanases by José H. B. De Araújo; Flávio F. de Moraes; Gisella M. Zanin (pp. 713-722).
The perform ance of commercial xylanases in totally chlorine-free bleaching of kraft pulp from conifer was tested with Pulpzyme HC (Novo Nordisk) and Cartazyme NS-10 (Sandoz/Clariant), at 500 U/kg of dry pulp, respectively. The treatment with Pulpzyme (Xp) or Cartazyme (Xc) has been combined with stages of bleaching using: oxygen (O), sulfuric acid (A), and extraction with hydrogen peroxide (Eop). The following sequences have been tested: OXpAEop, OXcAEop, XpOAEop, XcOAEop and OAEop, Kraft pulp bleached at the Klabin industrial plant using the sequence, CEH (chlorine, alkaline extreaction, and hypochlorination) was, used for comparison. The following average values were obtained: 1. Kappa number: OXpAEop, 4.8; OXcAEop, 4.9; XpOAEop, 5.0; XcOAEop, 4.9; OAEop, 5.6, and CEH, 1.9; 2. Brightness (% ISO values): OXpAEop, 68.4; OXcAEop, 70.1; XpOAEop, 67.9; XcOAEop, 26.9; XpOAEop, 23.4; XcOAEop, 23.1; OAEop, 25.4, and CEH, 25.2. Pulps that were treated with xylanases, before or affer the delignification with oxygen, have shown reduced kappa number and higher brightness than the pulp OAEop, Enzyme treatment before delignification with oxygen reduces pulp viscosity. Brightness obtained for pulp produced with bleaching sequences containing the enzymatic treatment, when compared with the control, CEH, shows that the xylanases enhance the action of the bleaching agents.

Keywords: Chlorine free bleaching; kraft pulp bleaching; oxidative peroxide extraction; xylanase

Laccase from Trametes versicolor by Maria E. A. De Carvalho; M. C. Monteiro; G. L. Sant'Anna Jr. (pp. 723-733).
The enzyme laccase was produced by the white-rot fungus Trametes versicolor in repeated batches cultures with immobilized mycelium. Two different culture conditions were used. Enzymes produced were evaluated regarding their stability a thigh temperatures (55°C and 65°C) and at alkaline conditions (pH 7.0 and pH 8.0) having in view the application of these enzymes in biobleaching of hardwood Kraft pulp.Biobleaching experiments were divided in two parts, enzymatic prebleaching followed by chemical bleaching. In the enzymatic prebleaching the enzyme laccase was used at two conditions of pH and temperature, whereas the reaction time was fixed at 1h in all pretreatments. In the chemical bleaching the DEDED and DEpDED sequences were used.The enzyme action was evaluated by Kappa number, viscosity, and brightness at the end of bleaching sequences. There were obtained values of Kappa numbers lower than control assays, viscosities compatible with industrial pulps, and brightness higher than controls, when pulps were pretreated for 1 h with laccase at pH 8.0 and 55°C.

Keywords: Trametes versicolor ; laccase; biobleaching; kappa number reduction; brightness increase

Lignin peroxidase and protease production by Streptomyces viridosporus T7A in the presence of calcium carbonate by Jacyara M. B. Macedo; Leda M. F. Gottschalk; Elba P. S. Bon (pp. 735-744).
Streptomyces are good producers of enzymes of industrial interest, such as lignin peroxidase (LiP) and proteases. To optimize production of these enzymes by Streptomyces viridosporus T7A, two parameters were evaluated: carbon sources and calcium carbonate. Shake-flask fermentations were performed using culture media, with and without CaCO3, contained yeast extract, mineral salts and either glucose, lactose, galactose, or corn oil. In the absence of calcium carbonate, the maximum values for LiP and protease activities occurred during the idiophase with LiP activity being favored by glucose, corn oil, and galactose, and protease activity being favored only by corn oil. Calcium carbonate affected the cell morphology by reducing the size of the pellets. Moreover, in the presence of the salt, LiP production was growth-associated in all media but the glucose medium. Higher enzyme levels were observed when galactose and glucose were used as carbon sources. Protease activity was repressed by both glucose and galactose, whereas corn oil was the best carbon source for the enzyme production. Calcium carbonate increased LiP production by up to 2.6-fold. Such improvement was not observed for protease production, suggesting a selective effect of CaCO3 on LiP activity.

Keywords: Streptomyces viridosporus ; lignin peroxidase; protease; carbon sources; calcium carbonate

Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica by Cleide M. F. Soares; Heizir F. De Castro; Flávio F. De Moraes; Gisella M. Zanin (pp. 745-757).
Candida rugosa lipase was immobilized by covalent binding on controlled poresilica (CPS) using glutaraldehyde ascross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5–8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10°C higher than that for the free lipase. The thermal stability of the CPS lipase was alsogreater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.

Keywords: Lipase; immobilization; controlled poresilica; cross-linking; characterization; hydrolysis; esterification

Enzymatic synthesis of medium-chain triglycerides in a solvent-free system by Marta A. P. Langone; Geraldo L. Sant'Anna Jr. (pp. 759-770).
The synthesis of tricaprylin, tricaprin, trilaurin, and trimyristin in a solvent-freesystem was conducted by mixing a commercial immobilized lipase (Lipozyme IM 20, Novo Nordisk, Bagsvaerd, Denmark) with the organic reactants (glycerol and fatty acids) in a 20-mL batch reactor with constant stirring. In a first set of experiments, the effect of water concentration (0–6%) on the reaction conversion was shown to be negligible. In a second set of experiments, the effects of temperature (70–90°C), fatty acid/glycerol molar ratio (1–5), and enzyme concentration (1–9%[w/w]) on the reaction conversion were determined by the application of a 3×3 experimental design. The reactions were carried out for 26 h and the nonpolar phase was analyzed by gas chromatography (GC). Appreciable levels of medium-chain triglycerides were achieved, except for tricaprylin. For the triglyceride production, higher selectivity was attained under the following conditions: molar ratio of 5, enzyme concentration of 5 or 9% (w/w) and temperatures of 70°C (Tricaprin), 80°C (trilaurin), and 90°C (trimyristin). Statistical analysis indicated that the fatty acid/glycerol molar ratio was the most significant variable affecting the synthesis of triglycerides.

Keywords: Medium-chain triglycerides; immobilized lipase; esterification

Lignin peroxidase production by Streptomyces viridosporus T7A by Leda M. F. Gottschalk; Jacyara M. B. Macedo; Elba P. S. Bon (pp. 771-778).
Lignin peroxidase (LiP) production cost should be reduced to justify its use in the control of environmental pollution. In this work, we studied the enzyme production by Streptomyces viridosporus T7A using glucose or corn oil as a carbon source having 0.65% yeast extract as a nitrogen source. Enzyme activity, observed using either 0.65% glucose or corn oil at 0.1, 0.5, and 1.0% concentration, was 300, 150, 300, and 200 U/L, respectively. Although higher enzyme activity was obtained in both media containing 0.65% glucose and 0.5% corn oil, the use of corn oil resulted in a better LiP stability. When combined carbon sources were used, higher values of enzyme activity (360, 350, and 225 U/L) were observed in media with 0.65% glucose and supplemented with 0.1, 0.5, and 1.0% corn oil, respectively. Although the presence of both glucose and 0.5% corn oil is favorable for LiP production, satisfactory results in terms of enzyme production and stability could be also observed using 0.5% corn oil as a sole carbon source, which may lead to reduced production costs of the LiP enzyme.

Keywords: Streptomyces viridosporus ; Lignin peroxidase; enzyme production; corn oil; medium optimization

Screening of variables in xylananese recovery using BDBAC reversed micelles by E. M. G. Rodrigues; A. Pessoa Jr.; A. M. F. Milagres (pp. 779-788).
Xylanase recovery by reversed micelles using cationic surfactants (N-benzyl-N-dodecyl-N-bis(2-hydroxyethhyl)ammonium chloride) BDBAC was evaluated under different experimental conditions. A full factorial design with center point was employed to verify the in fluence of different factors on the recovery. A mathematical model was found to represent the xylanese yield (Y) as a function of BDBAC and hexanol: Y=4.32+5.1B+2.64D+0.83B2+1.46D2, where B=BDBAC and D=hexanol. The highest xylanase recovery (27%), indicated by the model was attained at pH=8.1, BDBAC=0.38 M and hexanol=8.6%. Under these conditions, and to test the model, a new xylanase extraction was performed in laboratory, giving 29.4% recovery yield, this value was similar to that predicted by the model.

Keywords: Reversed micelles; xylanase; liquid-liquid extraction; statistical design

Modeling cellobiose hydrolysis with integrated kinetic models by Luiza P. V. Calsavara; Flávio F. De Moraes; Gisella M. Zanin (pp. 789-806).
The enzyme cellobiase Novozym 188, which is used for improving hydrolysis of bagasse with cellulase, was characterized in its commercial available form and integrated kinetic models were applied to the hydrolysis of cellobiose. The specific activity of this enzyme was determined for pH values from 3.0–7.0, and temperatures from 40–75°C, with cellobiose at 2 g/L. Thermal stability was measured at pH 4.8 and temperatures from 40–70°C. Substrate inhibition was studied at the same pH, 50°C, and cellobiose concentrations from 0.4–20 g/L. Product inhibition was determined at 50°C, pH 4.8, cellobiose concentrations of 2 and 20 g/L, and initial glucose concentration nearly zero or 1.8 g/L. The enzyme has shown the greatest specific activity, 17.8 U/mg, at pH 4.5 and 65°C. Thermal activation of the enzyme followed Arrhenius equation with the Energy of Activation being equal to 11 kcal/mol for pH values 4 and 5. Thermal deactivation was adequately modeled by the exponential decay model with Energy of Deactivation giving 81.6 kcal/mol. Kinetics parameters for substrate uncompetitive inhibition were: Km=2.42 mM, V max=16.31 U/mg, Ks=54.2 mM. Substrate inhibition was clearly observed above 10 mM cellobiose. Product inhibition at the concentration studied has usually doubled the time necessary to reach the same conversion at the lower temperature tested.

Keywords: Cellobiase; cellobiose; kinetic modeling; thermal stability; energy of deactivation; energy of activation

Recovery of cellulase by HPMC-salt precipitation by Sebastião Avelino; Adriano R. Azzoni; Paulo T. V. Rosa; Everson A. Miranda; Cesar C. Santana (pp. 807-815).
Production of industrial enzymes including cellulases requires minimum cost with the down stream processing. The objective of this work was toanalyze the precipitation of cellulases by ammonium sulfate in the presence of hydroxypropyl(methylcellulose) as a co-precipitant through the use of statistical experimental design. The model generated with the experimental results showed that high protein recovery can be achieved at high levels of temperature, aging times, and rate of salt-solution addition, and at a low mixing level. The results also allowed the observation that activity recovery was improved at high levels of temperature, rate of salt addition and mixing level, and a low level of aging time.

Keywords: Hydroxypropyl (methylcellulose); precipitation; down-stream processing; cellulase

Fusel oil as precursor for aroma generation by biotransformation using lipase by Heizir F. De Castro; Regina Y. Moriya; Pedro C. Oliveira; Cleide M. F. Soares (pp. 817-826).
The feasibility of using mixtures of C4 and C5 chain-length aliphatic alcohols from fusel oil as the bulk organic media for lipase-mediated synthesis of laurate esters was assessed. Reaction mixtures consisted of lauric acid, lipase, solvent (if added), and appropriate amount of fusel oil (previously dehydrated with inorganic salts and molecular sieves). The influence of the reaction conditions such as substrate concentrations and temperature were investigated. Increased molar ratio of acyl donor to acyl acceptor allowed the esterification to proceed with no need for solvent addition.

Keywords: Fusel oil; lipase; esterification; laurate esters

Effect of growth substrates on production of new soluble glucose 3-dehydrogenase in Halomonas (Deleya) sp. α-15 by Katsuhiro Kojima; Wakako Tsugawa; Tetsuro Hamahuji; Yoshihumi Watazu; Koji Sode (pp. 827-834).
Halomonas (Deleya) sp. α-15 produces new co-factor binding soluble glucose 3-dehydrogenase (G3DH), which oxidizes the third hydroxy group of pyranose. This study investigated the condition of efficient production of G3DH using Halomonas (Deleya) sp. α-15. This enzyme was inducible, and α-methyl-D-glucoside, isopropyl-thioga lactopyranoside (IPTG) and lactose were revealed to be suitable carbon sources for G3DH induction. Maximum G3DH production was achieved by using minimal medium containing 0,8% (w/v) lactose with a productivity of 470U/1.

Keywords: glucose dehydrogenase; marine bacteria; Halomonas sp

Enzymatic alcoholysis of palm and palm kernel oils by Debora Oliveira; Tito Livio M. Alves (pp. 835-844).
The use of lipases as biocatalysts in ester synthesis has been the object of growing interest, owing to the importance of esters as emulsifiers, intermediates to produce oleochemicals, and fuel alternatives. We consider in this report the application of lipases in the ethanolysis of palm and palm kernel oils to produce fatty-acid esters, using n-hexane assolvent. In order to maximize ester production, wea dopted a Taguchi design and built an empirical model. Using this procedure, we determined the optimal condition for each system and established the influence of process variables in the conversion.

Keywords: Alcoholysis; lipases; experimental design; vegetable oils; fatty acid esters

Mathematical modeling of lipase and protease production by Penicillium restrictum in a batch fermenter by Denise M. G. Freire; Geraldo L. Sant'Anna Jr.; Tito Livio M. Alves (pp. 845-855).
This work presents a mathematical model that describes time course variations of extracellular lipase and protease activities for the batch fermentation of the fungus Penicillium restrictum, a new and promising strain isolated from soil and wastes of a Brazilian babassu coconutoil industry. The fermentation process was modeled by an unstructured model, which considered the following dependent variables: cells, fat acid, dissolved oxygen concentrations, lipase and protease activities, and cell lysate concentration. The last variable represents the amount of cells that has been lysed by the shear stress and natural cell death. Proteases released to the medium, as consequence of this process, enhance lipase inactivation. The model is able to predict the effects of some operation variables such as air flow rate and agitation speed. The mathematical model was validated against batch-fermentation data obtained under several operating conditions. Because substrate concentration has antagonistic effects on lipase activity, a typical optimization scheme should be developed in order to minimize these deleterious effects while maximizing lipase activity.

Keywords: Lipase; protease; unstructured model; kinetics; Penicillium restrictum

Modification of the thermoresistance to spray-drying of a cold-adapted subtilisin by genetic engineering by Ghislain Bare; Aaron Diakiese; Slim Zgoulli; Ahmed Sabri; Charles Gerday; Philippe Thonart (pp. 857-865).
The thermoresistance of a cold-adapted subtilisin dried by spray-drying was studied. Proteolytic activity of this enzyme was measured before and after spray-drying. Without chemical additives, spray-drying yields ranged from 2–13%. The use of arabic gum and lactose in the composition of the enzyme solutions allowed the strengthening of the enzyme structures and increased water mobility in the product. Increase of water mobility led to a shorter residence time of the product in the spray-drier and a net yield increase was obtained (yield higher than 50%). The effect of two selective mutations on the thermoresistance to spray-drying of the cold-adapted subtilisin was also investigated. Mutation T85D (introduction of an additional link with an ion Ca2+ necessary for enzyme activity, by substitution of Asp for Thr 85) had no effect on the thermoresistance of the subtilisin to spray-drying. Mutation H121W (introduction of an additional aromatic link by substitution of Trp for His 121) reduced the drying yield from 66% (not modified subtilisin) to 52%. This higher thermosensitivity could be explained by an increase of the hygroscopic character of the modified subtilisin (mutation H121W).

Keywords: Subtilisin; mutations; cold-adapted enzyme; thermo-resistance; spray-drying

The nature of lignin from steam explosion/enzymatic hydrolysis of softwood by Sergey M. Shevchenko; Rodger P. Beatson; John N. Saddler (pp. 867-876).
Effective utilization of the lignin by-product is a prerequisite to the commercial viability of ethanol production from softwood wastes using a steam explosion (SE)/enzymatic hydrolysis (EH)/fermentation process. Changes in the chemical composition of Douglas fir wood on SO2-catalyzed SE followed by EH were assessed using conventional analytical methods and new halogen-probetechniques. A significant solubilization of hemicelluloses was observed in the SE stage, the severity of which affected subsequent fermentation of cellulose and sorption of enzymes. SE of softwood resulted in dramatic changes in the chemical structure of lignin in the residual material involving chemical reactions via the benzyl cation. This leads to a more condensed lignin with partly blocked α-reaction centres. Possible uses for this lignin are discussed.

Keywords: Lignin; steam explosion; enzymatic hydrolysis; softwood

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