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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.113, #1-3)

Designing an effective federal biomass program by David Morris (pp. 5-12).

This article addresses two questions: Has the effectiveness of the US governments federal research and development (R&D) spending suffered from the post-1980 strategic change from freely shared and publicly owned to privately owned scientific advances? What criteria would a federal R&D program use to design a strategy that most effectively enhances the well-being of farmers and rural communities? Several studies found that the pre-1980 US Department of Agriculture research strategy was very effective. No comparable studies have analyzed the comparative effectiveness of the post-1980 strategy of restricting access to the results of public research. Recent experience and several analytical studies suggest that to significantly enhance the health of rural economies from an expanded use of plant matter as an industrial material, federal policy should channel scientific and engineering research into small- and medium-sized production and processing technologies and should encourage farmer-owned, value-added enterprises.

Keywords: Ethanol; scale; ownership; research and development; effectiveness

Methodology for estimating removable quantities of agricultural residues for bioenergy and bioproduct use by Richard G. Nelson; Marie Walsh; John J. Sheehan; Robin Graham (pp. 13-26).

A methodology was developed to estimate quantities of crop residues that can be removed while maintaining rain or wind erosion at less than or equal to the tolerable soil-loss level. Six corn and wheat rotations in the 10 largest corn-producing states were analyzed. Residue removal rates for each rotation were evaluated for conventional, mulch/reduced, and no-till field operations. The analyses indicated that potential removable maximum quantities range from nearly 5.5 million dry metric t/yr for a continuous corn rotation using conventional till in Kansas to more than 97 million dry metric t/yr for a corn-wheat rotation using no-till in Illinois.

Keywords: Corn stover; wheat straw; rainfall erosion; wind erosion; tolerable soil loss

Pipeline transport of biomass by Amit Kumar; Jay B. Cameron; Peter C. Flynn (pp. 27-39).

The cost of transporting wood chips by truck and by pipeline as a water slurry was determined. In a practical application of field delivery by truck of biomass to a pipeline inlet, the pipeline will only be economical at large capacity (>0.5 million dry t/yr for a one-way pipeline, and >1.25 million dry t/yr for a two-way pipeline that returns the carrier fluid to the pipeline inlet), and at medium to long distances (>75 km [one-way] and >470 km [two-way] at a capacity of 2 million dry t/yr). Mixed hardwood and softwood chips in western Canada rise in moisture level from about 50% to 67% when transported in water; the loss in lower heating value (LHV) would preclude the use of water slurry pipelines for direct combustion applications. The same chips, when transported in a heavy gas oil, take up as much as 50% oil by weight and result in a fuel that is >30% oil on mass basis and is about two-thirds oil on a thermal basis. Uptake of water by straw during slurry transport is so extreme that it has effectively no LHV. Pipeline-delivered biomass could be used in processes that do not produce contained water as a vapor, such as supercritical water gasification.

Keywords: Wood chips; pipeline; biomass; lower heating value; straw

Hydrodynamic separation of grain and stover components in corn silage by Philippe Savoie; Kevin J. Shinners; Benjamin N. Binversie (pp. 41-54).

Mixing fresh silage in water resulted in partial segregation of grain from stover. Grain concentration was 75% in the sunk material when silage was relatively dry (64% moisture content [MC]) and 41% when silage was relatively wet (74% MC). Partial drying to remove 20 percentage units of moisture prior to water separation increased grain concentration to 92%, and complete drying increased grain concentration to 99%. Sieving without drying followed by water separation resulted in a grain concentration of 79%. A byproduct of water separation is a large amount of soluble and deposited fine particles in the effluent: 18% of original dry matter after one separation, and between 21 and 26% after eight separations. In an industrial setting, hydrodynamic separation of silage with minimal pretreatment could provide a feedstock with a high concentration of grain (75–80%). In a laboratory setting, hydrodynamic separation with prior oven drying could provide a method to separate grain from stover in corn silage by reaching a grain concentration higher than 99%.

Keywords: Corn; stover; grain; separation; silage

A new class of plants for a biofuel feedstock energy crop by James Kamm (pp. 55-70).

Directly burnable biomass to be used primarily in steam boilers for power production has been researched and demonstrated in a variety of projects in the United states. The biomass typically comes from wood wastes, such as tree trimmings or the byproducts of lumber production, or from a cash crop, grown by farmers. Of this latter group, the main emphasis has been utilizing corn stover, or a prairie grass called switchgrass, or using tree seedlings such as willow. In this article, I propose an alternative to these energy crops that consists of several different herbaceous plants with the one consistent property that they annually generate and appreciable bulk of dried-down burnable mass. The fact that they are a set of plants (nine are offered as candidates) gives this energy crop a great deal of flexibility as far as growing condition and annual harvest time line. Their predicted yield is impressive and leads to speculation that they can be economically feasible.

Keywords: Biomass; biofuel; energy crop; sclerified stalked plants; stiff stalked plants

Fungal upgrading of wheat straw for straw-thermoplastics production by Tracy P. Houghton; David N. Thompson; J. Richard Hess; Jeffrey A. Lacey; Michael P. Wolcott; Anke Schirp; Karl Englund; David Dostal; Frank Loge (pp. 71-93).

Combining biologic pretreatment with storage is an innovative approach for improving feedstock characteristics and cost, but the magnitude of responses of such systems to upsets is unknown. Unsterile wheat straw stems were upgraded for 12 wk with Pleurotus ostreatus at constant temperature to estimate the variation in final compositions with variations in initial moisture and inoculum. Degradation rates and conversions increased with both moisture and inoculum. A regression analysis indicated that system performance was quite stable with respect to inoculum and moisture content after 6 wk of treatment. Scale-up by 150× indicated that system stability and final straw composition are sensitive to inoculum source, history, and inoculation method. Comparative testing of straw-thermoplastic composites produced from upgraded stems is under way.

Keywords: Fungal upgrading; engineered storage; biological preprocessing; Pleurotus ostreatus ; straw composite

Economic analysis of ethanol production in California using traditional and innovative feedstock supplies by Ellen I. Burnes; John Hagen; Dennis Wichelns; Kristen Callens (pp. 95-110).

In this article, we estimate the costs of using alternative feedstocks to produce ethanol in a 40 million-gal facility in California’s San Joaquin Valley. Feedstocks include corn imported from Midwestern states and locally grown agricultural products such as corn, grapes, raisins, oranges, and other tree fruits. The estimated feedstock costs per gallon of ethanol include $0.92 for Midwestern corn, $1.21 for locally grown corn, $6.79 for grapes, $3.36 for raisins, $3.92 for citrus, and $1.42 for other tree fruit. Adjusting for coproduct values lowers the estimated net feedstock costs to $0.67/gal of ethanol for Midwestern corn, $0.96 for locally grown corn, $6.53 for grapes, and $3.30 for raisins. We also examine the potential increases in net revenue to raisin producers, made possible by having an alternative outlet available for selling surplus raisins.

Keywords: Biofuels; renewable energy; raisins; ethanol; feedstocks

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose by Nóra Szijártó; Zsolt Szengyel; Gunnar Lidén; Kati Réczey (pp. 115-124).

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.

Keywords: Trichoderma reesei ; fermentation; cellulase; growth characterization; cellulose hydrolysis

Development and application of an integrated system for monitoring ethanol content of fuels by Eliana M. Alhadeff; Andrea M. Salgado; Nei Pereira Jr.; Belkis Valdman (pp. 125-136).

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-aminophenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05–1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05–1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.

Keywords: Biosensors; gasohol; immobilized enzymes; alcohol oxidase; horseradish peroxidase

Model based soft-sensor for on-line determination of substrate by Andréa M. Salgado; Rossana O. M. Folly; Belkis Valdman; Francisco Valero (pp. 137-144).

A software sensor for on-line determination of substrate was developed based on a model for fed-batch alcoholic fermentation process and on-line measured signals of ethanol, biomass, and feed flow. The ethanol and biomass signals were obtained using a colorimetric biosensor and an optical sensor developed in previous works that permitted determination of ethanol at a concentration of 0–40 g/L and biomass of 0–60 g/L. The volume in the fermentor could be continuously calculated using the total measured signal of the feed flow. The results obtained show that the model used is adequate for the proposed software sensor and determines continuously the substrate concentration with efficiency and security during the fermentation process.

Keywords: Soft-sensor; substrate; alcohol fermentation; ethanol; biomass

Screening of Dowex^® anion-exchange resins for invertase immobilization by Ester Junko Tomotani; Michele Vitolo (pp. 145-159).

Commercial yeast invertase (Bioinvert^®) was immobilized by adsorption on anion-exchange resins, collectively named Dowex^® (1×8:50–400, 1×4:50–400, and 1×2:100–400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1×4–200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The K m and V _max for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL.

Keywords: Invertase; immobilization; exchange resins; Dowex^®

Effects of carbon source on expression of alcohol oxidase activity and on morphologic pattern of YR-1 strain, a filamentous fungus isolated from petroleum-contaminated soils by Carmen Rodríguez Robelo; Vanesa Zazueta Novoa; Roberto Zazueta-Sandoval (pp. 161-171).

Soluble alcohol oxidase (AO) activity was detected in the supernatant fraction of a high-speed centrifugation procedure after ballistic cellular homogenization to break the mycelium from a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing different carbon sources, including alcohols and hydrocarbons but not in glucose. In previous work, zymogram analysis conducted with crude extracts from aerobic mycelium of YR-1 strain indicated the existence of two AO enzymes originally named AO-1 and AO-2. In the present study, we were able to separate the AO-1 band into two bands depending on culture conditions, carbon source, and polyacrylamide gel electrophoresis (PAGE) separation conditions; the enzyme activity pattern in zymograms from cell-free extracts exhibited three different bands after native PAGE. New nomenclature was used for upper bands AO-1 and AO-2 and lower band AO-3, respectively. The expression of AO activity was studied in the absence of glucose in the culture media and in the presence of hydrocarbons or petroleum as sole carbon source, suggesting that AO expression could be subjected to two regulatory possibilities: carbon catabolite regulation by glucose and induction by hydrocarbons. The possibility of catabolic inhibition of AO by glucose in the active enzyme was also tested, and the results confirm that this kind of regulatory mechanism is not present in AO activity.

Keywords: Alcohol oxidase; filamentous fungi; hydrocarbon biodegradation; petroleum contamination; YR-1 strain

Effect of temperature, moisture, and carbon supplementation on lipase production by solid-state fermentation of soy cake by Penicillium simplicissimum by Marco di Luccio; Fernando Capra; Najara P. Ribeiro; Gean D. L. P. Vargas; Denise M. G. Freire; Débora de Oliveira (pp. 173-180).

The production of lipases by Penicillium simplicissimum using solid-state fermentation and soy cake as substrate was investigated. The effects of temperature, cake moisture, and carbon supplementation on lipase production were studied using a two-level experimental plan. Moisture, pH, and lipase activity were followed during fermentation. Statistical analysis of the results was performed to evaluate the effect of the studied variables on the maximum lipase activity. Incubation temperature was the variable that most affected enzyme activity, showing a negative effect. Moisture and carbon supplementation presented a positive effect on activity. It was possible to obtain lipase activity as high as 21 U/g of dry cake in the studied range of process variables.

Keywords: Solid-state fermentation; lipase; Penicillium ; soy cake; olive oil; moisture

The effect of temperature, pressure, exposure time, and depressurization rate on lipase activity in SCCO₂ by Marcelo Lanza; Wagner Luís Priamo; José Vladimir Oliveira; Cláudio Dariva; Débora de Oliveira (pp. 181-187).

We investigated the influence of temperature, pressure, exposure time, and decompression rate on lipase activity in high-pressure CO₂ medium. A high-pressure, variable-volume view cell was employed in the experiments, varying the temperature from 30 to 70°C in the pressure range of 70–250 bar at various high-pressure exposure times (60–360 min) and adopting several decompression rates (10–200 kg/[m³·min]). The results obtained show that an increase in temperature and density led to an enhancement of enzyme activity losses while the decompression rates had a weak influence on enzyme inactivation.

Keywords: Lipase activity; high pressure; decompression rate; CO₂ ; SCCO₂ ; exposure time

Ester synthesis catalyzed by Mucor miehei lipase immobilized on magnetic polysiloxane-polyvinyl alcohol particles by Laura M. Bruno; José L. de Lima Filho; Eduardo H. de M. Melo; Heizir F. de Castro (pp. 189-199).

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding with high activity recovered. The performance of the resulting immobilized biocatalyst was evaluated in the synthesis of flavor esters using heptane as solvent. The impact on reaction rate was determined for enzyme concentration, molar ratio of the reactants, carbon chain length of the reactants, and alcohol structure. Ester synthesis was maximized for substrates containing excess acyl donor and lipase loading of 25 mg/mL. The biocatalyst selectivity for the carbon chain length was found to be different concerning the organic acids and alcohols. High reaction rates were achieved for organic acids with 8 or 10 carbons, whereas increasing the alcohol carbon chain length from 4 to 8 carbons gave much lower esterification yields. Optimal reaction rate was determined for the synthesis of butyl caprylate (12 carbons). Esterification performance was also dependent on the alcohol structure, with maximum activity occurring for primary alcohol. Secondary and tertiary alcohols decreased the reaction rates by more than 40%.

Keywords: Lipase; sol-gel matrix; polysiloxane-polyvinyl alcohol; esterification; activity

Effect of pH on cellulase production of Trichoderma ressei RUT C30 by Tamás Juhász; Zsolt Szengyel; Nóra Szijártó; Kati Réczey (pp. 201-211).

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.

Keywords: Cellulase production; Trichoderma reesei RUT C30; pH profiling; β-glucosidase; shake flask; fermentor

Quantitative analysis of cellulose-reducing ends by Sasithorn Kongruang; Myung Joo Han; Claudia Isela Gil Breton; Michael H. Penner (pp. 213-231).

Methods for the quantification of total and accessible reducing ends on traditional cellulose substrates have been evaluated because of their relevance to enzyme-catalyzed cellulose saccharification. For example, quantification of accessible reducing ends is likely to be the most direct measure of substrate concentration for the exo-acting, reducing end-preferring cellobiohydrolases. Two colorimetric assays (dinitrosalicylic acid [DNS] and bicinchoninic acid [BCA] assay) and a radioisotope approach (NaB³H₄ labeling) were evaluated for this application. Cellulose substrates included microcrystalline celluloses, bacterial celluloses, and filter paper. Estimates of the number of reducing ends per unit mass cellulose were found to be dependent on the assay system (i.e. the DNS and BCA assays gave strikingly different results). DNS-based values were several-fold higher than those obtained using the BCA assay, with fold-differences being substrate specific. Sodium borohydride reduction of celluloses, using cold or radiolabeled reagent under relatively mild conditions, was used to assess the number of surface (solvent-accessible) reducing ends. The results indicate that 30–40% of the reducing ends on traditional cellulose substrates are not solvent accessible; that is, they are buried in the interior of cellulose structures and thus not available to exo-acting enzymes.

Keywords: Cellulose; cellobiohydrolase; reducing sugarassays; insoluble reducing ends; solvent-accessible reducing ends

Properties of a recombinant β-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis by Xin-Liang Li; Lars G. Ljungdahl; Eduardo A. Ximenes; Huizhong Chen; Carlos R. Felix; Michael A. Cotta; Bruce S. Dien (pp. 233-250).

A β-glucosidase (BglA, EC 3.2.1.21) gene from the polycentric anaerobic fungus Orpinomyces PC-2 was cloned and sequenced. The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and bacterial β-glucosidases but lacked significant similarity to those from aerobic fungi. Neither cellulose- nor protein-binding domains were found in BglA. When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast cells. K m and V max values of the secreted BglA were 0.762 mM and 8.20 µmol/(min·mg), respectively, with p-nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate and 0.310 mM and 6.45 µmol/(min·mg), respectively, for the hydrolysis of cellobiose. Glucose competitively inhibited the hydrolysis of pNPG with a K i of 3.6 mM. β-Glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential for use in biofuel and feedstock chemical production.

Keywords: Cellulose; cellulase; β-glucosidase; Orpinomyces ; cellobiase

Characterization and performance of immobilized amylase and cellulase by Bradley A. Saville; Mikhail Khavkine; Gayathri Seetharam; Behzad Marandi; Yong-Li Zuo (pp. 251-259).

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7–10%. Immobilized amylase performance was assessed by batch kinetics in 100–300 g/L of corn flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost ∼55% of its activity, compared with ∼30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose.

Keywords: Amylase; cellulase; immobilization; inactivation; wastepaper

Immobilized enzyme studies in a microscale bioreactor by Francis Jones; Scott Forrest; Jim Palmer; Zonghuan Lu; John Elmore; Bill B. Elmore (pp. 261-272).

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of > 90% were observed.

Keywords: Microscale bioreactor; polydimethylsiloxane microreactor; immobilized enzymes; urease enzyme; silicon wafer

Performance of chloroperoxidase stabilization in mesoporous sol-gel glass using In situ glucose oxidase peroxide generation by Abhijeet Borole; Sheng Dai; Catherine L. Cheng; Miguel Rodriguez Jr.; Brian H. Davison (pp. 273-285).

A unique mesoporous sol-gel glass possessing a highly ordered porous structure (with three pore sizes of about 50, 150, and 200 Å diameter) was used as a support material for immobilization of the enzyme chloroperoxidase (CPO). CPO was bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal. In situ production of the cosubstrate, H₂O₂, was achieved using glucose oxidase. Solvent stability in acetonitrile mixtures was enhanced when a pore size larger than the size of CPO was used (i.e., 200) Å From these results, it appears that the glass-enzyme complex developed through the present work can be used as high-performance biocatalysts for various chemical-processing applications, particularly in harsh conditions.

Keywords: Sol-gel glass; chloroperoxidase; glucose oxidase; acetonitrile; horseradish peroxidase; thermostability

Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca by José M. Escovar-Kousen; David Wilson; Diana Irwin (pp. 287-297).

Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-α, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose.

Keywords: Thermonospora fusca ; Cel9A; cellulases; protein engineering; computer modeling

Kinetics of asparaginase II fermentation in Saccharomyces cerevisiae ure2dal80 mutant by Maria Antonieta Ferrara; Josiane M. V. Mattoso; Elba P. S. Bon; Nei Pereira Jr. (pp. 299-305).

Although the quality of nitrogen source affects fermentation product formation, it has been managed empirically, to a large extent, in industrial scale. Laboratory-scale experiments successfully use the high-cost proline as a nonrepressive source. We evaluated urea as a substitute for proline in Saccharomyces cerevisiae ure2dal80 fermentations for asparaginase II production as a model system for nitrogen-regulated external enzymes. Maximum asparaginase II levels of 265 IU/L were observed in early stationary-phase cells grown on either proline or urea, whereas in ammonium cells, the maximum enzyme level was 157 IU/L. In all cases, enzyme stability was higher in buffered cultures with an initial pH of 6.5.

Keywords: Saccharomyces cerevisiae ; ure2dal80 mutants; nitrogen sources; asparaginase II; fermentation kinetics

Studies on immobilizd lipase in hydrophobic sol-gel by Cleide M. F. Soares; Onelia A. dos Santos; Heizir F. de Castro; Flavio F. de Moraes; Gisella M. Zanin (pp. 307-319).

The hydrolysis of tetraethoxysilane using the sol-gel process was used to produce silica matrices, and these were tested for the immobilization of lipase from Candida rugosa by three methods: physical adsorption, covalent binding, and gel entrapment in the presence and absence of polyethylene glycol (PEG-1450). The silica matrices and their derivatives were characterized regarding particle size distribution, specific surface area, pore size distribution (Brunauer, Emmett, and Teller [B.E.T.] method), yield of grafting (thermogravimetric analyzer [TGA]), and chemical composition (Fourier transform infrared). Immobilization yields based on recovered lipase activity varied from 3.0 to 32.0%, and the highest efficiency was attained when lipase was encapsulated in the presence of PEG.

Keywords: Silica matrices; immobilization; lipase; additive; sol-gel

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.113, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.113, #1-3)