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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.129, #1-3)

Introduction to the Proceedings of the Twenty-Seventh Symposium on Biotechnology for Fuels and Chemicals by James D. McMillan (pp. iii-viii).

Introduction to session 1A by Peter C. Flynn (pp. 1-2).

Agricultural residue availability in the United States by Zia Haq; James L. Easterly (pp. 3-21).

The National Energy Modeling System (NEMS) is used by the Energy Information Administration (EIA) to forecast US energy production, consumption, and price trends for a 25-yr-time horizon. Biomass is one of the technologies within NEMS, which plays a key role in several scenarios. An endogenously determined biomass supply schedule is used to derive the price-quantity relationship of biomass. There are four components to the NEMS biomass supply schedule including: agricultural residues, energy crops, forestry residues, and urban wood waste/mill residues. The EIA'S Annual Energy Outlook 2005 includes updated estimates of the agricultural residue portion of the biomass supply schedule. The changes from previous agricultural residue supply estimates include: revised assumptions concerning corn stover and wheat straw residue availabilities, inclusion of non-corn and non-wheat agricultural residues (such as barley, rice straw, and sugarcane bagasse), and the implementation of assumptions concerning increases in no-till farming. This article will discuss the impact of these changes on the supply schedule.

Keywords: Agricultural residues; corn stover; wheat straw; feedstock cost; biomass supply

Canadian biomass reserves for biorefining by Warren E. Mabee; Evan D. G. Fraser; Paul N. McFarlane; John N. Saddler (pp. 22-40).

A lignocellulosic-based biorefining strategy may be supported by biomass reserves, created initially with residues from wood product processing or agriculture. Biomass reserves might be expanded using innovative management techniques that reduce vulnerability of feedstock in the forest products or agricultural supply chain. Forest-harvest residue removal, disturbance isolation, and precommercial thinnings might produce 20–33×10⁶ mt/yr of feedstock for Canadian biorefineries. Energy plantations on marginal Canadian farmland might produce another 9–20 mt. Biomass reserves should be used to support first-generation biorefining installations for bioethanol production, development of which will lead to the creation of future high-value coproducts. Suggestions for Canadian policy reform to support biomass reserves are provided.

Keywords: Biorefining; lignocellulosic biomass; forestry; agriculture; energy plantations; policy reform

Availability of crop residues as sustainable feedstock for bioethanol production in North Carolina by Abolghasem Shahbazi; Yebo Li (pp. 41-54).

The amount of corn stover and wheat straw that can be sustainably collected in North Carolina was estimated to be 0.64 and 0.16 million dry t/yr, respectively. More than 80% of these crop residues are located in the coastal area. The bioethanol potential from corn stover and wheat straw was estimated to be about 238 million L (63 million gal/yr) in North Carolina. The future location of ethanol plant in North Carolina was estimated based on feedstock demand and collection radius. It is possible to have four ethanol plants with feedstock demand of 400, 450, 500, and 640 dry t/d. The collection radii for these four ethanol plants are 46, 60, 42, and 67 km (28, 37, 26, and 42 miles), respectively. The best location for a bioethanol plant includes four counties (Beaufort, Hyde, Tyrrell, and Washington) with feedstock demand of 500 t/d and collection radius about 26 mile.

Keywords: Crop residues; ethanol; corn stover; wheat straw

Updates on softwood-to-ethanol process development by Warren E. Mabee; David J. Gregg; Claudio Arato; Alex Berlin; Renata Bura; Neil Gilkes; Olga Mirochnik; Xuejun Pan; E. Kendall Pye; John N. Saddler (pp. 55-70).

Softwoods are generally considered to be one of the most difficult lignocellulosic feedstocks to hydrolyze to sugars for fermentation, primarily owing to the nature and amount of lignin. If the inhibitory effect of lignin can be significantly reduced, softwoods may become a more useful feedstock for the bioconversion processes. Moreover, strategies developed to reduce problems with softwood lignin may also provide a means to enhance the processing of other lignocellulosic substrates. The Forest Products Biotechnology Group at the University of British Columbia has been developing softwood-to-ethanol processes with SO₂-catalyzed steam explosion and ethanol organosolv pretreatments. Lignin from the steam explosion process has relatively low reactivity and, consequently, low product value, compared with the highvalue coproduct that can be obtained through organosolv. The technical and economic challenges of both processes are presented, together with suggestions for future process development.

Keywords: SO₂ steam explosion; softwoods; ethanol; lignin; organosolv

Development of a multicriteria assessment model for ranking biomass feedstock collection and transportation systems by Amit Kumar; Shahab Sokhansanj; Peter C. Flynn (pp. 71-87).

This study details multicriteria assessment methodology that integrates economic, social, environmental, and technical factors in order to rank alternatives for biomass collection and transportation systems. Ranking of biomass collection systems is based on cost of delivered biomass, quality of biomass supplied, emissions during collection, energy input to the chain operations, and maturity of supply system technologies. The assessment methodology is used to evaluate alternatives for collecting 1.8×10⁶ dry t/yr based on assumptions made on performance of various assemblies of biomass collection systems is based on cost of delivered biomass, quality of biomass supplied, emissions during collection, energy input to the chain operations, and maturity of supply system technologies. The assessment methodology is used to evaluate alternatives for collecting 1.8×10⁶ dry t/yr based on assumptions made on performance of various assemblies of biomass collection systems. A proposed collection option using loafer/stacker was shown to be the best option followed by ensiling and baling. Ranking of biomass transport systems is based on cost of biomass transport, emissions during transport, traffic congestion, and maturity of different technologies. At a capacity of 4×10⁶ dry t/yr, rail transport was shown to be the best option, followed by truck transport and pipeline transport, respectively. These rankings depend highly on assumed maturity of technologies and scale of utilization. These may change if technologies such as loafing or ensiling (wet storage) methods are proved to be infeasible for large-scale collection systems.

Keywords: Multicriteria assessment; biomass collection systems; biomass transportation systems; PROMETHEE; ranking; bioenergy; IBSAL model

Rail vs truck transport of biomass by Hamed Mahmudi; Peter C. Flynn (pp. 88-103).

This study analyzes the economics of transshipping biomass from truck to train in a North American setting. Transshipment will only be economic when the cost per unit distance of a second transportation mode is less than the original mode. There is an optimum number of transshipment terminals which is related to biomass yield. Transshipment incurs incremental fixed costs, and hence there is a minimum shipping distance for rail transport above which lower costs/km offset the incremental fixed costs. For transport by dedicated unit train with an optimum number of terminals, the minimum economic rail shipping distance for straw is 170 km, and for boreal forest harvest residue wood chips is 145 km. The minimum economic shipping distance for straw exceeds the biomass draw distance for economically sized centrally located power plants, and hence the prospects for rail transport are limited to cases in which traffic congestion from truck transport would otherwise preclude project development. Ideally, wood chip transport costs would be lowered by rail transshipment for an economically sized centrally located power plant, but in a specific case in Alberta, Canada, the layout of existing rail lines precludes a centrally located plant supplied by rail, whereas a more versatile road system enables it by truck. Hence for wood chips as well as straw the economic incentive for rail transport to centrally located processing plants is limited. Rail transshipment may still be preferred in cases in which road congestion precludes truck delivery, for example as result of community objections.

Keywords: Biomass transportation; transportation economics; rail transport; truck transport; straw

Corn stover fractions and bioenergy by Danny E. Akin; W. Herbert Morrison III; Luanne L. Rigsby; Franklin E. Barton II; David S. Himmelsbach; Kevin B. Hicks (pp. 104-116).

Information is presented on structure, composition, and response to enzymes of corn stover related to barriers for bioconversion to ethanol. Aromatic compounds occurred in most tissue cell walls. Ferulic acid esterase treatment before cellulase treatment significantly improved dry weight loss and release of phenolic acids and sugars in most fractions over cellulase alone. Leaf fractions were considerably higher in dry weight loss and released sugars with esterase treatment, but stem pith cells gave up the most phenolic acids. Results help identify plant fractions more appropriate for coproducts and bioconversion and those more suitable as residues for soil erosion control.

Keywords: Zea mays L.; lignin; phenolic acids; esterase; cellulase

Separate and simultaneous enzymatic hydrolysis and fermentation of wheat hemicellulose with recombinant xylose utilizing Saccharomyces cerevisiae by L. Olsson; H. R. Soerensen; B. P. Dam; H. Christensen; K. M. Krogh; A. S. Meyer (pp. 117-129).

Fermentations with three different xylose-utilizing recombinant Saccharomyces cerevisiae strains (F12, CR4, and CB4) were performed using two different wheat hemicellulose substrates, unfermented starch free fibers, and an industrial ethanol fermentation residue, vinasse. With CR4 and F12, the maximum ethanol concentrations obtained were 4.3 and 4 g/L, respectively, but F12 converted xylose 15% faster than CR4 during the first 24 h. The comparison of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with F12 showed that the highest, maximum ethanol concentrations were obtained with SSF. In general, the volumetric ethanol productivity was initially, highest in the SHF, but the overall volumetric ethanol productivity ended up being maximal in the SSF, at 0.013 and 0.010 g/Lh, with starch free fibers and vinasse, respectively.

Keywords: Xylose conversion; ethanol; starch free fibers; vinasse

Biofiltration methods for the removal of phenolic residues by Luiz Carlos Martins Das Neves; Tábata Taemi Miazaki Ohara Miyamura; Dante Augusto Moraes; Thereza Christina Vessoni Penna; Attilio Converti (pp. 130-152).

Industrial effluents from the pharmaceutical industry often contain high concentrations of phenolic compounds. The presence of “anthropogenic” organic compounds in the environment is a serious problem for human health; therefore, it merits special attention by the competent public agencies. Different methods have been proposed in the last two decades for the treatment of this kind of industrial residues, the most important of which are those utilizing absorption columns, vaporization and extraction, and biotechnological methods. Biofiltration is a method for the removal of contaminants present in liquid or gaseous effluents by the use of aerobic microorganisms, which are immobilized on solid or porous supports. Although several bacteria can utilize aromatic compounds as carbon and energy source, only a few of them are able to make this biodegradation effectively and with satisfactory rate. For this reason, more investigation is needed to ensure an efficient control of process parameters as well as to select the suited reactor configuration. The aim of this work is to provide an overview on the main aspects of biofiltration for the treatment of different industrial effluents, with particular concern to those coming from pharmaceutical industry and laboratories for the production of galenicals.

Keywords: Biofiltration; phenolic residues; bioremediation

The BTL2 process of biomass utilization entrained-flow gasification of pyrolyzed biomass slurries by Klaus Raffelt; Edmund Henrich; Andrea Koegel; Ralph Stahl; Joachim Steinhardt; Friedhelm Weirich (pp. 153-164).

Forschungszentrum Karlsruhe has developed a concept for the utilization of cereal straw and other thin-walled biomass with high ash content. The concept consists of a regional step (drying, chopping, flash-pyrolysis, and mixing) and a central one (pressurized entrained-flow gasification, gas cleaning, synthesis of fuel, and production of byproducts). The purpose of the regional plant is to prepare the biomass by minimizing its volume and producing a stable and safe storage and transport form. In the central gasifier, the pyrolysis products are converted into syngas. The syngas is tar-free and can be used for Fischer-Tropsch synthesis after gas cleaning.

Keywords: Straw; flash-pyrolysis; slurry; entrained-flow gasification; synthesis gas

Emission profile of rapeseed methyl ester and its blend in a diesel engine by Gwi-Taek Jeong; Young-Taig Oh; Don-Hee Park (pp. 165-178).

Fatty acid methyl esters, also known as biodiesel, have been shown to have a great deal of potential as petro-diesel substitutes. Biodiesel comprise a renewable alternative energy source, the development of which would clearly reduce global dependence on petroleum and would also help to reduce air pollution. This paper analyzes the fuel properties of rapeseed biodiesel and its blend with petro-diesel, as well as the emission profiles of a diesel engine on these fuels. Fuels performance studies were conducted in order to acquire comparative data regarding specific fuel consumption and exhaust emissions, including levels of carbon monoxide (CO), carbon dioxide (CO₂), smoke density, and NO_x, in an effort to assess the performance of these biodiesel and blend. The fuel consumption amount of oil operations at high loads was similar or greater than that observed during petro-diesel operation. The use of biodiesel is associated with lower smoke density than would be seen with petro-diesel. However, biodiesel and its blend increased the emission of CO, CO₂, and nitrogen oxides, to a greater degree than was seen with petro-diesel. The above results indicate that rapeseed biodiesel can be partially substituted for petro-diesel under most operating conditions, regarding both performance parameters and exhaust, without any modifications having to be made to the engine.

Keywords: Biodiesel; engine performance; rapeseed oil; exhaust emission

Introduction to session 1B by Joel R. Cherry; Kevin Gray (pp. 179-179).

Properties and performance of glucoamylases for fuel ethanol production by Bradley A. Saville; Chunbei Huang; Vince Yacyshyn; Andrew Desbarats (pp. 180-194).

Studies were conducted on maltodextrin saccharification and on simultaneous saccharification and fermentation (SSF) with various commercial glucoamylases. In kinetics studies, none of the glucoamylases were able to completely convert maltodextrin into glucose. Typically, about 85% conversion was obtained, and glucose yields were about 75%. Typically, the kinetics were biphasic, with 1 h of rapid conversion, then a significant reduction in rate. Data were consistent with strong product inhibition and/or enzyme inactivation. Some glucoamylases followed first-order kinetics, initially slower at dextrin conversion, but eventually achieving comparable conversion and glucose concentrations. Most of the glucoamylases were more active at 55°C than at 35°C, but pH had little effect on activity. Screening studies in an SSF system demonstrated little difference between the glucoamylases, with a few exceptions. Subsequent targeted studies showed clear differences in performance, depending on the fermentation temperature and yeast used, suggesting that these are key parameters that would guide the selection of a glucoamylase.

Keywords: Glucoamylase; kinetics; fermentation; saccharification

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger by Christina Bohlin; Leif J. Jönsson; Robyn Roth; Willem H. van Zyl (pp. 195-214).

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A.

Keywords: Laccase; heterologous expression; Pichia pastoris ; Aspergillus niger

Lactose hydrolysis and formation of galactooligosaccharides by a novel immobilized β-galactosidase from the thermophilic fungus Talaromyces thermophilus by Phimchanok Nakkharat; Dietmar Haltrich (pp. 215-225).

β-Galactosidase from the fungus Talaromyces thermophilus CBS 236.58 was immobilized by covalent attachment onto the insoluble carrier Eupergit C with a high binding efficiency of 95%. Immobilization increased both activity and stability at higher pH values and temperature when compared with the free enzyme. Especially the effect of immobilization on thermostability is notable. This is expressed by the half-lifetime of the activity at 50°C, which was determined to be 8 and 27 h for the free and immobilized enzymes, respectively. Although immobilization did not significantly change kinetic parameters for the substrate lactose, a considerable decrease in the maximum reaction velocity V _max was observed for the artificial substrate o-nitrophenyl-β-d-galactopyranoside (oNPG). The hydrolysis of both oNPG and lactose is competitively inhibited by the end products glucose and galactose. However, this inhibition is only very moderate as judged from kinetic analysis with glucose exerting a more pronounced inhibitory effect. It was evident from bioconversion experiments with 20% lactose as substrate, that the immobilized enzyme showed a strong transgalactosylation reaction, resulting in the formation of galactooligosaccharides (GalOS). The maximum yield of GalOS of 34% was obtained when the degree of lactose conversion was roughly 80%. Hence, this immobilized enzyme can be useful both for the cleavage of lactose at elevated temperatures, and the formation of GalOS, prebiotic sugars that have a number of interesting properties for food applications.

Keywords: Immobilization; Eupergit; β-glycosidase; lactase; transgalactosylation

Evaluation of cell recycle on Thermomyces lanuginosus xylanase a production by Pichia pastoris GS 115 by Verônica Ferreira; Patricia C. Nolasco; Aline M. Castro; Juliana N. C. Silva; Alexandre S. Santos; Mônica C. T. Damaso; Nei Pereira Jr. (pp. 226-233).

This work aims to evaluate cell recycle of a recombinant strain of Pichia pastoris GS115 on the Xylanase A (XynA) production of Thermomyces lanuginosus IOC-4145 in submerged fermentation. Fed-batch processes were carried out with methanol feeding at each 12h and recycling cell at 24, 48, and 72 h. Additionally, the influence of the initial cell concentration was investigated. XynA production was not decreased with the recycling time, during four cell recycles, using an initial cell concentration of 2.5 g/L. The maximum activity was 14,050 U/L obtained in 24h of expression. However, when the initial cell concentration of 0.25 g/L was investigated, the enzymatic activity was reduced by 30 and 75% after the third and fourth cycles, respectively. Finally, it could be concluded that the initial cell concentration influenced the process performance and the interval of cell recycle affected enzymatic production.

Keywords: Xylanase A; Pichia pastoris ; cell reutilization; heterologous expression

Evaluation of solid and submerged fermentations for the production of cyclodextrin glycosyltransferase by Paenibacillus campinasensis H69-3 and characterization of crude enzyme by Heloiza Ferreira Alves-Prado; Eleni Gomes; Roberto Da Silva (pp. 234-246).

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Cyclodextrins have been shown to have a number of applications in the food, cosmetic, pharmaceutical, and chemical industries. In the current study, the production of CGTase by Paenibacillus campinasensis strain H69-3 was examined in submerged and solid-state fermentations. P. campinasensis strain H69-3 was isolated from the soil, which grows at 45°C, and is a Gramvariable bacterium. Different substrate sources such as wheat bran, soybean bran, soybean extract, cassava solid residue, cassava starch, corn starch, and other combinations were used in the enzyme production. CGTase activity was highest in submerged fermentations with the greatest production observed at 48–72 h. The physical and chemical properties of CGTase were determined from the crude enzyme produced from submerged fermentations. The optimum temperature was found to be 70–75°C, and the activity was stable at 55°C for 1 h. The enzyme displayed two optimum pH values, 5.5 and 9.0 and was found to be stable between a pH of 4.5 and 11.0.

Keywords: Cyclodextrin glycosyltransferase; Paenibacillus campinasensis ; submerged fermentation; solid-state fermentation

Effect of β-cyclodextrin in artificial chaperones assisted foam fractionation of cellulase by Vorakan Burapatana; Aleš Prokop; Robert D. Tanner (pp. 247-255).

Foam fractionation has the potential to be a low-cost protein separation process; however, it may cause protein denaturation during the foaming process. In previous work with cellulase, artificial chaperones were integrated into the foam fractionation process in order to reduce the loss of enzymatic activity. In this study, other factors were introduced to further reduce the loss of cellulase activity: type of cyclodextrin, cyclodextrin concentration, dilution ratio cyclodextrin to the foamate and holding time. α-Cyclodextrin was almost as effective as β-cyclodextrin in refolding the foamed cellulase-Cetyltrimethylammonium bromide mixture. β-Cyclodextrin (6.5 mM) was almost as effective as 13 mM β-cyclodextrin in refolding. The dilution ratio, seven parts foamate and three parts β-cyclodextrin solution, was found to be most effective among the three ratios tested (7∶3, 1∶1, and 3∶7). The activity after refolding at this dilution ratio is around 0.14 unit/mL The refolding time study showed that the refolding process was found to be most effective for the short refolding times (within 1 h).

Keywords: β-cyclodextrin; artificial chaperones; foam fractionation; cellulase; protein denaturation; protein refolding

RSM analysis of the effects of the oxygen transfer coefficient and inoculum size on the xylitol production by Candida guilliermondii by Mariana Peñuela Vásquez; Maurício Bezerra; De Souza Jr.; Nei Pereira Jr. (pp. 256-264).

Biotechnology production of xylitol is an excellent alternative to the industrial chemical process for the production of this polyalcohol. In this work the behavior of Candida guilliermondii yeast was studied when crucial process variables were modified. The K _La (between 18 and 40/h) and the initial cell mass (between 4 and 10 g) were considered as control variables. A response surface methodology was applied to the experimental design to study the resulting effect when the control variables were modified. A regression model was developed and used to determine an optimal value that was further validated experimentally. The optimal values determined for K _La and X ₀ were 32.85/h and 9.86 g, respectively, leading to maximum values for productivity (1.628 g/h) and xylitol yield (0.708 g/g).

Keywords: Xylitol; response surface methodology analysis; xylose

Enzymatic synthesis of sorbitan methacrylate according to acyl donors by Gwi-Taek Jeong; Hye-Jin Lee; Hae-Sung Kim; Don-Hee Park (pp. 265-277).

Recently, sugar polymers have been considered for use as biomaterials in medical applications. These biomaterials are already used extensively in burn dressings, artificial membranes, and contact lenses. In this study, we investigated the optimum conditions under which the enzymatic synthesis of sorbitan methacrylate can be affected using Novozym 435 in t-butanol from sorbitan and several acyl donors (ethyl methacrylate, methyl methacrylate, and vinyl methacrylate). The enzymatic synthesis of sorbitan methacrylate, catalyzed by Novozym 435 in t-butanol, reached an approx 68% conversion yield at 50 g/L of 1,4-sorbitan, 5% (w/v) of enzyme content, and a 1∶5 molar ratio of sorbitan to ethyl methacrylate, with a reaction time of 36 h. Using methyl methacrylate as the acyl donor, we achieved a conversion yield of approx 78% at 50 g/L of 1,4-sorbitan, 7% (w/v) of enzyme content, at a 1∶5 molar ratio, with a reaction time of 36 h. Sorbitan methacrylate synthesis using vinyl methacrylate as the acyl donor was expected to result in a superior conversion yield at 3% (w/v) of enzyme content, and at a molar ratio greater than 1∶2.5. Higher molar ratios of acyl donor resulted in more rapid conversion rates. Vinyl methacrylate can be applied to obtain higher yields than are realized when using ethyl methacrylate or methyl methacrylate as acyl donors in esterification reactions catalyzed by Novozym 435 in organic solvents. Enzyme recycling resulted in a drastic reduction in conversion yields.

Keywords: Immobilized enzyme; bioconversion; optimization; biocatalysis; sorbitan; esterification

Effect of inhibitors released during steam-explosion pretreatment of barley straw on enzymatic hydrolysis by Ma Prado García-Aparicio; Ignacio Ballesteros; Alberto González; José Miguel Oliva; Mercedes Ballesteros; Ma José Negro (pp. 278-288).

The influence of the liquid fraction (prehydrolysate) generated during steam-explosion pretreatment (210°C, 15 min) of barley straw on the enzymatic hydrolysis was determined. Prehydrolysate was analyzed for degradation compounds and sugars' content and used as a medium for enzymatic hydrolysis tests after pH adjusting to 4.8. Our results show that the presence of the compounds contained in the prehydrolysate strongly affects the hydrolysis step (a 25% decrease in cellulose conversion compared with control). Sugars are shown to be more potent inhibitors of enzymatic hydrolysis than degradation products.

Keywords: Enzymatic hydrolysis; barley straw; inhibition; steam explosion

Purification and characterization of two xylanases from alkalophilic and thermophilic Bacillus licheniformis 77-2 by Valquiria B. Damiano; Richard Ward; Eleni Gomes; Heloiza Ferreira Alves-Prado; Roberto Da Silva (pp. 289-302).

The alkalophilic bacteria Bacillus licheniformis 77-2 produces significant quantities of thermostable cellulase-free xylanases. The crude xylanase was purified to apparent homogeneity by gel filtration (G-75) and ionic exchange chromatography (carboxymethyl sephadex, Q sepharose, and Mono Q), resulting in the isolation of two xylanases. The molecular masses of the enzymes were estimated to be 17 kDa (X-I) and 40 kDa (X-II), as determined by SDS-PAGE. The K _m and V _max values were 1.8 mg/mL and 7.05 U/mg protein (X-I), and 1.05 mg/mL and 9.1 U/mg protein (X-II). The xylanases demonstrated optimum activity at pH 7.0 and 8.0–10.0 for xylanase X-I and X-II, respectively, and, retained more than 75% of hydrolytic activity up to pH 11.0. The purified enzymes were most active at 70 and 75°C for X-I and X-II, respectively, and, retained more than 90% of hydrolytic activity after 1 h of heating at 50°C and 60°C for X-I and X-II, respectively. The predominant products of xylan hydrolysates indicated that these enzymes were endoxylanases.

Keywords: Xylanase; Bacillus licheniformis ; xylanase purification; alkalophilic bacteria; xylanase characterization

Oxidation capacity of laccases and peroxidases as reflected in experiments with methoxy-substituted benzyl alcohols by Feng Hong; Leif J. Jönsson; Knut Lundquist; Yijun Wei (pp. 303-319).

A set of methoxy-substituted benzyl alcohol (MBA) congeners were examined regarding susceptibility to oxidation by Trametes versicolor laccase, T. versicolor lignin peroxidase and horseradish peroxidase: 2,4,5-trimethoxybenzyl alcohol (DMBA), 3,4,5-TMBA, 2,3,4-TMBA, 2,5-dimethoxybenzyl alcohol (DMBA), 3,4-DMBA, and 2,3-DMBA. The corresponding methoxysubstituted benzaldehydes were strongly predominant as products on enzymic oxidation. This together with different reaction rates and redox potentials makes the MBAs suitable as substrates in the characterization of ligninolytic enzymes. For fungal laccase, the reaction rate order was: 2,4,5-TMBA≫2,5-DMBA>3,4-DMBA>3,4,5-TMBA∼2,3,4-TMBA∼2,3-DMBA. Horseradish peroxidase displayed a similar reactivity order. Oxidation of some of the MBAs with laccase and horseradish peroxidase was only observed when the reactions were carried out at low pH and with relatively high-substrate concentration. 3,4-DMBA (veratryl alcohol) was the best substrate for lignin peroxidase and the reaction rate order was: 3,4-DMBA>2,4,5-TMBA∼3,4,5-TMBA>2,5-DMBA>2,3,4-TMBA∼2,3-DMBA. The oxidation experiments with different MBAs elucidate the potential of the enzymes as oxidants in various applications.

Keywords: Lignin peroxidase; horseradish peroxidase; laccase; methoxy-substituted benzyl alcohols

Obtainment of chelating agents through the enzymatic oxidation of lignins by phenol oxidase by Gabriela M. M. Calabria; Adilson R. Gonçalves (pp. 320-325).

Oxidation of lignin obtained from acetosolv and ethanol/water pulping of sugarcane bagasse was performed by phenol oxidases: tyrosinase (TYR) and laccase (LAC), to increase the number of carbonyl and hydroxyl groups in lignin, and to improve its chelating capacity. The chelating properties of the original and oxidized lignins were compared by monitoring the amount of Cu²⁺ bound to lignin by gel permeation chromatography. The Acetosolv lignin oxidized with TYR was 16.8% and with LAC 21% higher than that of the original lignin. For ethanol/water lignin oxidized with TYR was 17.2% and with LAC 18% higher than that of the original lignin.

Keywords: Lignin; enzymatic oxidation; polyphenoloxidase; chelating agents

Reuse of the xylanase enzyme in the biobleaching process of the sugarcane bagasse acetosolv pulp by Luís R. M. Oliveira; Regina Y. Moriya; Adilson R. Gonçalves (pp. 326-333).

In this work, pretreatment-enzymatic series of the bagasse-sugarcane pulp and alkaline extraction of enzyme treated pulp were carried out. In the pretreatment an enzyme dose was utilized and acetosolv pulp suspension of 3% (w/v) with different solvents (distilled water, 0.05 mol/L acetate buffer pH 5.5 and 0.05 mol/L phosphate buffer pH 7.25) stirred at 85 rpm for 2 or 4 h. The enzymes used were pulpzyme and cartazyme, both commercial. The accompaniment of the enzymatic activity was carried out through measurement in initial and finish of each enzymatic pretreatment. The xylanase-treated pulps and xylanase-alkaline-extracted pulps were analyzed regarding kappa number and viscosity. Pulpzyme recovery was better in phosphate buffered medium (84, 46, and 23% for first, second, and third enzymatic treatment, respectively) although in aqueous medium reached only 2% for every treatments. However, the improvement of pulp properties was evidenced only in aqueous medium for pulpzyme. Cartazyme recovery was similar for both solvents (water and acetate buffer), reaching values around 19% for first enzymatic treatment and 9% for second one. Nevertheless, the pulp properties increased only in acetate buffered medium.

Keywords: Enzymatic pretreatment; xylanase; acetosolv pulping; sugarcane bagasse

Detection of nisin expression by Lactococcus lactis using two susceptible bacteria to associate the effects of nisin with EDTA by Thereza Christina Vessoni Penna; Angela Faustino Jozala; Thomas Rodolfo Gentille; Adalberto Pessoa Jr.; Olivia Cholewa (pp. 334-346).

Nisin, a bacteriocin produced during the exponential growth phase of Lactococcus lactis ATCC 11454, inhibits the growth of a broad range of Grampositive bacteria. Gram-negative bacteria can also be inhibited by nisin with EDTA. In this study, nisin production was assayed by the agar diffusion method using Lactobacillus sake ATCC 15521 and a recombinant Escherichia coli DH5-α expressing the recombinant green fluorescent protein as the nisin-susceptible test organisms. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/mL of medium) and converted to standard nisin concentration (Nisaplin^®, 25 mg of pure nisin with an activity of 1×10⁶ AU/mL). The expression and release of nisin by L. lactis in skimmed milk (9.09% total solids) with Man Rugosa Shepeer-Bacto Lactobacilli broth (1∶1) was monitored in a 5 L New Brunswick fermentor. Combining EDTA with nisin increased the bactericidal effect of nisin on the bacteria examined. The presence of EDTA was necessary to inhibit E. coli growth with nisin. L. sake was shown to be a good indicator for the evaluation of nisin release in the culture media, including with the addition of EDTA.

Keywords: Nisin; Lactococcus lactis ; Lactobacillus sake ; EDTA; Escherichia coli ; recombinant green fluorescent protein

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.129, #1-3)