Applied Biochemistry and Biotechnology (v.146, #1-3)

Introduction to Session 2: Enzyme Catalysis and Engineering by Stephen R. Decker; Sarah Teter (1-2).

Production of Cyclodextrins by CGTase from Bacillus clausii Using Different Starches as Substrates by H. F. Alves-Prado; A. A. J. Carneiro; F. C. Pavezzi; E. Gomes; M. Boscolo; C. M. L. Franco; R. da Silva (3-13).
Cyclodextrins (CDs) are cyclic oligasaccharides composed by d-glucose monomers joined by α-1,4-d glicosidic linkages. The main types of CDs are α-, β- and γ-CDs consisting of cycles of six, seven, and eight glucose monomers, respectively. Their ability to form inclusion complexes is the most important characteristic, allowing their wide industrial application. The physical property of the CD-complexed compound can be altered to improve stability, volatility, solubility, or bio-availability. The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme capable of converting starch into CD molecules. In this work, the CGTase produced by Bacillus clausii strain E16 was used to produce CD from maltodextrin and different starches (commercial soluble starch, corn, cassava, sweet potato, and waxy corn starches) as substrates. It was observed that the substrate sources influence the kind of CD obtained and that this CGTase displays a β-CGTase action, presenting a better conversion of soluble starch at 1.0%, of which 80% was converted in CDs. The ratio of total CD produced was 0:0.89:0.11 for α/β/γ. It was also observed that root and tuber starches were more accessible to CGTase action than seed starch under the studied conditions.
Keywords: CGTase; Cyclodextrin; Bacillus clausii ; Soluble starch; Corn starch; Cassava starch; Sweet potato starch; Waxy corn starch

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability by Fei Jiang; Puapong Kongsaeree; Karl Schilke; Curtis Lajoie; Christine Kelly (15-27).
The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.
Keywords: Manganese peroxidase; Yeast; Pichia ; Biofuels; Proteases

Xylanase Production by Bacillus circulans D1 Using Maltose as Carbon Source by D. A. Bocchini; E. Gomes; R. Da Silva (29-37).
Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbon sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that d-maltose is the best inducer of the enzyme synthesis (7.05 U/mg dry biomass at 48 h), while d-glucose and d-arabinose lead to the production of basal levels of xylanase. The crude enzyme solution is free of cellulases, even when the microorganism was cultivated in a medium with d-cellobiose. When oat spelt xylan was supplemented with d-glucose, the repressive effect of this sugar on xylanase production was observed at 24 h, only when used at 5.0 g/L, leading to a reduction of 60% on the enzyme production. On the other hand, when the xylan medium was supplemented with d-xylose (3.0 or 5.0 g/L), this effect was more evident (80 and 90% of reduction on the enzyme production, respectively). Unlike that observed in the xylan medium, glucose repressed xylanase production in the maltose medium, leading to a reduction of 55% on the enzyme production at 24 h of cultivation. Xylose, at 1.0 g/L, induced xylanase production on the maltose medium. On this medium, the repressive effect of xylose, at 3.0 or 5.0 g/L, was less expressive when compared to its effect on the xylan medium.
Keywords: Xylanase; Maltose; Induction; Repression; Bacillus circulans

Immobilization of Fungal β-Glucosidase on Silica Gel and Kaolin Carriers by Hakob K. Karagulyan; Vardan K. Gasparyan; Stephen R. Decker (39-47).
β-Glucosidase is a key enzyme in the hydrolysis of cellulose for producing feedstock glucose for various industrial processes. Reuse of enzyme through immobilization can significantly improve the economic characteristics of the process. Immobilization of the fungal β-glucosidase by covalent binding and physical adsorption on silica gel and kaolin was conducted for consequent application of these procedures in large-scale industrial processes. Different immobilization parameters (incubation time, ionic strength, pH, enzyme/support ratio, glutaric aldehyde concentration, etc.) were evaluated for their effect on the thermal stability of the immobilized enzyme. It was shown that the immobilized enzyme activity is stable at 50 °C over 8 days. It has also been shown that in the case of immobilization on kaolin, approximately 95% of the initial enzyme was immobilized onto support, and loss of activity was not observed. However, covalent binding of the enzyme to silica gel brings significant loss of enzyme activity, and only 35% of activity was preserved. In the case of physical adsorption on kaolin, gradual desorption of enzyme takes place. To prevent this process, we have carried out chemical modification of the protein. As a result, after repeated washings, enzyme desorption from kaolin has been reduced from 75 to 20–25% loss.
Keywords: β-glucosidase; Immobilization; Silica gel; Kaolin; Thermostability; Corn stover; Enzymatic hydrolysis

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques by Aline G. Cunha; Gloria Fernández-Lorente; Juliana V. Bevilaqua; Jacqueline Destain; Lúcia M. C. Paiva; Denise M. G. Freire; Roberto Fernández-Lafuente; Jose M. Guisán (49-56).
Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.
Keywords: Immobilized lipase; Y. lipolytica ; Biocatalysis; Glutaraldehyde; Hydrophobic supports

Heterologous Expression of Aspergillus niger β-d-Xylosidase (XlnD): Characterization on Lignocellulosic Substrates by Michael J. Selig; Eric P. Knoshaug; Stephen R. Decker; John O. Baker; Michael E. Himmel; William S. Adney (57-68).
The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T max was determined using differential scanning microcalorimetry (DSC) to be 78.2 °C; the K m and k cat were found to be 255 μM and 13.7 s−1, respectively, using pNP-β-d-xylopyranoside as substrate. End-product inhibition by d-xylose was also verified and shown to be competitive; the K i for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.
Keywords: Xylosidase; Xylanase; Xylo-oligomer; Pretreatment; Lignocellulose; Cellobiohydrolase

Cloning, Expression and Characterization of a Glycoside Hydrolase Family 39 Xylosidase from Bacillus Halodurans C-125 by Kurt Wagschal; Diana Franqui-Espiet; Charles C. Lee; George H. Robertson; Dominic W. S. Wong (69-78).
The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag, and the recombinant gene product termed BH1068(His)6 was expressed in Escherichia coli. Of the artificial substrates tested, BH1068(His)6 hydrolyzed nitrophenyl derivatives of β-d-xylopyranose, α-l-arabinofuranose, and α-l-arabinopyranose. Deviation from Michaelis−Menten kinetics at higher substrate concentrations indicative of transglycosylation was observed, and k cat and K m values were measured at both low and high substrate concentrations to illuminate the relative propensities to proceed along this alternate reaction pathway. The pH maximum was 6.5, and under the conditions tested, maximal activity was at 47°C, and thermal instability occurred above 45°C. BH1068(His)6 was inactive on arabinan, hydrolyzed xylooligosaccharides, and released only xylose from oat, wheat, rye, beech, and birch arabinoxylan, and thus, can be classified as a xylosidase with respect to natural substrate specificity. The enzyme was not inhibited by up to 200 mM xylose. The oligomerization state was tetrameric under the size-exclusion chromatography conditions employed.
Keywords: Alkaliphile; Xylosidase; Glycoside hydrolase family 39; Hemicellulose degradation

Heterologous Expression of Two Ferulic Acid Esterases from Penicillium funiculosum by Eric P. Knoshaug; Michael J. Selig; John O. Baker; Stephen R. Decker; Michael E. Himmel; William S. Adney (79-87).
Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.
Keywords: Ferulic acid esterase; Hemicellulose; Cellulose; Biomass digestion; Heterologous expression

Evaluation of a Hypocrea jecorina Enzyme Preparation for Hydrolysis of Tifton 85 Bermudagrass by E. A. Ximenes; S. K. Brandon; J. Doran-Peterson (89-100).
Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.
Keywords: Bermudagrass; Trichoderma ; Hypocrea ; Cellulases; Hemicellulases

A Novel Technique that Enables Efficient Conduct of Simultaneous Isomerization and Fermentation (SIF) of Xylose by Kripa Rao; Silpa Chelikani; Patricia Relue; Sasidhar Varanasi (101-117).
Of the sugars recovered from lignocellulose, d-glucose can be readily converted into ethanol by baker’s or brewer’s yeast (Saccharomyces cerevisiae). However, xylose that is obtained by the hydrolysis of the hemicellulosic portion is not fermentable by the same species of yeasts. Xylose fermentation by native yeasts can be achieved via isomerization of xylose to its ketose isomer, xylulose. Isomerization with exogenous xylose isomerase (XI) occurs optimally at a pH of 7–8, whereas subsequent fermentation of xylulose to ethanol occurs at a pH of 4–5. We present a novel scheme for efficient isomerization of xylose to xylulose at conditions suitable for the fermentation by using an immobilized enzyme system capable of sustaining two different pH microenvironments in a single vessel. The proof-of-concept of the two-enzyme pellet is presented, showing conversion of xylose to xylulose even when the immobilized enzyme pellets are suspended in a bulk solution whose pH is sub-optimal for XI activity. The co-immobilized enzyme pellets may prove extremely valuable in effectively conducting “simultaneous isomerization and fermentation” (SIF) of xylose. To help further shift the equilibrium in favor of xylulose formation, sodium tetraborate (borax) was added to the isomerization solution. Binding of tetrahydroxyborate ions to xylulose effectively reduces the concentration of xylulose and leads to increased xylose isomerization. The formation of tetrahydroxyborate ions and the enhancement in xylulose production resulting from the complexation was studied at two different bulk pH values. The addition of 0.05 M borax to the isomerization solution containing our co-immobilized enzyme pellets resulted in xylose to xylulose conversion as high as 86% under pH conditions that are suboptimal for XI activity. These initial findings, which can be optimized for industrial conditions, have significant potential for increasing the yield of ethanol from xylose in an SIF approach.
Keywords: Xylose; Xylulose; Urea; Urease; Borate; Simultaneous isomerization and fermentation; Ethanol

The effects of the starch, protein, and soluble oligosaccharides contents in wheat bran on the extracellular biomass-hydrolyzing enzymes activities released by Penicillium decumbens mycelia grown in batch fermentations have been examined. The results showed increased starch content correlated directly with an increase in released amylase activity but inversely with the levels of secreted cellulase and xylanase. High amounts of protein in wheat bran also reduced the activities of cellulase, xylanase and protease in the culture medium. The effects of the soluble and insoluble components of wheat bran and cello-oligosaccharides supplements on production of extracellular cellulase and xylanase were compared. The soluble cello-oligosaccharides compositions in wheat bran were proved to be one of the most significant factors for cellulase production. According to the results of this research, determining and regulating the composition of wheat bran used as a fermentation supplement may allow for improved induction of cellulase and xylanase production.
Keywords: Penicillium decumbens ; Wheat bran; Biomass; Cellulase; Xylanase

Integrated Biosensor Systems for Ethanol Analysis by Eliana M. Alhadeff; Andrea M. Salgado; Oriol Cós; Nei Pereira Jr; Francisco Valero; Belkis Valdman (129-136).
Different integrated systems with a bi-enzymatic biosensor, working with two different methods for ethanol detection—flow injection analysis (FIA) or sequential injection analysis (SIA)—were developed and applied for ethanol extracted from gasohol mixtures, as well as for samples of alcoholic beverages and fermentation medium. A detection range of 0.05–1.5 g ethanol/l, with a correlation coefficient of 0.9909, has been reached when using FIA system, working with only one microreactor packed with immobilized alcohol oxidase and injecting free horseradish peroxidase. When using both enzymes, immobilized separately in two microreactors, the detection ranges obtained varied from 0.001 to 0.066 g ethanol/l, without on-line dilution to 0.010–0.047 g ethanol/l when a 1:7,000 dilution ratio was employed, reaching correlation coefficients of 0.9897 and 0.9992, respectively. For the integrated biosensor SIA system with the stop–flow technique, the linear range was 0.005–0.04 g/l, with a correlation coefficient of 0.9922.
Keywords: Flow injection analysis; Sequential injection analysis; Ethanol; Alcohol oxidase; Horseradish peroxidase; Biosensor

Catalytically efficient β-d-xylosidase from Selenomonas ruminantium (SXA) exhibits pK as 5 and 7 (assigned to catalytic base, D14, and catalytic acid, E186) for k cat/K m with substrates 1,4-β-d-xylobiose (X2) and 1,4-β-d-xylotriose (X3). Catalytically inactive, dianionic SXA (D14E186) has threefold lower affinity than catalytically active, monoanionic SXA (D14E186H) for X2 and X3, whereas D14E186 has twofold higher affinity than D14E186H for 4-nitrophenyl-β-d-xylopyranoside (4NPX), and D14E186 has no affinity for 4-nitrophenyl-α-l-arabinofuranoside. Anomeric isomers, α-d-xylose and β-d-xylose, have similar affinity for SXA. 4-Nitrophenol competitively inhibits SXA-catalyzed hydrolysis of 4NPX. SXA steady-state kinetic parameters account for complete progress curves of SXA-catalyzed hydrolysis reactions.
Keywords: Fuel ethanol; Glycoside hydrolase; GH43; Hemicellulose; pH dependence; Stereochemistry; Inhibitor; Assay method

Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH3: 1 biomass, 100 °C, 30 min) or with NH4OH (0.5 g NH4OH of a 28% [v/v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.
Keywords: Ammonia pretreatment; Sugar cane bagasse; Hydrolysis; Cellulase; Hemicellulase

Monoglycerides and Diglycerides Synthesis in a Solvent-Free System by Lipase-Catalyzed Glycerolysis by Patricia Bogalhos Lucente Fregolente; Leonardo Vasconcelos Fregolente; Gláucia Maria F. Pinto; Benedito César Batistella; Maria Regina Wolf-Maciel; Rubens Maciel Filho (165-172).
Five lipases were screened (Thermomyces lanuginosus free and immobilized forms, Candida antarctica B, Candida rugosa, Aspergillus niger, and Rhizomucor miehei) to study their ability to produce monoglycerides (MG) and diglycerides (DG) through enzymatic glycerolysis of soybean oil. Lipase from C. antarctica was further studied to verify the enzyme load (wt% of oil mass), the molar ratio glycerol/oil, and the water content (wt% of glycerol) on the glycerolysis reaction. The best DG and MG productions were in the range 45–48% and 28–30% (w/w, based on the total oil), respectively. Using immobilized lipases, the amount of free fatty acids (FFA) produced was about 5%. However, the amount of FFA produced when using free lipases, with 3.5% extra water in the system, is equivalent to the MG yield, about 23%. The extra water content provides a competition between hydrolysis and glycerolysis reactions, increasing the FFA production.
Keywords: Monoglycerides; Diglycerides; Lipase-catalyzed glycerolysis; Soybean oil

Immobilization of Candida antarctica Lipase B by Adsorption to Green Coconut Fiber by Ana I. S. Brígida; Álvaro D. T. Pinheiro; Andrea L. O. Ferreira; Luciana R. B. Gonçalves (173-187).
An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t 1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction.
Keywords: Candida antarctica lipase B; Enzyme immobilization; Coconut fiber; Physical adsorption; Methyl butyrate hydrolysis; Butyl butyrate synthesis

Methods and Supports for Immobilization and Stabilization of Cyclomaltodextrin Glucanotransferase from Thermoanaerobacter by Ana Elisa Amud; Gercio Rodrigo Presa da Silva; Paulo Waldir Tardioli; Cleide Mara Faria Soares; Flavio Faria Moraes; Gisella Maria Zanin (189-201).
Thermoanaerobacter cyclomaltodextrin glucanotransferase (CGTase) was immobilized using different supports and immobilization methods to study the effect on activity recovery. The enzyme covalently attached into glyoxyl-silica showed low activity recovery of 1.5%. The hydrophobic adsorption of the enzyme on Octadecyl-Sepabeads yielded also low activity recovery, 3.83%, and the enzyme could easily leak from the support at low ionic strength, although the immobilization yield was satisfactory, approximately 76%. The CGTase encapsulated in a sol–gel matrix gave an activity recovery of 6.94% and maximum cyclization activity at 60 °C, at pH 6.0. The half-time life at 60 °C, pH 6.0, in the presence of substrate was 100 min, which was lower than that of the free enzyme. The best activity recovery in this work (6.94%) is approximately five times smaller than that obtained previously using glyoxyl-agarose as support and covalent immobilization. Thus, the best support and method we tested so far for immobilization of CGTase is covalent attachment on glyoxyl-agarose.
Keywords: CGTase; Toruzyme®; Glyoxyl-silica; Octadecyl-sepabeads; Sol–gel encapsulation; Multipoint attachment; Hydrophobic adsorption

Response Surface Methodology as an Approach to Determine Optimal Activities of Lipase Entrapped in Sol–Gel Matrix Using Different Vegetable Oils by Rubiane C. Pinheiro; Cleide M. F. Soares; Heizir F. de Castro; Flavio F. Moraes; Gisella M. Zanin (203-214).
The conditions for maximization of the enzymatic activity of lipase entrapped in sol–gel matrix were determined for different vegetable oils using an experimental design. The effects of pH, temperature, and biocatalyst loading on lipase activity were verified using a central composite experimental design leading to a set of 13 assays and the surface response analysis. For canola oil and entrapped lipase, statistical analyses showed significant effects for pH and temperature and also the interactions between pH and temperature and temperature and biocatalyst loading. For the olive oil and entrapped lipase, it was verified that the pH was the only variable statistically significant. This study demonstrated that response surface analysis is a methodology appropriate for the maximization of the percentage of hydrolysis, as a function of pH, temperature, and lipase loading.
Keywords: Lipase; Canola oil; Soybean oil; Olive oil; Sol–gel encapsulation

Improving Activity of Salt-Lyophilized Enzymes in Organic Media by Abhijeet P. Borole; Brian H. Davison (215-222).
Lyophilization with salts has been identified as an important method of activating enzymes in organic media. Using salt-activated enzymes to transform molecules tethered to solid surfaces in organic phase requires solubilization of enzymes in the solvents. Methods of improving performance of salt-lyophilized enzymes, further, via chemical modification, and use of surfactants and surfactants to create fine emulsions prior to lyophilization are investigated. The reaction system used is transesterification of N-acetyl phenylalanine ethyl ester with methanol or propanol. Initial rate of formation of amino acid esters by subtilisin Carlsberg (SC) was studied and found to increase two to sevenfold by either chemical modification or addition of surfactants in certain solvents, relative to the salt (only)-lyophilized enzyme. The method to prepare highly dispersed enzymes in a salt-surfactant milieu also improved activity by two to threefold. To test the effect of chemical modification on derivatization of drug molecules, acylation of bergenin was investigated using chemically modified SC.
Keywords: Subtilisin Carlsberg; Salt lyophilization; Nonaqueous media; Drug derivatization; Bergenin; Acylation; Surfactant; PEG

Protease Production by Different Thermophilic Fungi by Mariana M. Macchione; Carolina W. Merheb; Eleni Gomes; Roberto da Silva (223-230).
A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi – Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 – using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.
Keywords: Protease; Thermophilic fungi; Solid state fermentation; Submerged fermentation; Wheat bran

Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass by Yi Zheng; Zhongli Pan; Ruihong Zhang; Donghai Wang; Bryan Jenkins (231-248).
Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.
Keywords: Saline Creeping Wild Ryegrass; Cellulase; β-Glucosidase; Tween 20; Tween 80; Bovine serum albumin; Enzymatic hydrolysis; Avicel PH101; Lignaceous residue