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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.155, #1-3)
Purification and Characterization of a Glycoside Hydrolase Family 43 β-xylosidase from Geobacillus thermoleovorans IT-08 by Kurt Wagschal; Chamroeun Heng; Charles C. Lee; George H. Robertson; William J. Orts; Dominic W. S. Wong (pp. 1-10).
The gene encoding a glycoside hydrolase family 43 β-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis–Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-β-d-xylopyranose (4NPX) and p-nitrophenyl-α-l-arabinofuranose (4NPA), and it was found that the ratio k cat/K m 4NPA/k cat/K m 4NPX was ∼7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k cat/K m muA/k cat/K m muX was ∼5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K i of 6.8 ± 0.62 mM and xylose K i of 76 ± 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 °C, with a t 1/2 of 35 min at 57.5 °C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.
Keywords: Arabinofuranosidase; Xylosidase; Glycoside hydrolase family 43; Hemicellulose degradation; Substrate inhibition
An α-Glucuronidase Enzyme Activity Assay Adaptable for Solid Phase Screening by Charles C. Lee; Kurt Wagschal; Rena E. Kibblewhite-Accinelli; William J. Orts; George H. Robertson; Dominic W. S. Wong (pp. 11-17).
Glucuronic acid is a common chemical moiety that decorates the xylan polymer of hemicellulose. This chemical substituent impairs both enzymatic and acidic hydrolysis of xylosidic bonds. The α-glucuronidase enzyme hydrolyzes the 1,2-linked glucuronic acid from the terminal, non-reducing xylose of xylo-oligosaccharides. There are relatively few α-glucuronidase genes in the public databases. We have developed an assay with commercially available reagents that can be used to search DNA libraries for α-glucuronidase genes in a high-throughput, solid phase activity screen.
Keywords: Glucuronidase; Glucuronic acid; Hemicellulase; Activity assay
Aspergillus fumigatus Thermophilic and Acidophilic Endoglucanases by A. L. Grigorevski-Lima; F. N. M. Da Vinha; D. T. Souza; A. S. R. Bispo; E. P. S. Bon; R. R. R. Coelho; R. P. Nascimento (pp. 18-26).
This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer’s spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 °C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L-1 and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 °C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g-1 of dry substrate (wheat bran and sugarcane bagasse) within 3 days.
Keywords: Aspergillus fumigatus ; Thermophilic endoglucanase; Acidophilic endoglucanase; CMCase; Agro-industrial by-products
β-d-Xylosidase from Selenomonas ruminantium: Thermodynamics of Enzyme-Catalyzed and Noncatalyzed Reactions by Douglas B. Jordan; Jay D. Braker (pp. 27-43).
β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and 1,4-β-d-xylobiose (X2) was determined on and off (k non) the enzyme at pH 5.3, which lies in the pH-independent region for k cat and k non. Rate enhancements (k cat/k non) for 4NPX, 4NPA, and X2 are 4.3 × 1011, 2.4 × 109, and 3.7 × 1012, respectively, at 25 °C and increase with decreasing temperature. Relative parameters k cat 4NPX/k cat 4NPA, k cat 4NPX/k cat X2, and (k cat/K m)4NPX/(k cat/K m)X2 increase and (k cat/K m)4NPX/(k cat/K m)4NPA, (1/K m)4NPX/(1/K m)4NPA, and (1/K m)4NPX/(1/K m)X2 decrease with increasing temperature.
Keywords: Fuel ethanol; Glycoside hydrolase; GH43; k non ; pH dependence; Temperature dependence; Activation energy
Enzymatic Synthesis of Biodiesel via Alcoholysis of Palm Oil by André L. F. Matassoli; Igor N. S. Corrêa; Márcio F. Portilho; Cláudia O. Veloso; Marta A. P. Langone (pp. 44-52).
The enzymatic alcoholysis of crude palm oil with methanol and ethanol was investigated using commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM). The effect of alcohol (methanol or ethanol), molar ratio of alcohol to crude palm oil, and temperature on biodiesel production was determined. The best ethyl ester yield was about 25 wt.% and was obtained with ethanol/oil molar ratio of 3.0, temperature of 50 °C, enzyme concentration of 3.0 wt.%, and stepwise addition of the alcohol after 4 h of reaction. Experiments with 1 and 3 wt.% of KOH and 3 wt.% of MgO were carried out to compare their catalytic behavior with the enzymatic transesterification results. The commercial immobilized lipase, Lipozyme TL IM, showed the best catalytic performance.
Keywords: Enzyme; Ethanol; Crude palm oil; Immobilized lipase; Biodiesel
Banana as Adjunct in Beer Production: Applicability and Performance of Fermentative Parameters by Giovani B. M. Carvalho; Daniel P. Silva; Camila V. Bento; António A. Vicente; José A. Teixeira; Maria das Graças A. Felipe; João B. Almeida e Silva (pp. 53-62).
Traditionally, the raw materials for beer production are barley, hops, water, and yeast, but most brewers use also different adjuncts. During the alcoholic fermentation, the contribution of aroma compounds from other ingredients to the final beer flavor depends on the wort composition, on the yeast strain, and mainly on the process conditions. In this context, banana can also be a raw material favorable to alcoholic fermentation being rich in carbohydrates and minerals and providing low acidity. In this work, the objective was to evaluate the performance of wort adjusted with banana juice in different concentrations. For this, static fermentations were conducted at 15 °C at pilot scale (140 L of medium). The addition of banana that changed the concentration of all-malt wort from 10 °P to 12 and 15 °P were evaluated (°P is the weight of the extract or the sugar equivalent in 100 g solution, at 20 °C). The results showed an increase in ethanol production, with approximately 0.4 g/g ethanol yield and 0.6 g/L h volumetric productivity after 84 h of processing when concentrated wort was used. Thus, it was concluded that banana can be used as an adjunct in brewing methods, helping in the development of new products as well as in obtaining concentrated worts.
Keywords: Beer; Raw materials; Adjunct; Banana; Brewing; Fermentation
Evaluation of Cashew Apple Juice for Surfactin Production by Bacillus subtilis LAMI008 by Maria Valderez Ponte Rocha; Raphaela V. Gomes Barreto; Vânia Maria M. Melo; Luciana Rocha Barros Gonçalves (pp. 63-75).
Bacillus subtilis LAMI008 strain isolated from the tank of Chlorination at the Wastewater Treatment Plant on Campus do Pici in Federal University of Ceará, Brazil has been screened for surfactin production in mineral medium containing clarified cashew apple juice (MM-CAJC). Results were compared with the ones obtained using mineral medium with glucose PA as carbon source. The influence on growth and surfactin production of culture medium supplementation with yeast extract was also studied. The substrate concentration analysis indicated that B. subtilis LAMI008 was able to degrade all carbon sources studied and produce biosurfactant. The highest reduction in surface tension was achieved with the fermentation of MM-CAJC, supplemented with yeast extract, which decreased from 58.95 ± 0.10 to 38.10 ± 0.81 dyn cm−1. The biosurfactant produced was capable of emulsifying kerosene, achieving an emulsification index of 65%. Surfactin concentration of 3.5 mg L−1 was obtained when MM-CAJC, supplemented with yeast extract, was used, thus indicating that it is feasible to produce surfactin from clarified cashew apple juice, a renewable and low-cost carbon source.
Keywords: Biosurfactants; Raw material; Cashew apple juice; B. subtilis LAMI008; Surfactin; Fermentation
Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts by Mark S. Ou; Nazimuddin Mohammed; L. O. Ingram; K. T. Shanmugam (pp. 76-82).
Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g−1 cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g−1 cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.
Keywords: Cellulase; Cellulose SSF; Bacillus coagulans ; Saccharomyces ; Zymomonas ; Lactic acid bacteria
Pretreatment of Reed by Wet Oxidation and Subsequent Utilization of the Pretreated Fibers for Ethanol Production by Nóra Szijártó; Zsófia Kádár; Enikő Varga; Anne Belinda Thomsen; Maria Costa-Ferreira; Kati Réczey (pp. 83-93).
Common reed (Phragmites australis) is often recognized as a promising source of renewable energy. However, it is among the least characterized crops from the bioethanol perspective. Although one third of reed dry matter is cellulose, without pretreatment, it resists enzymatic hydrolysis like lignocelluloses usually do. In the present study, wet oxidation was investigated as the pretreatment method to enhance the enzymatic digestibility of reed cellulose to soluble sugars and thus improve the convertibility of reed to ethanol. The most effective treatment increased the digestibility of reed cellulose by cellulases more than three times compared to the untreated control. During this wet oxidation, 51.7% of the hemicellulose and 58.3% of the lignin were solubilized, whereas 87.1% of the cellulose remained in the solids. After enzymatic hydrolysis of pretreated fibers from the same treatment, the conversion of cellulose to glucose was 82.4%. Simultaneous saccharification and fermentation of pretreated solids resulted in a final ethanol concentration as high as 8.7 g/L, yielding 73% of the theoretical.
Keywords: Lignocellulose; Phragmites australis ; Pretreatment; Wet oxidation; Fermentation; SSF; Ethanol
The Effect of Lignin Removal by Alkaline Peroxide Pretreatment on the Susceptibility of Corn Stover to Purified Cellulolytic and Xylanolytic Enzymes by Michael J. Selig; Todd B. Vinzant; Michael E. Himmel; Stephen R. Decker (pp. 94-103).
Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.
Keywords: Lignin; Pretreatment; Corn stover; Enzyme accessibility; Cellulase; Xylanase
Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production by Maria Valderez Ponte Rocha; Tigressa Helena Soares Rodrigues; Gorete Ribeiro de Macedo; Luciana R. B. Gonçalves (pp. 104-114).
The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82 ± 2 mg/g CAB-H and 730 ± 20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44 ± 2 mg/g CAB-H, and 450 ± 50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0 ± 0.2 g L−1 and 3.33 g L−1 h−1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L−1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L−1), ethanol concentration and productivity were 8.2 ± 0.1 g L−1 and 2.7 g L−1 h−1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.
Keywords: Ethanol; Cashew apple bagasse; Pretreatment diluted acid sulfuric; Enzymatic hydrolysis; Saccharomyces cerevesiae ; Cellulase
High Xylose Yields from Dilute Acid Pretreatment of Corn Stover Under Process-Relevant Conditions by Noah D. Weiss; Nicholas J. Nagle; Melvin P. Tucker; Richard T. Elander (pp. 115-125).
Pretreatment experiments were carried out to demonstrate high xylose yields at high solids loadings in two different batch pretreatment reactors under process-relevant conditions. Corn stover was pretreated with dilute sulfuric acid using a 4-l Steam Digester and a 4-l stirred ZipperClave® reactor. Solids were loaded at 45% dry matter (wt/wt) after sulfuric acid catalyst impregnation using nominal particle sizes of either 6 or 18 mm. Pretreatment was carried out at temperatures between 180 and 200 °C at residence times of either 90 or 105 s. Results demonstrate an ability to achieve high xylose yields (>80%) over a range of pretreatment conditions, with performance showing little dependence on particle size or pretreatment reactor type. The high xylose yields are attributed to effective catalyst impregnation and rapid rates of heat transfer during pretreatment.
Keywords: Pretreatment; Dilute acid; Batch reactors; Particle size; Corn stover; Xylose and severity
Session 9: Advances in Bioprocessing and Related Separations Technology
by Timothy C. Dodge; Brian H. Davison (pp. 126-127).