Applied Biochemistry and Biotechnology (v.76, #2)

Pretreatment and enzymatic saccharification of corn fiber by Badal C. Saha; Rodney J. Bothast (65-77).
Corn fiber consists of about 20% starch, 14% cellulose, and 35% hemicellulose, and has the potential to serve as a low-cost feedstock for production of fuel ethanol. Several pretreatments (hot water, alkali, and dilute, acid) and enzymatic saccharification procedures were evaluated for the conversion of corn fiber starch, cellulose, and hemicellulose to monomeric sugars. Hot water pretreatment (121°C, 1 h) facilitated the enzymatic sacch arification of starch and cellulose but not hemicellulose. Hydrolysis of corn fiber pretreated with alkali un dersimilar conditions by enzymatic means gave similar results. Hemicellulose and starch components were converted to monomeric sugars by dilute H2SO4 pretreatment (0.5–1.0%, v/v) at 121°C. Based on these findings, a method for pretreatment and enzymatic saccharification of corn fiber is presented. It in volves the pretreatment of corn fiber (15% solid, w/v) with dilute acid (0.5% H2SO4, v/v) at 121°C for 1 h, neutralization to pH 5.0, then saccharification of the pretreated corn fiber material with commercial cellulase and β-glucosidase preparations The yield of monomeric sugars from corn fiber was typically 85–100% of the theoretical yield.
Keywords: Corn fiber; pretreatment; enzymatic saccharification; fuel ethanol

The metabolic capabilities of subsurface, jet-fuel-contaminated, aquifer microbial communities were characterized using an ecological approach to biotreatment assessment. A multifaceted experimental design was used that incorporated quantification of metabolite formation and toxicity screening along with the typical microbial activity measurements and pollutant degradation measurements used for assessing biorem ediation potential. Forselect experiments, dissolved oxygen levels and pH in microcosm systems were also monitored. Results suggest that a sizable, metabolically active microbial community exists in both contaminated and uncontaminated areas of the study site. Time course metabolism analyses indicated that the microbial communities were capable of degrading all three test compounds (amino acids, decane, and toluene) without any apparent adaptation period. Measurements of mineralization, cellular uptake, and metabolite formation indicated that metabolite formation was the predominant fate of the target pollutants in the microcosms. The results of toxicity screening time courses indicated that under oxygen-limiting conditions, the potential for the accumulation of toxic, acidic metabolites that could adversely affect the rates and extent of bioremediation existed. The experimental results indicate that the microbial communities at the site possess the metabolic potential for in situ biodegradation of the jet fuel. Care must be taken in the design of an in situ biodegradation treatment system (for this site and perhaps other petroleum-contaminated aquifers) to avoid the development of microaerophilic or oxygen-depleted zones; which could result in possible self-poisoning owing to acidic metabolite accumulation
Keywords: Aquifer; bioremediation; jetfuel; decane; toluene; microcosm

On-line dialysis of organic acids from a Propionibacterium freudenreichii fermentation by José P. L. C. da Costa; Christian Schorm; Adolfo Quesada-Chanto; Karl W. Böddeker; Rainer Jonas (99-105).
The efficiency of an ion exchange system coupled to a bioreactor to extract on-line inhibitory organic acids produced was evaluated. Batch fermentations without dialysis and fed-batch experiments with and without dialysis of Propio nibacterium freudenreichii were conducted. It was possible to keep the propionic acid concentration in the reactor at a low level to avoid complete growth inhibition. Improvements in biomass and propionic acid productivities were achieved when the dialysis system was used. The performance of the dialysis system was improved when a new pH control strategy evaluated in this study was used.
Keywords: Dialysis; propionic acid; Propionibacterium ; membranes; pH control strategy

Met-Lys-human proinsulin could be converted into insulin in vitro with the treatment of trypsin and carboxypeptides B (CPB). Under less effective conditions, the enzymatic reaction does not proceed perfectly, and twomain bands have been identified by native-polyacrylamide gel electrophoresis (PAGE) analysis. These two main products were thus separated and purified by DEAE-Sephadex A25 chromatography in a Tris-isopropanol system with an NaCl gradient. The isopropanol and NaCl were removed by a second DEAE-Sephadex column. Native-PAGE, mass spectrometric, and amino acid composition analyses indicate that one fraction of these two major products contains human insulin and des B30-insulin and that the other fraction is a mixture of human insulin analogs, which have one more basic amino acid than human in sulin owing to the unsuitable amount of proteases, especially the lack of CPB. Furthermore, both receptor binding assay and radioimmunoassay have been utilized for the activity determination, and both fractions display almost full biological activity with porcine insulin as the standard. Present results provide further evidence for the quality control of recombinant human insulin production.
Keywords: Insulin analog; proinsulin; enzymatic conversion; MS analysis; activity

Laccase of Coriolus zonatus by O. V. Koroljova (Skorobogat'ko); E. V. Stepanova; V. P. Gavrilova; V. I. Biniukov; A. I. Jaropolov; S. D. Varfolomeyev; F. Scheller; A. Makower; A. Otto (115-127).
Laccase is one of the lignolytic enzymes found in liquid cultures of the fungus Coriolus zonatus in defined medium. The enzyme was isolated from culture liquid and characterized. Laccase from C. zonatus is a single-chain protein with a molecular mass of 60 kDa. Carbohydrate moiety of enzyme consisted of mannose, galactose and N-acetyl-glucosamine in a ratio of 6:2:0,6 respectively, and comprised 10% of the entiremolecule lsoelectric point was detected at pH 4.6. Laccase was found to have a pH optimum of 4.9 and temperature optimum of 55°C. Substrate specificity studies were conducted with catechol, K-ferrocyanide, hydroquinone, and sinapinic acid as substrates. The highest efficiency of catalysis was observed with sinapic acid as the substrate. The kinetic constants k cat and K28 of this reaction were 624 s−1 and 7 μM, respectively.
Keywords: Laccase; enzyme purification; Coriolus zonatus ; substrate specificity; thermostability; pH stability

Cyclic AMP and low molecular weight effector(s) present in yeast extract are involved in pectin lyase production by Penicillium griseoroseum cultured on sucrose by Maria Cristina Baracat-Pereira; Jorge Luiz Cavalcante Coelho; Rosana Cristina Minussi; Virgínia Maria Chaves-Alves; Rogelio Lopes Brandão; Daison Olzany Silva (129-141).
Pectin lyase (PL) induction by organic and inorganic components of yeast extract (YE) was evaluated in Penicillum griseoroseum, cultured in a mineral medium containing sucrose, by determining PL activity (A 235) and mycelial growth (mycelial dry weight). The lowest YE concentration that promoted significant PL induction without acting as a carbon source for the fungus corresponded to 0.0075%. Neither calcined YE nor a nutrient solution containing micronutrients induced PL production, indicating that the inducer was an organic compound. Vitamins, phospholipid components, amino acids, and nitrogenous bases were tested in place of YE and promoted no significant PL induction. APL inducer compound was found to be soluble in the nucleotide fraction obtained during extraction of YE. The inducer was shown to be a thermostable polar substance dialyzable at 2000 Daltons, hydrolyzable by HCl, and activated by boiling for up to 60 min. Cyclic AMP (cAMP) exogenously added to the culture medium at 5 and 10 mM was capable of inducing PL in P. griseoroseum grown on sucrose, suggesting that at least one compound may be present in YE acting in a cooperative fashion for the maintenance of high levels of cAMP in to the cell. PL activity and the level of cAMP inside the fungal cells increased after the addition of YE to the culture medium, suggesting the participation of this messenger in this enzyme's synthesis.
Keywords: Cyclic AMP; yeast extract; pectin lyase; pectinases; P. griseoroseum ; sucrose

A shear-enhanced, cross-flow ultrafiltration module was used to separate cells and proteins from the fermentation broth. Three (fermented) media were studied: rich medium, rich medium with hydrolytic enzymes added after fermentation, and wheat flour hydrolysate. To find a membrane with as high a flux as possible, but still capable of separating cells and proteins from the lactic acid containing broth, the performance of three hydrophilic membranes of varying cutoffs (10,000, 20,000, and 30,000) and one hydrophobic membrane (cutoff 25,000) was investigated. The proteins produced by the lactic acid bacteria during fermentation and the hydrolytic proteins were retained by the hydrophilic membrane with a cutoff of 20,000, whereas wheat flour proteins were detected in the permeate. In the permeates from the hydrophobic membrane (cutoff 25,000), almost no proteins were detected. The flux of the whole-wheat flour hydrolysate was significantly lower than that of rich medium, for both the hydrophilic and the hydrophobic membranes. The flux was, in all cases, higher for the hydrophilic membrane (12–85 L/[m2·h], depending, on which medium was treated) than for the hydrophobic one (8–45 L/[m2·h]), even though the nominal cutoffs of the hydrophobic and hydrophilic membranes were almost the same. However, the difference in flux was smaller when the whole-wheat flour hydrolysate was processed (12 vs 8 L/[m2·h]) than when the rich medium was processed (85 vs 45 L/[m2·h]). Protein retention was higher for the hydrophobic membrane than for the hydrophilic membrane (cutoff 20,000) owing to blocking of the pores by proteins adsorbed on to the hydrophobic membrane surface.
Keywords: Lacticacid; membrane; separation; ultrafiltration; wheat flour