Applied Biochemistry and Biotechnology (v.149, #3)

Extraction of Inulinase Obtained by Solid State Fermentation of Sugarcane Bagasse by Kluyveromyces marxianus NRRL Y-7571 by João Paulo Bender; Marcio Antônio Mazutti; Marco Di Luccio; Helen Treichel (195-203).
Production of inulinase by solid state fermentation always involves an extraction step, which dictates enzyme recovery yield and is related to cultivation conditions and control of process parameters. This work is focused on the study of extraction conditions aiming to maximize yield of an inulinase obtained by solid state fermentation of sugar cane bagasse and Kluyveromyces marxianus NRRL Y-7571. Kinetics of extraction was followed varying the kind of solvent used. After determining the best solvent, an experimental design was carried out to study the effect of the solid/liquid ratio (1:10–1:20), extraction temperature (20–53°C), and stirring rate (50–177 rpm). Results showed that maximum yield was obtained when sodium acetate buffer 0.1 M pH 4.8 was used, using a solid/liquid ratio of 1:10, at 53°C and 150 rpm for 40 min.
Keywords: Inulinase; Solid state fermentation; Extraction; Experimental design; Kluyveromyces ; Sugarcane bagasse

Characterization of an Exopolygalacturonase from Aspergillus niger by Afaf S. Fahmy; Fawkia M. El-beih; Saleh A. Mohamed; Somia S. Abdel-Gany; Engy A. Abd-Elbaky (205-217).
Polygalacturonase (PGI) from Aspergillus niger NRRL3 was purified about 12.0-fold from the cell-free broth using diethylaminoethyl-Sepharose and Sephacryl S-200 columns. The molecular weight of the PGI was 32,000 Da as estimated by gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. PGI had an isoelectric point of 7.6 and an optimum pH of 5.0. PGI was active on polygalacturonic acid and esterified pectins, but the activity on pectin decreased with an increase in degree of esterification. PGI had higher affinity (low K m) and turnover number (V max /K m and K cat /K m) toward polygalacturonic acid. PGI was found to have a temperature optimum at 40°C and was approximately stable up to 30 °C. All the examined metal cations had partial inhibitory effects on PGI, while Mn+2 at 5 mM caused a complete inhibition for the enzyme. Comparison of viscosity reduction rates with release of reducing sugars indicated that the enzyme from A. niger is exoacting. The storage stability study of PGI showed that the enzyme in powder form retained 56% of its activity after 9 months of storage at 4 °C. The above properties of PGI may be suitable for food processing.
Keywords: Pectin; Polygalacturonic acid; Polygalacturonase; Aspergillus niger ; Purification; Characterization; Mode of action

The imperative role of functionalized natural alginate in immobilization of Lactobacillus delbrucekii (NCIM 2365) cells in production of optically pure l (+) lactic acid was studied. L. delbrucekii cells were immobilized in alginate, succinylated alginate and carrageenan to evaluate the bead stability and selectivity towards production of optically pure l (+) lactic acid. The scanning electron microscopic studies of free and immobilized cells show little morphological changes. The present study highlights the use of functionalized alginate-immobilized L. delbrucekii cells in production of l (+) lactic acid in higher yields (0.93 Yp/s in grams) with an improved enantioselectivity (99%). In addition, they further revealed decreased by-product formation (acetic and propionic acid) when compared to free and other immobilized cell fermentation.
Keywords: Alginate; Derivatization; Immobilization; Lactic acid; Lactobacillus delbrucekii

Purification and Biochemical Characterization of Two Xylanases from Aspergillus sydowii SBS 45 by Suprabha G. Nair; R. Sindhu; Shankar Shashidhar (229-243).
Two xylanases were isolated and purified from crude culture filtrate of Aspergillus sydowii SBS 45 after 9 days of growth on wheat bran containing 0.5% (w/v) birch wood xylan as the carbon source under solid-state fermentation. After a three-step purification scheme involving ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-200), and anion exchange chromatography (DEAE-Sephadex A-50), xylanase I was purified 93.41 times, and xylanase II was purified 77.40 times with yields of 4.49 and 10.46, respectively. Molecular weights of xylanase I and II were 20.1 and 43 kDa, respectively, in sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Optimum temperature was 50 °C, and optimum pH was 10.0 for both xylanase I and II. The K m value of xylanase I for birch wood xylan was 3.18 mg ml−1 and for oat spelt xylan 6.45 mg ml−1, while the K m value of xylanase II for birch wood xylan was 6.51 mg ml−1 and for oat spelt xylan 7.69 mg ml−1. Metal ions like Al3+, Ba2+, Ca2+, Na+, and Zn2+ enhanced the activity of xylanase I and II at 10 mM concentration. Among the additives, l-tryptophan enhanced the activity of xylanase I and II at 10-, 20-, and 30-mM concentrations. Both xylanases appeared to be glycoproteins.
Keywords: Aspergillus sydowii ; Xylanase I; Xylanase II; Purification; Characterization; Solid-state fermentation

Chitopearl beads were used as immobilization supports for d-tagatose production from d-galactose by l-arabinose isomerase from Thermotoga neapolitana because chitopearl beads were more stable than alginate beads at temperatures above 60 °C. The pH and temperature for the maximum isomerization of galactose were 7.5 and 90 °C, respectively. In thermostability experiments, the half-lives of the immobilized enzyme at 70, 75, 80, 85, and 90 °C were 388, 106, 54, 36, and 22 h, respectively. The reaction temperature was determined to be 70 °C because the enzyme is highly stable up to 70 °C during the reaction. When the reaction time, galactose concentration, and temperature were increased, the pH of a mixture containing enzyme and galactose decreased by the Maillard reaction, resulting in decreased tagatose production. With pH control at 7.5, tagatose production (138 g/L) at 70 °C in a stirred tank reactor containing immobilized enzyme and 300 g/L galactose increased two times higher, comparing that without pH control.
Keywords: l-Arabinose isomerase; Tagatose; Immobilization; Thermotoga neapolitana ; Stirred tank reactor; pH control

The objective of this study was to exploit the decolorization potential of a newly isolated white-rot fungus Schizophyllum commune IBL-6 for the biodegradation of reactive textile dye Cibacron Red FN-2BL. In the initial decolorization study of 10 days, it was observed that S. commune IBL-6 was a better decolorizer of Cibacron Red FN-2BL. Various process parameters like composition of basal nutrient medium, pH, temperature, additional carbon and nitrogen sources, and initial dyestuff concentration were optimized to develop an economic decolorization process. The optimum dye decolorization was achieved in basal nutrient medium II containing 0.1% Cibacron Red FN-2BL and supplemented with 1% glucose after 3 days incubation at pH 4.5 and 30 °C. All the additional carbon sources were found to enhance decolorization process, whereas most of the nitrogen supplements caused fungal-growth inhibition. The pattern of enzymes involved in the biodegradation of this dye was studied, and manganese peroxidase was found to be the major peroxidase with minor lignin peroxidase and laccase activities.
Keywords: Reactive dyestuff; Schizophyllum commune IBL-6; Decolorization; Optimization; Ligninase profile

Cultivations of filamentous fungi in stirred tank reactors (STRs) to produce metabolites are often limited by insufficient mixing and mass transfer because of the formation of mycelial clumps inside the reactors. This study developed an acid-adapted preculture approach to control the morphology of filamentous Rhizopus arrhizus in a STR, consequently to enhance the production yield and productivity of L(+)-lactic acid efficiently using waste potato starch as substrate. Using the acid-adapted precultures as inoculum, the morphology of R. arrhizus was maintained as large clumps, coalesced loose small pellets, and freely dispersed small pellets. The highest lactic acid concentration of 85.7 g/L with a yield of 86% was obtained in association with the formation of coalesced loose small pellets. The results indicate that the use of the acid-adapted precultures as inoculum is a promising approach for lactic acid production in STRs.
Keywords: L(+)-lactic acid; Rhizopus arrhizus ; Pellets; Acid-adapted precultures; Morphology; Stirred tank reactor

Thermostability of Selected Enzymes in Organic Wastes and in their Humic Extract by Keiji Jindo; José L. Moreno; Teresa Hernández; Carlos García (277-286).
The objective of this study was to evaluate the thermostability up to 70 °C for 1 h of selected enzymes present in fresh and composted sewage sludge (SS and SSC) or municipal solid wastes (MSW and MSWC) and their humic extract. After a thermal treatment at 70 °C, no β-glucosidase activity in any humic extract was detected, whereas in SS, SSC, MSW, and MSWC, it was respectively, 35%, 68%, 17%, and 12% compared to thermally untreated samples. By contrast, o-diphenol oxidase activity was even stimulated by thermal treatment in SS samples, but in the humic extracts, this activity decreased by 75–81%. Urease activity in all humic extracts decreased by 70% or more just at 40 °C, whereas for organic wastes, this decrease was observed after treatment at 70 °C. Alkaline phosphatase (AP) activity was affected by thermal treatment only in MSW and MSWC. In humic extracts, AP activity decreased gradually to zero except for the MSW extract, where 45% activity was retained after treatment at 70 °C. In general, thermostability of enzymes in humic extracts was lower than the materials they were extracted from.
Keywords: Enzyme stabilization; Compost; Municipal solid wastes; Sewage sludge; Thermostability; Humus–enzyme complexes

Potential Application of Alkaline Pectinase from Bacillus subtilis SS in Pulp and Paper Industry by Sonia Ahlawat; R. P. Mandhan; Saurabh Sudha Dhiman; Rakesh Kumar; Jitender Sharma (287-293).
Pectinase production from Bacillus subtilis SS was optimized under solid-state fermentation (5,943 U/g of dry bacterial bran). The pectinase produced was stable in neutral to alkaline pH range at 70 °C; therefore, the suitability of this pectinase in pulp and paper industry was investigated. The enzyme pretreatment process was optimized, and a pectinase dose of 5 IU/g of oven-dried pulp (10% consistency) at pH 9.5 temperature 70 °C after 150 min of treatment gave the best pretreatment to the pulp. An increase of 4.3% in brightness along with an increase of 14.8 and 65.3% in whiteness and fluorescence, respectively, whereas a 15% decrease in the yellowness of the pretreated pulp were observed. There was a 5.85% reduction in kappa number and 6.1% reduction in permanganate number along with a reduction in the chemical oxygen demand value. Significant characteristics showed by pectinase open new possibilities of application of this cellulase-free enzyme in the pulp and paper industry by reducing the negative environmental impact of chemicals apart from improving the properties of paper.
Keywords: Cellulase-free alkaline pectinase; Bacillus subtilis ; Solid-state fermentation; Pulp and paper