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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.118, #1-3)

Lactic acid bacteria used in inoculants for silage as probiotics for ruminants by Zwi G. Weinberg; Richard E. Muck; Paul J. Weimer; Yaira Chen; Mira Gamburg (pp. 1-9).

Many studies have shown the beneficial effects on ruminant performance of feeding them with silages inoculated with lactic acid bacteria (LAB). These benefits might derive from probiotic effects. The purpose of the current study was to determine whether LAB included in inoculants for silage can survive in rumen fluid (RF), as the first step in studying their probiotic effects. Experiments were conducted in the United States and Israel with clarified (CRF) and strained RF (SRF) that were inoculated at 10⁶–10⁸ microorganisms/mL with and without glucose at 5 g/L. RF with no inoculants served as control. Ten commercial inoculants were used. The RF was incubated at 39°C and sampled in duplicates at 6, 12, 24, 48, 72, and 96 h for pH and LAB counts. The results indicate that with glucose the pH of the RF decreased during the incubation period. In the SRF, the pH of the inoculated samples was higher than that of the controls in most cases. This might be a clue to the mechanism by which LAB elicit the enhancement in animal performance. LAB counts revealed that the inoculants survived in the RF during the incubation period. The addition of glucose resulted in higher LAB counts.

Keywords: Lactic acid bacteria; silage inoculants; rumen fluid; ruminant performance; probiotics

Reductive alkylation of lipase by Raja Noor Zaliha A. Rahman; Bimo Ario Tejo; Mahiran Basri; Mohd Basyaruddin A. Rahman; Farid Khan; Sharifuddin M. Zain; Teruna J. Siahaan; Abu Bakar Salleh (pp. 11-20).

Candida rugosa lipase was modified via reductive alkylation to increase its hydrophobicity to work better in organic solvents. The free amino group of lysines was alkylated using propionaldehyde with different degrees of modification obtained (49 and 86%). Far-ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent showed that such chemical modifications at the enzyme surface caused a loss in secondary and tertiary structure that is attributed to the enzyme unfolding. Using molecular modeling, we propose that in an aqueous environment the loss in protein structure of the modified lipase is owing to disruption of stabilizing salt bridges, particularly of surface lysines. Indeed, molecular modeling and simulation of a salt bridge formed by Lys-75 to Asp-79, in a nonpolar environment, suggests the adoption of a more flexible alkylated lysine that may explain higher lipase activity in organic solvents on alkylation.

Keywords: Lipase; Candida rugosa ; enzyme modification; circular dichroism; molecular modeling

Fermentative production of curdlan by Parag S. Saudagar; Rekha S. Singhal (pp. 21-31).

Curdlan was produced by pure culture fermentation using Agrobacterium radiobacter NCIM 2443. Three different carbon sources (glucose, sucrose, maltose) were selected for study. Sucrose was found to be the most efficient. Utilization of sugar during the course of fermentation was studied, and the data were correlated to the production of curdlan. Curdlan mimics a secondary metabolite, in that its synthesis is associated with the poststationary growth phase of nitrogen-depleted batch culture. This was inferred from the results obtained from utilization of nitrogen. Regulation of pH at 6.1±0.3 resulted in an increased yield of curdlan from 2.48 to 4.8 g/L, and the corresponding increase in succinoglucan production was from 1.78 to 2.8 g/L. An attempt was made to increase curdlan production by the addition of the uridine nucleotides UMP and UDP-glucose to the fermentation broth. It was found that UDP-glucose at 0.8 µg/mL and UMP at 0.6 µg/mL served as precursors for curdlan and succinoglucan production when added after 18 h of nitrogen depletion in the fermentation broth.

Keywords: Curdlan; succinoglucan; fermentation; UDP-glucose; UMP

Evolutionary operation-factorial design technique for optimization of conversion of mixed agroproducts into gallic acid by Gargi Mukherjee; Rintu Banerjee (pp. 33-46).

This article presents the optimization of gallic acid production using filamentous fungi from tannin-rich mixed substrates taking into account the interaction effects of six variable process parameters. The methodology adopted for optimization was the evolutionary operation (EVOP)-factorial design technique. This technique combines the factorial method for designing experiments with the EVOP methodology for analyzing the experimental results systematically and arriving at conclusions according to its decision-making procedure. Standard deviation and error limits based on 95% confidence were calculated according to the relationship given in the literature. It was found that the best combinations of the process parameters at the optimum levels were 30°C, 80% relative humidity, pH 5.0, 48-h incubation period, 3 mL of induced inoculum, and 35 g of mixed substrate, resulting in a gallic acid yield of 94.8% under modified solid-state fermentation.

Keywords: Mixed substrates; modified solid-state fermentation; optimization; evolutionary operation-factorial design; filamentous fungi; gallic acid

Microbial desulfurization of different coals by C. Acharya; R. N. Kar; L. B. Sukla (pp. 47-63).

Coal is the most important nonrenewable energy source of fossil origin. It is also the most common fuel in thermal power plants. However, during coal incineration in power plants, high sulfur content of coal poses serious environmental problems owing to sulfur dioxide emission. We studied the application of microbial methods for removal of sulfur from three types of high sulfur coals—two samples collected from Assam and Rajasthan in India and one from Libiaz, Poland. These coal samples were desulfurized using indigenous Acidithiobacillus sp. After investigation of the effect of various parameters, the conditions optimized for the maximum removal of total sulfur (91.87% for lignite, 63.13% for Polish coal, and only 9.44% for Assam coal) were as follows: initial pH of 1.5 (2.5 in the case of Assam coal), particle size of 45 µ, pulp density of 2% (w/v), incubation period of 30 d at −35°C in presence of 44.2 g/L of ferrous sulfate in the media with shaking at 140 rpm. Poor removal of sulfur in the case of Assam coal was owing to extensive precipitation of jarosites. In addition, the sulfur in Assam coal is mostly found in organic form, which is difficult to remove with Acidithiobacillus sp. The removal of sulfur from the three coal samples was demonstrated with photomicrographic studies.

Keywords: Coal; Acidithiobacillus ; sulfur; desulfurization; lignite

Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp. MMRR₇ by K. Rajeshkumar; K. Jayachandran (pp. 65-72).

Physicochemical and biologic analysis of dairy wastewater showed that the effluent had a high organic load (chemical oxygen demand [COD]: 5095 mg/L), an acidic pH (6.4), and a high probability of coliforms (most probable number [MPN]>1100). The various bacterial strains isolated and purified were identified as Sporolactobacillus sp., Citrobacter sp., Pseudomonas sp., Alcaligenes sp., Bacillus sp., Staphylococcus sp., and Proteus sp., as per the Bergey’s manual of systematic bacteriology. Among the five selected bacterial strains, the strain designated as MMRR₇ and identified as Alcaligenes sp. was found to give a maximum reduction in COD (62%) in 5 d of incubation. Chemical coagulation using alum at a concentration of 0.5 g/100 mL was found to be effective in the primary treatment of the effluent. Studies on free-cell treatment of the coagulated effluent with the selected bacterial strain Alcaligenes sp. MMRR₇ gave a maximum COD reduction of 91% in 120 h. This study clearly indicates the possibility of using Alcaligenes sp. MMRR₇ for the effective treatment of dairy wastewater.

Keywords: Dairy waste; wastewater treatment; biologic methods; free-cell treatment; Alcaligenes sp.

Enzyme production and profile by Aspergillus niger during solid substrate fermentation using palm kernel cake as substrate by L. G. A. Ong; S. Abd-Aziz; S. Noraini; M. I. A. Karim; M. A. Hassan (pp. 73-79).

The oil palm sector is one of the major plantation industries in Malaysia. Palm kernel cake is a byproduct of extracted palm kernel oil. Mostly palm kernel cake is wasted or is mixed with other nutrients and used as animal feed, especially for ruminant animals. Recently, palm kernel cake has been identified as an important ingredient for the formulation of animal feed, and it is also exported especially to Europe, South Korea, and Japan. It can barely be consumed by nonruminant (monogastric) animals owing to the high percentages of hemicellulose and cellulose contents. Palm kernel cake must undergo suitable pretreatment in order to decrease the percentage of hemicellulose and cellulose. One of the methods employed in this study is fermentation with microorganisms, particularly fungi, to partially degrade the hemicellulose and cellulose content. This work focused on the production of enzymes by Aspergillus niger and profiling using palm kernel cake as carbon source.

Keywords: Palm kernel cake; hemicellulose; solid-state fermentation; Aspergillus niger ; mannanase

Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita by Débora Brand; Sevastianos Roussos; Ashok Pandey; Paulo C. Zilioli; Jorge Pohl; Carlos Ricardo Soccol (pp. 81-88).

Root-knot disease caused by Meloidogyne incognita is a matter of grave concern because it affects several economically important crop plants. The use of solid-state fermentation (SSF) may help to elaborate efficient formulations with fungi to be employed in the biologic control of nematodes. Attempts were made to select low-cost substrates for spore production of a strain of Paecilomyces lilacinus with known nematicide capacity. Coffee husks, cassava bagasse, and defatted soybean cake were utilized as substrates, and sugarcane bagasse was used as support. Fermentations were carried out in flasks covered with filter paper at 28°C for 10 d. The products obtained by SSF were evaluated for their nematicide activity in pot experiments containing one seedling of the plant Coleus inoculated with the nematode M. incognita. The plants were evaluated 2 mo after inoculation. Fermented products showed a reduction in the number of nematodes. The best results were obtained with defatted soybean cake, which showed almost 100% reduction in the number of nematodes; the reduction with coffee husk was 80% and with cassava bagasse was about 60%.

Keywords: Biocontrol; Paecilomyces lilacinus ; Meloidogyne incognita ; coffee husks; cassava bagasse; defatted soybean cake; sugarcane bagasse; solid-state fermentation

Derepressed 2-deoxyglucose-resistant mutants of Aspergillus niger with altered hexokinase and acid phosphatase activity in hyperproduction of β-fructofuranosidase by B. Ashokkumar; S. R. Senthilkumar; P. Gunasekaran (pp. 89-96).

Aspergillus niger NRRL330 produces extracellular β-fructofuranosidase (Ffase), and its production is subject to repression by hexoses in the medium. After ultraviolet mutagenization and selection, seven derepressed mutants resistant to 2-deoxyglucose (2-DG) were isolated on Czapek’s minimal medium containing glycerol. One of the mutants, designated DGRA-1, produced higher levels of Ffase. A considerable difference occurred in the mutants with reference to hexokinase and intracellular acid phosphatase activities. The hexokinase activity of the mutant DGRA-1 (0.69 U/mg) was 1.8-fold higher than the wild type (0.38U/mg). Intracellular acid phosphatase activity of the mutant DGRA-1 (0.83 U/g of mycelia) was twofold higher than that of the wild type (0.42U/g of mycelia), suggesting that phosphorylation and dephosphorylation steps could attribute to the 2-DG resistance of A. niger. However, additional mutations could account for the increased production of Ffase in the mutant DGRA-1.

Keywords: Aspergillus niger ; hexokinase; acid phosphatase; β-fructo-furanosidase; 2-deoxyglucose

Control of life cycle of mouse adipogenic 3T3-L1 cells by dietary lipids and metabolic factors by Kohji Nishimura; Yoko Hatano; Tsutomu Setoyama; Hirohumi Tsumagari; Kazuo Miyashita; Shan Lu; Mitsuo Jisaka; Tsutomu Nagaya; Kazushige Yokota (pp. 97-114).

Adipocytes function not only as in the storage and mobilization of lipids but also as endocrine cells by secreting tumor necrosis factor-α (TNF-α), free fatty acids, and other cytokines. To study the effects of dietary lipids and metabolic factors on the control of the life cycle of adipocytes, we utilized mouse 3T3-L1 preadipocytes that could be induced to differentiate into adipocytes. To evaluate the role of endogenous prostaglandins (PGs) in the adipogenic changes, we examined the effect of specific inhibitors of cyclooxygenase (COX). SC-560, a specific COX-1 inhibitor, suppressed adipogenesis dose dependently, suggesting a role of constitutive COX-1 in the endogenous synthesis of PGs, including PGJ₂ derivatives formed by mature adipocytes with the ability to promote adipogenesis. NS-398, a COX-2 inhibitor, had little influence on the maturation processes. Both COX inhibitors were effective in stimulating apoptosis of preadipocytes induced by TNF-α, indicating that both PGE₂ and PGF_2α produced by preadipocytes through the action of both COX isoforms serve as survival factors. However, the effect of both inhibitors was negligible for the proliferation of preadipocytes. Moreover, conjugated linolenic acid from bitter gourd at lower concentrations that was without effects by itself synergistically stimulated TNF-α-induced apoptosis. Therefore, dietary lipid factors are capable of controlling the life cycle of adipocytes together with metabolic factors.

Keywords: Adipocyte; apoptosis; arachidonate cascade; conjugated linolenic acid; cyclooxygenase; lipoxygenase; obesity; peroxisome proliferator-activated receptor γ

Characterization of metabolic pathway of linoleic acid 9-hydroperoxide in cytosolic fraction of potato tubers and identification of reaction products by Hideto Kimura; Kazushige Yokota (pp. 115-132).

Potato tubers are shown to contain a unique lipoxygenase pathway to form 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) from linoleic acid. Here, we report the metabolic pathway of 9-HPODE in the cytosolic fraction and the characterization of enzymes involved in the conversion of metabolites. The analysis of enzymatic reaction products at pH 5.5 revealed the formation of 9-keto-10,12-octadecadienoic acid, 9-hydroxy-10,12-octadecadienoic acid, 9,10-epoxy-11-hydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid. The cytosolic enzymes were separated by anion-exchange chromatography into two fractions E1 and E2, having molecular masses of 66 and 54 kDa, respectively. The enzyme fraction E1 only produced 9-keto-10,12-octadecadienoic acid, whereas E2 formed other products. The enzyme E1 showed higher reactivity with 13- and 9-hydroperoxide of α-linolenic acid than 9-HPODE, but no reaction with hydroxy fatty acids. In contrast, the enzyme E2 showed the highest reactivity with 9-HPODE, followed by hydroperoxides of α-linolenic acid and arachidonic acid. We also evaluated the antibacterial activity of hydroxy fatty acids against Erwinia carotovora T-29, a bacterium infecting potato tubers. Growth of the bacteria was suppressed more potently with 9- or 13-hydroxy fatty acids than dihydroxy or trihydroxy fatty acids, suggesting a role for the metabolites in the resistance of bacterial infection.

Keywords: Potato tuber; linoleic acid 9-hydroperoxide; hydroxy fatty acids; Erwinia carotovora T-29; bacterial infection

Regulation and role of arachidonate cascade during changes in life cycle of adipocytes by Shan Lu; Kohji Nishimura; Mohammad A. Hossain; Mitsuo Jisaka; Tsutomu Nagaya; Kazushige Yokota (pp. 133-153).

Although some eicosanoids serve as potent natural ligands to activate peroxisome proliferator-activated receptor (PPARγ), the ability of adipocytes to produce eicosanoids and regulate PPARγ remains unclear. Here, adipogenic 3T3-L1 cells were employed to determine the gene expression of isoforms of biosynthetic enzymes in the arachidonate cyclooxygenase (COX) pathway and the synthesis of prostaglandins (PGs). The expression of COX-2 was induced transiently in a biphasic manner upon the triggering of the differentiation and maturation phases while COX-1 was constitutive. The exclusive expression of lipocalin-type PGD synthase occurred and gradually increased during the maturation process along with the stable expression of PPARγ. Moreover, we confirmed the formation of PGD₂ from arachidonic acid by the mature adipocytes, suggesting conversion into PGJ₂ derivatives. Even though cytosolic and membrane-associated subtypes of PGE synthase were expressed at relatively constant levels, the ability of preadipocytes to produce PGE₂ was greater than that of mature adipocytes in the cell response. The treatment of the mature adipocytes with exogenous PGD₂, 15-deoxy-Δ^12,14-PGJ₂ and PGE₂, in the presence of aspirin, enhanced the adipogenesis. These findings imply the specific roles of prostanoids produced by the mature adipocytes in the maintenance of terminal differentiation through an autocrine control mechanism.

Keywords: Adipocyte; life cycle; peroxisome proliferator-activated receptor γ; adipogenesis; arachidonate cascade; eicosanoid; cyclooxygenase; prostaglandin; isoforms

Lipases and their industrial applications by Alain Houde; Ali Kademi; Danielle Leblanc (pp. 155-170).

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are part of the family of hydrolases that act on carboxylic ester bonds. The physiologic role of lipases is to hydrolyze triglycerides into diglycerides, monoglycerides, fatty acids, and glycerol. These enzymes are widely found throughout the animal and plant kingdoms, as well as in molds and bacteria. Of all known enzymes, lipases have attracted the most scientific attention. In addition to their natural function of hydrolyzing carboxylic ester bonds, lipases can catalyze esterification, interesterification, and transesterification reactions in nonaqueous media. This versatility makes lipases the enzymes of choice for potential applications in the food, detergent, pharmaceutical, leather, textile, cosmetic, and paper industries. The most significant industrial applications of lipases have been mainly found in the food, detergent, and pharmaceutical sectors. Limitations of the industrial use of these enzymes have mainly been owing to their high production costs, which may be overcome by molecular technologies, enabling the production of these enzymes at high levels and in a virtually purified form.

Keywords: Lipases; industrial applications; detergent; protein engineering; rational protein design; directed evolution

Insight into catalytic mechanism of papain-like cysteine proteinases by E. M. Papamichael; L. G. Theodorou; J. G. Bieth (pp. 171-175).

We studied the role of D¹⁵⁸ in papain-like cysteine proteinases by using subtilisin Carlsberg, and its chemically modified analog thiolsubtilisin, by applying the proton inventory (PI) method and also by taking into account the pH profiles of the k cat/K m parameter. In the case of thiolsubtilisin, we estimated large inverse solvent isotope effects for k cat/K m, as in papain, whereas for subtilisin we found “dome-shaped” PI, suggesting a completely different mechanism. Finally, the kinetic behavior of thiolsubtilisin presented similarities as well as differences, compared to papain, suggesting a possible role for D¹⁵⁸ as part of a catalytic triad in papain-like cysteine proteinases.

Keywords: Cysteine proteinases; proton inventory; catalytic mechanism; aspartate¹⁵⁸ ; thiolsubtilisin

Differential thermal and thermogravimetric analyses of bound water content in cellulosic substrates and its significance during cellulose hydrolysis by alkaline active fungal cellulases by Santosh Vyas; S. D. Pradhan; N. R. Pavaskar; Anil Lachke (pp. 177-188).

Various cellulosic substrates were examined for bound water content by differential thermal analysis (DTA) and thermogravimetry (TG). Samples were heated in the range of 30–100°C at a rate of 3°/min. DTA vaporization curves for different cellulose samples indicated that the bound water (W b) was vaporized at higher temperature than free water (W f) at the surface. Weight loss was observed in two stages, corresponding to W f and W b in TG curves. The bound water content was dependent on the degree of crystallinity of cellulose. Among different cellulosic substrates, Walseth cellulose showed the highest bound water content, and it also was found to be the least crystalline. The alkaline-active, alkali-stable cellulase was obtained from the alkalotolerant Fusarium sp. The substrate specificity and viscometric characteristics confirmed the enzyme to be an endoglucanase. The W b content of Walseth cellulose was lowered during the enzymatic hydrolysis. The possible application of bound water analysis in understanding the hydrolysis of cellulosic substrates of different crystallinity is discussed.

Keywords: Cellulose; endoglucanase; differential thermal analysis; thermogravimetry; bound water; vaporization

Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation by Krisztina Kovacs; Gyorgy Szakacs; Tunde Pusztahelyi; Ashok Pandey (pp. 189-204).

Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0–5.0), and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, α-amylase, and β-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55°C, respectively. The enzyme totally lost its activity at 70°C in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70°C after a 60-min incubation in the presence of solid substrate fermentation solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities between pI 5.9 and 4.8, where N-acetyl-β-d-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI.

Keywords: Chitinase; chitinolytic enzymes; N-acetyl-β-d-hexosaminidase; solid substrate fermentation; solid-state fermentation; Trichoderma longibrachiatum

Thermostable phytase production by Thermoascus aurantiacus in submerged fermentation by K. Madhavan Nampoothiri; G. Jino Tomes; Krishnan Roopesh; George Szakacs; Viviana Nagy; Carlos Ricardo Soccol; Ashok Pankey (pp. 205-214).

Phytases act on phytic acid, an antinutrient factor present in animal feeds, and release inorganic phosphate. We optimized the production parameters for phytase production using Thermoascus aurantiacus (TUB F 43), a thermophilic fungal culture, by submerged fermentation. A semisynthetic medium containing glucose, starch, peptone, and minerals supplemented with 3.75% (w/v) wheat bran particles was found to be the best production medium among the various combinations tried. Further supplementation of this medium with surfactants such as Tween-20 and Tween-80 considerably enhanced the enzyme yield. A maximum phytase activity (468.22 U/mL) was obtained using this production medium containing 2% (v/v) Tween-20 after 72 h of fermentation at 45°C in shake-flask cultures with a rotation of 150 rpm. Herein we present details of a few of the process parameter optimizations. The phytase enzyme was found to be thermostable, and the optimal temperature for phytase activity was found to be 55°C. However, 80% of the activity still remained when the temperature was shifted to 70°C.

Keywords: Thermoascus aurantiacus ; thermostable phytase; submerged fermentation; surfactants; phytic acid

Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays by Tino Polen; Volker F. Wendisch (pp. 215-232).

DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Grampositive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli.

Keywords: Genomewide gene expression analysis; DNA chips; DNA microarrays; cluster analysis; global regulatory mechanisms; amino acid production; Corynebacterium glutamicum ; Escherichia coli.

In vitro multiplication in Kaempferia galanga linn by T. S. Swapna; M. Binitha; T. S. Manju (pp. 233-241).

Kaempferia galanga is an important medicinal plant that is facing threat of extinction owing to indiscriminate and unsustainable harvesting in the wild. Conventional breeding is difficult in this plant, and in vitro multiplication is important to conservation and propagation. Leaf and rhizome explants of Kaempferia were aseptically cultured on MS medium with various combinations of indole-3-acetic acid (IAA), benzyl amino purine (BAP), napthalene acetic acid (NAA), 2-4-dichlorophenoxy acetic acid (2,4-D) and kinetin at concentrations ranging from 0.5 to 2.5 mg/L. High-frequency organogenesis and multiple shoot regeneration was induced from rhizome explants on MS medium supplemented with 0.5 mg/L of IAA and 2.5 mg/L of BAP. Rooting was induced in MS medium with 0.5 mg/L of IAA and 2 mg/L of BAP.

Keywords: Medicinal plants; Kaempferia galanga ; in vitro propagation; multiple shooting; organogenesis

Siderophore production by a marine Pseudomonas aeruginosa and its antagonistic action against phytopathogenic fungi by A. V. Manwar; S. R. Khandelwal; B. L. Chaudhari; J. M. Meyer; S. B. Chincholkar (pp. 243-251).

A marine isolate of fluorescent Pseudomonas sp. having the ability to produce the pyoverdine type of siderophores under low iron stress (up to 10 µM iron in the succinate medium) was identified as Pseudomonas aeruginosa by using BIOLOG Breathprint and siderotyping. Pyoverdine production was optimum at 0.2% (w/v) succinate, pH 6.0, in an iron-deficient medium. Studies carried out in vitro revealed that purified siderophores and Pseudomonas culture have good antifungal activity against the plant deleterious fungi, namely, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium oxysporum, and Sclerotium rolfsii. Siderophore-based maximum inhibition was observed against A. niger. These in vitro antagonistic actions of marine Pseudomonas against phytopathogens suggest the potential of the organism to serve as a biocontrol agent.

Keywords: Marine fluorescent pseudomonas ; siderophores; phytopathogens; biocontrol agent; pyoverdine

Structural analysis of mycobacterial lipoglycans by Jérôme Nigou; Martine Gilleron; Thérèse Brando; Germain Puzo (pp. 253-267).

Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan (LAM), one of the most potent mycobacterial immunomodulatory molecules. The importance of lipoarabinomannan (LAM) in the immunopathogenesis of tuberculosis has incited structural studies on this molecule to (1) establish a precise structural model of the molecule and (2) decipher the structure/function relationships. In recent years, we have focused on the two domains essential for LAM biologic activities: the mannosyl-phosphatidyl-myo-inositol anchor and the caps. We review here the recent procedures developed for the structural analysis of these domains.

Keywords: Mycobacterium; lipoglycan; lipoarabinomannan; structure; capillary electrophoresis; nuclear magnetic resonance

Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II by Preeti Bachhawat; Saroj Mishra; Yukti Bhatia; V. S. Bisaria (pp. 269-282).

The biosynthetic activity of yeast Pichia etchellsii β-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate: 1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethylacetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

Keywords: β-Glucosidase; Pichia etchellsii ; cellooligosaccharides; alkyl glucosides; monoterpenyl glucosides

Synthesis, characterization, and antifungal studies of transition metal complexes of ω-bromoacetoacetanilide isonicotinylhydrazone by K. P. Deepa; K. K. Aravindakshan (pp. 283-292).

Isonicotinic acid hydrazide or isonicotinylhydrazide, commonly known as isoniazid, is an antibacterial agent that has been used to treat tuberculosis. It interacts with microbial cell walls. Schiff’s bases or anils are the compounds having >C=N−N< linkages, which have immense applications as catalysts, stabilizers, pigments, dyes, and drugs. They have good ability to form chelates with many metal ions. Isoniazid can form Schiff’s bases with diketones such as acetoacetanilide. Acetoacetanilide isonicotinylhydrazone and its metal chelates exhibit anticancer activity. Our studies on N-methyl-acetoacetanilide isonicotinylhydrazone and its metal chelates revealed that they are active against pathogenic fungal strains. Hence, it is worthwhile to synthesize new complexes of ligands having different substituents on the acetoacetanilide moiety. We synthesized five new metal chelates of ω-bromoacetoacetanilide isonicotinylhydrazone. The ligand behaved as a tridentate monoanion or as a tridentate dianion in the complexes. These compounds were characterized mainly by elemental analysis; conductivity measurements; and electronic, infrared, and nuclear magnetic resonance spectral studies. We also carried out antifungal studies of these compounds against four selected pathogenic fungal strains using a cup-plate technique. Both the ligand and its metal chelates were active against all fungal strains investigated. However, the chelates were found to be more active than the ligand.

Keywords: Metal chelates; ω-bromoacetoacetanilide isonicotinylhydrazone; synthesis; characterization; antifungal studies

Comparison of citric acid production by solid-state fermentation in flask, column, tray, and drum bioreactors by Luciana P. S. Vandenberghe; Carlos R. Soccol; F. C. Prado; Ashok Pandey (pp. 293-303).

Studies were conducted to evaluate citric acid production by solid-state fermentation (SSF) using cassava bagasse as substrate employing a fungal culture of Aspergillus niger LPB 21 at laboratory and semipilot scale. Optimization of the process parameters temperature, pH, initial humidity, aeration, and nutritive composition was conducted in flasks and column fermentors. The results showed that thermal treatment of cassava bagasse enhanced fungal fermentation efficacy, resulting in 220 g of citric acid/kg of dry cassava bagasse with only treated cassava bagasse as substrate. The results obtained from the factorial experimental design in a column bioreactor showed that an aeration rate of 60 mL/min (3 mL/[g·min]) and 60% initial humidity were optimum, resulting in 265.7 g/kg of dry cassava bagasse citric acid production. This was almost 1.6 times higher than the quantities produced under unoptimized conditions (167.4 g of citric acid/kg of dry cassava bagasse). The defined parameters were transferred to semipilot scale, which showed high promise for large-scale citric acid production by SSF with cassava bagasse. Respirometry assays were carried out in order to follow indirectly the biomass evolution of the process. Citric acid production reached 220, 309, 263, and 269 g/kg of dry cassava bagasse in Erlenmeyer flasks, column fermentors, a tray bioreactor, and a horizontal drum bioreactor, respectively.

Keywords: Citric acid; solid-state fermentation; respirometry; column fermentors; horizontal drum

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen by Adenise L. Woiciechowski; Carlos R. Soccol; Saul N. Rocha; Ashok Pandey (pp. 305-312).

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).

Keywords: Cassava bagasse; fermentative process; xanthan gum; nitrogen sources; Xanthomonas campestris

Immobilization of lipase from Candida rugosa on layered double hydroxides for esterification reaction by Mohd Basyaruddin A. Rahman; Mahiran Basri; Mohd Zobir Hussein; Raja Nor Zaliha A. Rahman; Dara Hatira Zainol; Abu Bakar Salleh (pp. 313-320).

Synthesis of layered double hydroxides (LDHs) of Zn/Al-NO₃ ⁻ hydrotalcite (HIZAN) and Zn/Al-diocytyl sodium sulfosuccinate (DSS) nanocomposite (NAZAD) with a molar ratio of Zn/Al of 4:1 were carried out by coprecipitation through continuous agitation. Their structures were determined using X-ray diffractometer spectra, which showed that basal spacing for LDH synthesized by both methods was about 8.89 Å. An expansion of layered structure of about 27.9 Å was observed to accommodate the surfactant anion between the interlayer. This phenomenon showed that the intercalation process took place between the LDH interlayer. Lipase from Candida rugosa was immobilized onto these materials by physical adsorption method. It was found that the protein loading onto NAZAD is higher than HIZAN. The activity of immobilized lipase was investigated through esterification of oleic acid and 1-butanol in hexane. The effects of pore size, surface area, reaction temperature, thermostability of the immobilized lipases, storage stability in organic solvent, and leaching studies were investigated. Stability was found to be the highest in the nanocomposite NAZAD.

Keywords: Immobilization; lipase; layered double hydroxides; esterification; Candida rugosa

Sorbitol can be produced not only chemically but also biotechnologically by Rainer Jonas; Mauricio M. Silveira (pp. 321-336).

Sorbitol, a polyol found in many fruits, is attracting increasing industrial interest as a sweetener, humectant, texturizer, and softener. It is principally produced by chemical means. The bacterium Zymomonas mobilis is able to produce sorbitol together with gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via the enzyme glucose-fructose oxidoreductase, so far only known from Z. mobilis. The possibilities for the production of sorbitol by Z. mobilis are discussed also under the aspect of an industrial process and compared with the current chemical as well as other microbiologic processes. The production process by Z. mobilis shows economic possibilities for certain countries, such as Brazil, considering only the products sorbitol and ethanol as an important byproduct. For the other byproduct, gluconic acid, further studies for its partial substitution must be conducted.

Keywords: Sorbitol; Zymomonas mobilis ; glucose-fructose oxidoreductase; glucono-δ-lactone; gluconic acid

Effect of vitamin E on autolysis and sporulation of Aspergillus nidulans by Tamás Emri; Zsolt Molnár; Tünde Pusztahelyi; Stefan Rosén; István Pócsi (pp. 337-348).

The morphologic and physiologic effects of vitamin E, a powerful antioxidant, on the autolysis and sporulation of Aspergillus nidulans FGSC26 were studied. In carbon-depleted submerged cultures, reactive oxygen species (ROS) accumulated in the cells and, concomitantly, progressing autolysis was observed, which was characterized by decreasing dry cell masses and pellet diameters as well as by increasing extracellular chitinase activities. Vitamin E supplemented at a concentration of 1 g/L hindered effectively the intracellular accumulation of ROS, the autolytic loss of biomass, the disintegration of pellets, and the release of chitinase activities. In surface cultures, vitamin E inhibited autolysis of both A. nidulans FGSC26 and a loss-of-function FlbA autolytic phenotype mutant. In addition, supplementation of the culture medium with this antioxidant also had a negative effect on the sporulation of strain FGSC26 and the FadA G203R hypersporulating phenotype mutant. These results suggest that accumulation of ROS was involved in the initiation of both sporulation and autolysis in this filamentous fungus, but that FadA/FlbA signaling was not involved in this vitamin E-dependent regulation. Vitamin E can be recommended as a supplement in fermentations in which the disintegration of pellets and gross autolysis should be avoided.

Keywords: Aspergillus nidulans ; vitamin E; menadione; sporulation; autolysis; fadA ; flbA

Influence of fadA G203R and ΔflbA mutations on morphology and physiology of submerged Aspergillus nidulans cultures by Zsolt Molnár; Edina Mészáros; Zsolt Szilágyi; Stefan Rosén; Tamás Emri; István Pócsi (pp. 349-360).

Morphologic and physiologic changes taking place in carbon-limited submerged cultures of Aspergillus nidulans ΔflbA and fadA G203R strains were studied. Loss-of-function mutation of the flbA gene resulted in an altered germination with unusually thick germination tubes, “fluffy” pellet morphology, as well as a reduced fragmentation rate of hyphae during autolysis. In the fadA G203R mutant strain, conidiophores formed in the stationary phase of growth, and the size of pellets shrank considerably. There were no significant differences in the generation of reactive oxygen species (ROS) and in the specific catalase and superoxide dismutase activities by the tested mutants and the appropriate parental strains. Therefore, the participation of ROS or antioxidative enzymes in FadA/FlbA signaling pathways seems to be unlikely in submerged cultures. On the other hand, earlier increases in the extracellular protease and ammonia production were recorded with the ΔflbA strain, whereas the protease and ammonia production of the fadA G203R mutant lagged behind those of the wild-type strains. Similar changes in the time courses of the induction of γ-glutamyltranspeptidase and the degradation of glutathione were observed. These results suggest that FadA/FlbA signaling may be involved in the mobilization of protein and peptide reserves as energy sources during carbon starvation.

Keywords: Aspergillus nidulans ; fadA ; flbA ; autolysis; chitinase; glutathione

Entrapment of enzyme in water-restricted microenvironment for enzyme-mediated catalysis under microemulsion-based organogels by Datta Madamwar; Amit Thakar (pp. 361-369).

Nonaqueous enzymology has emerged as a major area of biotechnology research and development. Enzymes in organic solvents offer great potential for the biocatalysis of a wide range of chemical processes that cannot occur in water. One of the most commonly used methods for carrying out enzymatic conversions in organic solvents is enzymes solubilized in water-in-oil (w/o) microemulsions or water containing reverse micelles. In reverse micelles, enzyme molecules are solubilized in discrete hydrated micelles formed by surfactants within a continuous phase, i.e., nonpolar organic solvent. Under appropriate conditions, these solutions are homogeneous, thermodynamically stable, and optically transparent. However, there are very few examples of preparative-scale enzyme-catalyzed synthesis in water-in-oil microemulsion. One reason is that despite the advantages offered by microemulsion media, product isolation and enzyme reuse from such singlephase liquid medium is more complex than in competing methodologies in which the catalyst is present as a separate solid phase. Therefore, the approach simplifying product isolation, and enzyme reuse from microemulsion-based media, has been the use of a gelled microemulsion system, especially gelatin silica nanocomposite.

Keywords: Reverse micelles; microemulsion organogels; lipase; organic solvents; esterification; enzymes

Decolorization of ranocid fast blue dye by bacterial consortium SV5 by Sini Mathew; Datta Madamwar (pp. 371-381).

Synthetic dyes are not uniformly susceptible to degradation in conventional wastewater treatment processes. A number of biotechnological processes have been suggested as of potential interest in combating these pollutants in an ecofriendly manner. We determined the optimal parameters necessary for the bacterial consortium SV5 to decolorize Ranocid Fast Blue dye. The best results were obtained with a 0.1% (w/v) concentration of both starch and yeast extract supplemented in Bushnell Hass Medium under static conditions at a temperature of 37°C in less than 24 h with an initial dye concentration of 100 ppm.

Keywords: Ranocid Fast Blue dye; decolorization; bacterial consortium; biomass; microorganisms

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.118, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.118, #1-3)