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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.102-103, #1-6)

New horizons in biotechnology—An introduction (pp. 1-3).

Effect of ecological factors on conjugal transfer of chromium-resistant plasmid in Escherichia coli isolated from tannery effluent by Tuhina Verma; P. W. Ramteke; S. K. Garg (pp. 5-20).

The influence of total organic carbon (TOC), pH, and mating temperature on transfer of chromium-resistant plasmid between Escherichia coli strains in terms of variation in the number of transconjugants formed and variation in transfer frequency was investigated. In vitro transfer was studied in five chromate-tolerant E. coli strains isolated from tannery effluent using E. coli K12 J62 (Nal^r Lac⁻) as a recipient. Conjugal transfer of different selection markers was observed in three strains. The study was carried out in sterile wastewater. A gradual decrease was observed both in the number of transconjugants and in transfer frequencies as the concentration of TOC in the mating medium descended from 10,095 to 1.2 mg of C/L, obtaining the maximum values with a TOC concentration of 10,095 mg of C/L. The number of transconjugants and the transfer frequency were maximum at 30°C. However, neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. The strains were also found resistant to different heavy metals and antibiotics. Curing of these strains resulted in loss of one or more resistance markers indicating the plasmid-borne resistance. It is inferred that plasmid transfer by conjugation occurs in wastewater bodies within a wide range of conditions.

Keywords: Chromium; Escherichia coli ; R-plasmid; conjugation; wastewaters; total organic carbon; mating temperature

Isolation, characterization, and plasmid pUPI126-mediated indole-3-acetic acid production in Acinetobacter strains from rhizosphere of wheat by S. B. Huddedar; A. M. Shete; J. N. Tilekar; S. D. Gore; D. D. Dhavale; B. A. Chopade (pp. 21-39).

Thirty-seven strains of Acinetobacter isolated and characterized from rhizosphere of wheat were screened for indole-3-acetic acid (IAA) production. Only eight Acinetobacter strains showed IAA production. The genus Acinetobacter was confirmed by chromosomal DNA transformation assay. Biotyping of eight strains was carried out and they were found to be genospecies of A. junii, A. baumannii, A. genospecies 3, and A. haemolyticus. Five of eight strains produced IAA at the early stationary phase: A. haemolyticus (A19), A. baumannii (A18, A16, A13), and Acinetobacter genospecies 3 (A15). A. junii A6 showed maximum IAA production at log phase and A. genospecies 3 and A. baumannii (A28, A30) at late stationary phase. IAA was extracted by ethyl acetate and purified by preparative thin-layer chromatography. Purified IAA was confirmed by ¹H-nuclear magnetic resonance and infrared spectrum analysis. Pot experiments showed a significant increase in plant growth inoculated with eight Acinetobacter genospecies as compared to control plants. IAA production was found to be encoded by plasmid pUPI126. All eight strains of Acinetobacter contain a plasmid pUPI126 with a molecular weight of 40 kb. Plasmid pUPI126 was transformed into Escherichia coli HB101 at a frequency of 5 × 10⁻⁵, and E. coli HB101 (pUPI126) transformants also showed IAA activity. PUPI126 also encoded resistance to selenium, tellurium, and lead. This is the first report of plasmid-encoded IAA production in the genus Acinetobacter.

Keywords: Acinetobacter spp; wheat rhizosphere; tryptophan; indole-3-acetic acid production; plasmid pUPI126; transformation; infrared analysis; ¹H-nuclear magnetic resonance; plant growth promotion

Synthetic peptides for study of human immunodeficiency virus infection by Monica Dettin; Claudia Scarinci; Antonella Pasquato; Carlo Di Bello (pp. 41-47).

The formation of a complex among gp120, CD4, and CCR5/CXCR4 represents a key step in human immunodeficiency virus (HIV) infection. The use of synthetic peptides reproducing sequences of these surface proteins has increased knowledge about the interactions that determine the penetration of HIV viruses into target cells. The final aim of such investigations is the design of molecules able to inhibit the initial step of infection and the development of high-sensitivity in vitro assays for detection of HIV. In particular, the studies presented herein concern the role of the gp120 V3 loop in the CD4 binding, the importance of the N-terminal sequence of HIV-coreceptor CCR5, the sequences patterned on CXCR4 natural ligand (stromal-derived factor 1 [SDF-1]) as inhibitory peptides, and the importance of substrate secondary structure in determining the enzymatic processing of gp120 precursor (gp160).

Keywords: Synthetic peptides; human immunodeficiency virus; gp120-CD4 binding; SDF-1; CCR5; CXCR4; drug design

Fermentation diagnosis by multivariate statistical analysis by Silvio Bicciato; Andrea Bagno; Marco Soldà; Riccardo Manfredini; Carlo Di Bello (pp. 49-62).

During the course of fermentation, online measuring procedures able to estimate the performance of the current operation are highly desired. Unfortunately, the poor mechanistic understanding of most biologic systems hampers attempts at direct online evaluation of the bioprocess, which is further complicated by the lack of appropriate online sensors and the long lag time associated with offline assays. Quite often available data lack sufficient detail to be directly used, and after a cursory evaluation are stored away. However, these historic databases of process measurements may still retain some useful information. A multivariate statistical procedure has been applied for analyzing the measurement profiles acquired during the monitoring of several fed-batch fermentations for the production of erythromycin. Multivariate principal component analysis has been used to extract information from the multivariate historic database by projecting the process variables onto a low-dimensional space defined by the principal components. Thus, each fermentation is identified by a temporal profile in the principal component plane. The projections represent monitoring charts, consistent with the concept of statistical process control, which are useful for tracking the progress of each fermentation batch and identifying anomalous behaviors (process diagnosis and fault detection).

Keywords: Fermentation processes; process identification; process diagnosis; multiway principal component analysis; statistical process control; database mining

Invertase production on solid-state fermentation by Aspergillus niger strains improved by parasexual recombination by Alba Mónica Montiel-González; Francisco José Fernández; Gustavo Viniegra-González; Octavio Loera (pp. 63-70).

Invertase production by Aspergillus niger grown by solid-state fermentation was found to be higher than by conventional submerged fermentation. The haploid mutant strains Aw96-3 and Aw96-4 showed better productivity of various enzymes, as compared to wild-type parental strain A. niger C28B25. Here we use parasexual crosses of those mutants to increase further the productivity of invertase in solid-state fermentation. We isolated both a diploid (DAR2) and an autodiploid (AD96-4) strain, which were able to grow in minimal medium after mutation complementation of previously isolated haploid auxotrophic strains. Invertase production was measured in solid-state fermentation cultures, using polyurethane foam as an inert support for fungal growth. Water activity value (Aw) was adjusted to 0.96, since low Aw values are characteristic in some solid-state fermentation processes. Such diploid strains showed invertase productivity levels 5–18 times higher than levels achieved by the corresponding haploid strains. For instance, values for C28B25, Aw96-3, Aw96-4, DAR2, and AD96-4 were 441, 254, 62, 1324, and 2677 IU/(L·h), respectively. These results showed that genetic recombination, achieved through parasexual crosses in A. niger, results in improved strains with potential applications for solid-state fermentation processes.

Keywords: Aspergillus niger ; diploids; invertase; solid-state fermentation; parasexual recombination

Continuous production of extracellular l-glutaminase by ca-alginate-immobilized marine Beauveria bassiana BTMF S-10 in packed-bed reactor by Abdulhameed Sabu; Sukumaran Rajeev Kumar; Muthusamy Chandrasekaran (pp. 71-79).

l-Glutamine amidohydrolase (l-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on l-glutaminase. In this article, we report the continuous production of extracellular l-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl₂ for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for l-glutaminase production were incorporated into the continuous production studies. Beads with 12×10⁸ spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of l-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilized spores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of l-glutaminase.

Keywords: Glutaminase; Beauveria bassiana ; immobilization; packed-bed reactor

Selection of human isolates of bifidobacteria for their use as probiotics by Mayur R. Acharya; Rajiv K. Shah (pp. 81-98).

An attempt was made to isolate human strains of Bifidobacteria, all together 36, from fecal samples of 15 breast-fed infants ages 1–6 mo. These isolates were checked for their ability to grow in the presence of 1–3% bile, 0.2–0.4% phenol, and low pH (3.0–5.0) in vitro, to evaluate their capacity to grow under hostile and unfavorable conditions of the human digestive tract. Because milk is to be used as a carrier medium, their ability to grow in 10% sterile skim milk was also evaluated. The bifidobacteria count of the cultured milk samples (0, 24, and 48 h) was taken on tryptone yeast extract agar after 48 h of incubation in the presence of 10% CO₂ at 37°C. The changes in pH and developed titratable acidity were also recorded up to 96 h of incubation. The results indicated that all the isolates obtained had reasonable resistance to pH, bile, and phenol and were capable of growing well in milk. Among the 36 isolates, Bifidobacterium bifidum (isolates no. 4, 8, and 17) and B. breve (isolates no. 25 and 26) were the most tolerant to unfavorable conditions, and they may therefore be recommended for use in fermented milk or baby food formulations as probiotic dietary adjuncts.

Keywords: Bifidobacteria; growth characteristics in milk; acidity; count; pH; bile; phenol tolerance; probiotics

Transformation of textile dyes by white-rot fungus Trametes versicolor by Haresh Keharia; Datta Madamwar (pp. 99-108).

We have investigated transformation of eight industrial dyes by a whiterot fungus, Trametes versicolor. The fungus was found to decolorize Reactive Golden Yellow R, Procion Red, Reactive Violet 5, Reactive Blue 28, and Ponceau Red 4R at an initial dye concentration of 80 ppm within 72 h of incubation, whereas it took 5 d to completely decolorize Reactive Black 5 (40 ppm). However, it did not significantly decolorize Reactive Red 152 and Novatic Blue BC S/D. During decolorization in liquid medium, laccase and manganese-independent peroxidase (MiP) activities were detected in culture filtrate of T. versicolor. Dye-decolorizing activity of the culture was found to be associated with H₂O₂-dependent activity of the culture filtrate. Furthermore, dye-decolorizing activity of the culture filtrate was not influenced by Mn²⁺ or veratryl alcohol, thus suggesting a role of extracellular MiP in decolorization of synthetic dyes by T. versicolor.

Keywords: Decolorization; Trametes versicolor ; dyes; manganese-independent peroxidase; laccase

Production of ligninolytic enzymes for dye decolorization by cocultivation of white-rot fungi Pleurotus ostreatus and Phanerochaete chrysosporium under solid-state fermentation by Pradeep Verma; Datta Madamwar (pp. 109-118).

Lignocellulosic wastes such as neem hull, wheat bran, and sugarcane bagasse, available in abundance, are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. A ligninolytic enzyme system with high activity showing enhanced decomposition was obtained by cocultivation of Pleurotus ostreatus and Phanerochaete chrysosporium on combinations of lignocellulosic waste. Among the various substrate combinations examined, neem hull and wheat bran wastes gave the highest ligninolytic activity. A maximum production of laccase of 772 U/g and manganese peroxidase of 982 U/g was obtained on d 20 and lignin peroxidase of 656 U/g on d 25 at 28±1 °C under solid-state fermentation. All three enzymes thus obtained were partially purified by acetone fractionation and were exploited for decolorizing different types of acid and reactive dyes.

Keywords: Neem hulls; white-rot fungi; solid-state fermentation; ligninolytic enzymes; lignocellulosic waste

Optimization of media by evolutionary algorithms for production of polyols by S. V. Patil; V. K. Jayaraman; B. D. Kulkarni (pp. 119-128).

Biotransformation of sucrose-based medium to polyols has been reported for the first time using osmophilic yeast, Hansenula anomala. A new, real coded evolutionary algorithm was developed for optimization of fermentation medium in parallel shake-flask experiments. By iteratively employing the nature-inspired techniques of selection, crossover, and mutation for a fixed number of generations, the algorithm obtains the optimal values of important process variables, namely, inoculum size and sugar, yeast extract, urea, and MgSO₄ concentrations. Maximum polyols yield of 76.43% has been achieved. The method is useful for reducing the overall development time to obtain an efficient fermentation process.

Keywords: Media optimization; polyols yield; genetic algorithm

Effect of environmental factors and carbohydrate on gellan gum production by Basundhara Kanari; Ratindra Ram Banik; Siddha Nath Upadhyay (pp. 129-140).

Submerged culture fermentation studies were carried out in batch mode for optimizing the environmental parameters and carbon source requirement by Pseudomonas elodea for the production of gellan gum. The maximum production of gellan gum was obtained with 16-h-old culture and 8% inoculum at 30°C and pH 7.0 after 52 h of incubation (6.0 g/L). Of the various carbon sources tested, 2% sucrose, glucose, and soluble starch yielded considerably high amounts of gellan. Studies on the concentration of various carbohydrates on gellan gum production indicated that the optimum concentration of glucose and starch was 3%, whereas for sucrose it was 4%. The addition of glucose in the medium above 3% had a detrimental effect on gellan yield. The investigation of intermediate two-step addition of glucose under identical conditions of fermentation showed an enhanced production of gellan (8.12 g/L) as compared with the control (6.0 g/L). To optimize the recovery of gellan from fermented broth, different solvents were tested for precipitation of gellan gum. Among the various solvents tested, tetrahydrofuran gave better recovery of gellan (82%) as compared with the conventional solvent isopropanol (49%).

Keywords: Exopolysaccharide; gellan gum; Pseudomonas elodea ; batch fermentation

A biotechnological process involving filamentous fungi to produce natural crystalline vanillin from maize bran by Laurence Lesage-Meessen; Anne Lomascolo; Estelle Bonnin; Jean-Francois Thibault; Alain Buleon; Marc Roller; Michele Asther; Eric Record; Benoit Colonna Ceccaldi; Marcel Asther (pp. 141-153).

A new process involving the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus has been designed for the release of ferulic acid by enzymic degradation of a cheap and natural agricultural byproduct (autoclaved maize bran) and its biotransformation into vanillic acid and/or vanillin with a limited number of steps. On the one hand, the potentialities of A. niger I-1472 to produce high levels of polysaccharide-degrading enzymes including feruloyl esterases and to transform ferulic acid into vanillic acid were successfully combined for the release of free ferulic acid from autoclaved maize bran. Then vanillic acid was recovered and efficiently transformed into vanillin by P. cinnabarinus MUCL 39533, since 767 mg/L of biotechnologic vanillin could be produced in the presence of cellobiose and XAD-2 resin. On the other hand, 3-d-old high-density cultures of P. cinnabarinus MUCL39533 could be fed with the autoclaved fraction of maize bran as a ferulic acid source and a. niger I-1472 culture filtrate as an extracellular enzyme source. Under these conditions, P. cinnabarinus MUCL39533 was shown to directly biotransform free ferulic acid released from the autoclaved maize bran by A. niger I-1472 enzymes into 584 mg/L of vanillin. These processes, involving physical, enzymic, and fungal treatments, permitted us to produce crystallin vanillin from autoclaved maize bran without any purification step.

Keywords: Aroma; maize bran; enzyme; ferulic acid; vanillic acid; vanillin; Aspergillus niger ; Pycnoporus cinnabarinus

Siderophoregenic Bradyrhizobia boost yield of soybean by S. R. Khandelwal; A. V. Manwar; B. L. Chaudhari; S. B. Chincholkar (pp. 155-168).

After screening for siderophore (microbial iron chelator) production, of seven available cultures of soybean (Glycine max L.) root nodule bradyrhizobia, one strain, Bradyrhizobium japonicum NCIM 2746, was selected to confirm its phytopathogenic suppression and soybean growth promotion. Based on chromatographic and spectrophotometric studies, two different siderophores, a hydroxamate type (MW 734) and another catecholate type (MW 1000), were observed. Randomized block design (RBD) analysis of sickpot studies (soil inoculated with phytopathogens) with an MACS 124 variety of soybean, bacterized with siderophoregenic B. japonicum, showed a marked increase in the percentage of germination, nodulation, chlorophyll, oil, protein content, and number of pods. Field trial study confirmed these pot results, which were evident from enhancement in shoot length, number of branches, chlorophyll content, number of nodules, root length, and number of pods. These results suggest the possibility of exploiting B. japonicum NCIM 2746 as a potential bioinoculant.

Keywords: Bradyrhizobium japonicum ; siderophores; soybean; growth promotion

Relationship between coffee husk caffeine degradation and respiration of Aspergillus sp. LPBx in solid-state fermentation by Débora Brand; Ashok Pandey; Jose A. Rodriguez-Leon; Sevastianos Roussos; Ivo Brand; Carlos R. Soccol (pp. 169-177).

Studies were carried out in a packed-bed column fermentor using coffee husk as substrate in order to verify a relationship between caffeine degradation and the respiration of Aspergillus sp. LPBx. Fermentation conditions were optimized by using factorial design experiments. The kinetic study showed that the caffeine degradation was related to the development of mold and its respiration and also with the consumption of reducing sugars present in coffee husk. From the values obtained experimentally for oxygen uptake rate and CO₂ evolved, we determined a biomass yield of 3.811 g of biomass/g of consumed O₂ and a maintenance coefficient of 0.0031 g of consumed O₂/(g of biomass·h). The maximum caffeine degradation achieved was 90%.

Keywords: Coffee husk; factorial design; surface response methodology; solid-state fermentation; column fermentor

Gibberellic acid production by solid-state fermentation in coffee husk by Cristina M. M. Machado; Carlos R. Soccol; Brás H. de Oliveira; Ashok Pandey (pp. 179-191).

Five strains of Gibberella fujikuroi and one of Fusarium moniliforme were screened for the production of gibberellic acid (GA₃) in coffee husk, and based on the results, one strain, G. fujikuroi LPB-06, was selected. The comparative production of GA₃ by solid-state fermentation and submerged fermentation indicated better productivity with the former technique, mainly with pretreated substrate. The GA₃ accumulation was 6.1 times higher in the case of solid-state fermentation. Considering the C:N relation, higher yields of GA₃ were achieved using a mixed substrate comprising coffee husk and cassava bagasse (7:3, dry wt), increasing the results twice. Supplementation of an optimized saline solution containing 0.03% FeSO₄ and 0.01% (NH₄)₂SO₄ enhanced the accumulation of GA₃ 1.7 times in the fermented substrate. Under the finally optimized condition, the culture gave a maximum of 492.5 mg of GA₃/kg of dry substrate, with a pH of 5.3, moisture of 75%, and incubation temperature of 29°C. GA₃ yield was almost 13 times more than the initial results.

Keywords: Gibberellic acid; Gibberella fujikuroi ; biosynthesis; solidstate fermentation; coffee husk; alkali hydrolysis

Effect of pH on simultaneous saccharification and isomerization by glucoamylase and glucose isomerase by Abha Mishra; Meera Debnath (Das) (pp. 193-199).

pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum temperature for glucoamylase and glucose isomerase was 50 and 60°C, respectively. When both the enzymatic conversions were performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity of glucose isomerase at an acidic pH of 5.0.

Keywords: Glucoamylase; glucose isomerase; Aspergillus oryzae ; Streptomyces griseus

Toxicity assessment of nickel using Aspergillus niger and its removal from an industrial effluent by P. Rajendran; B. Ashokkumar; J. Muthukrishnan; P. Gunasekaran (pp. 201-206).

Chemical analysis of electroplating effluent revealed the presence of very high concentrations of nickel (393 ppm) in the effluent. Bioassay was carried out to test the toxicity of nickel chloride to Aspergillus niger. In contrast to 50% conidial inhibition at 1.7 mM nickel, hyphal extension was affected even at a lower concentration (0.4 mM), suggesting that hyphae are more sensitive than conidia to nickel. An increase in nickel concentration resulted in a proportionate decrease in the hyphal extension. Nickel (II)-resistant mutants of A. niger M1, M2, and M3, were obtained using direct selection, stepwise adaptation, and ultraviolet mutation techniques. Biosorption of Ni (II) by the mutant M3 was 50% more than that of its parent strain.

Keywords: Aspergillus niger ; nickel toxicity; bioremediation; biosorption

Remediation of textile effluent using agricultural residues by Chandrashekar B. Chandran; Dalel Singh; Poonam Nigam (pp. 207-212).

The sorption of artificial dye effluent made from two different dyes, Solar orange 7 GLL and Solar Jade Green FFB from Clariant, onto three different agricultural residues—barley husk, sugarcane bagasse, and wheat straw—was studied. Twenty percent of 600-µm particle size of these agricultural residues was used as substrates and studied individually. The percentage of dye removal was observed in concentrations of dye varying from 50 to 200 mg/L. The effect of temperature ranging from 25 to 50°C and pH from 6.0 to 12.0 on the dye removal by the substrates was also studied. The effective adsorption of the substrates was calculated.

Keywords: Agricultural residues; textile dyes; remediation; barley husk; sugarcane bagasse; wheat straw; Solar orange; Solar Jade Green

Phenotypic and genetic diversity of Bacillus thuringiensis strains isolated in India active against Spodoptera litura by S. R. Prabagaran; S. Jonathan Nimal; S. Jayachandran (pp. 213-226).

Bacillus thuringiensis strains isolated from different agroclimatic regions of India were found to harbor cry1 family genes. Of 831 strains 18 that were found to produce 130- and 68-kDa mol wt proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis were subjected to bioassay against second instar larvae of Spodoptera litura. According to the time response curve, while the highest toxic activity against S. litura was observed in PBT-782 with an LT₅₀ of 25.46 h, strains PBT-372, PBT-574, PBT-801, and PBT-716 in descending order of merit had LT₅₀ values of 36.81, 48.18, 50.35, and 73.53 h. The results of the field experiment testing the efficacy of different B. thuringiensis strains in controlling S. litura larvae infecting peanut plants showed that the chemical insecticide chlorpyriphos was the most effective in controlling S. litura throughout the study period. However, among B. thuringiensis strains, PBT-372 was superior. All the B. thuringiensis strains except PBT-689 were found to contain cry1Ac1-type gene. However, only nine strains contained cry1Aa1 gene. While cry1Ab1 was present only in PBT-372 and PBT-689, cry1Ca1 was present in PBT-574, PBT-688, PBT-689, and PBT-695. cry1Da1 was detected only in PBT-688 and PBT-692. None of the strains contained cry1Ba1 and cry1Ea1 genes. When polymerase chain reaction analysis using cry1Ca1 primer was performed, PBT-695 produced an unexpected 739-bp product, which showed 33% homology with cry1Ca1 gene between nucleotides 1819 and 2107. Our results indicated that among the field-collected B. thuringiensis strains, PBT-372 harbors multiple cry-type genes and could be employed for biological control of insects.

Keywords: Bacillus thuringiensis ; Indian isolates; polymerase chain reaction; cry genes; Spodoptera litura ; bioassays

Heavy metal removal by biosorption using Phanerochaete chrysosporium by M. Gopal; K. Pakshirajan; T. Swaminathan (pp. 227-237).

Biosorption using microbial cells as adsorbents is being seen as a cost-effective method for the removal of heavy metals from wastewaters. Biosorption studies with Phanerochaete chrysosporium were performed for copper (II), lead (II), and cadmium (II) to evaluate the effectiveness and to optimize the operational parameters using response surface methodology. The operational parameters chosen were initial metal ion concentration, pH, and biosorbent dosage. Using this method, the metal removal could be correlated to the operational parameters, and their values were optimized. The results showed fairly high adsorptive capacities for all the metals within the settings of the operational parameters. The removal efficiencies followed the order Pb>Cu>Cd. As a general trend, metal removal efficiency decreased as the initial metal ion concentration increased, and the results fitted the Langmuir and Freundlich isotherms well.

Keywords: Biosorption; Phanerochaete chrysosporium ; heavy metals; heavy metal removal; response surface methodology; adsorptive capacity

Regulation of apoptosis through arachidonate cascade in mammalian cells by Kohji Nishimura; Hirohumi Tsumagari; Asami Morioka; Yukiko Yamauchi; Kazuo Miyashita; Shan Lu; Mitsuo Jisaka; Tsutomu Nagaya; Kazushige Yokota (pp. 239-250).

The arachidonate cascade includes the cyclooxygenase (COX) pathway to form prostanoids and the lipoxygenase (LOX) pathway to generate several oxygenated fatty acids, collectively called eicosanoids. Eicosanoids are suggested to play a dual role in regulating cell survival and apoptosis in various types of cells through an unknown mechanism. We found apoptosis in cultured Madin-Darby canine kidney (MDCK) cells treated with 12-O-tetradecanoylphorbol β-acetate (TPA), a potent tumor promoter, and nordihydroguaiaretic acid (NDGA), a LOX inhibitor. The effect of TPA was synergistically stimulated along with NDGA. Aspirin, a COX inhibitor, was not effective. The target of NDGA might be different from the mechanism involving a LOX activity in some kinds of carcinoma cells because the increased expression of 12-LOX was not detected in MDCK cells treated with TPA. Caspase and poly(ADP-ribose) metabolites were found to be involved in the signal transduction pathway of the TPA- and NDGA-induced apoptosis in MDCK cells. Alternatively, hydrogen peroxide-induced apoptosis was not affected by NDGA. Thus, the TPA-induced response involved the mechanism independent of the oxidative stress. Obesity is a risk factor for severe diseases including noninsulin-dependent diabetes and atherosclerosis characterized by the changes of cell properties of adipocytes. We found that conjugated linolenic acid from bitter gourd was able to induce apoptosis in mouse preadipogenic 3T3-L1 cells. The findings provide the potential use of conjugated fatty acids to regulate obesity.

Keywords: Adipocyte; apoptosis; arachidonate cascade; conjugated linolenic acid; cyclooxygenase; lipoxygenase; Madin-Darby canine kidney; nordihydroguaiaretic acid; obesity; 12-O-tetradecanoylphorbol β-acetate

Solid-state fermentation for production of phytase by Rhizopus oligosporus by Abdulhameed Sabu; Saradamma Sarita; Ashok Pandey; Barbara Bogar; George Szakacs; Carlos Ricardo Soccol (pp. 251-260).

Solid-state fermentation of coconut oil cake has been carried out with Rhizopus oligosporus for the production of phytase. Phytase is used commercially in the animal feed industry to improve animal performance because there is a substantial and growing interest among swine and poultry producers in the application of phytase to improve the nutritional quality in animal feeds. Demonstrated benefits include improved feed yield ratios and reduction in the environmental costs associated with the disposal of animal wastes. We report the production of extracellular phytase by R. oligosporus under solid-state fermentation using coconut oil cake as substrate. Maximal enzyme production (14.29 U/g of dry substrate) occurred at pH 5.3, 30°C, and 54.5% moisture content after 96 h of incubation. The addition of extra nutrients to the substrate resulted in inhibition of product formation. The results indicate the scope for production of phytase using coconut oil cake as solid substrate without additional nutrients.

Keywords: Phytase; Rhizopus oligosporus ; coconut oil cake; solid-state fermentation

Cloning and sequencing of a phenol hydroxylase gene of Pseudomonas pseudoalcaligenes strain MH1 by H. Zouari; S. Moukha; M. Labat; S. Sayadi (pp. 261-276).

The phenol-degrading strain Pseudomonas pseudoalcaligenes MH1, identified by the rRNA approach, was isolated from wastewater enrichment culture. It utilized phenol up to 1.5 g/L as the sole source of carbon and energy. In addition, cresols (o-, m-, p-), 4-hydroxybenzoic acid, syringic acid, and vanillic acid were metabolized as sole substrates by phenol-grown cells of strain MH1. Using primers, designed on the basis of the sequence of the dmp operon of P. putida strain CF600, a gene encoding phenol hydroxylase, which catalyzes the hydroxylation of phenol to catechol, was detected in strain MH1. The whole phenol hydroxylase operon of strain MH1 was amplified in a polymerase chain reaction fragment of 5.207 kb that was cloned and sequenced. The total sequence revealed a cluster of six ATG starting open reading frames (ORFs). Analysis of the regulatory signals showed a putative promoter region, 40 bp upstream from the transcriptional start of ORF1, which have a strong homology to a set of positively controlled promoters. Comparison of the MH1 phenol hydroxylase gene sequence with those of other Pseudomonas strains revealed higher homology except in the 5’ region. Thus, the deduced amino acid sequence of the first subunit of phenol hydroxylase of P. pseudoalcaligenes strain MH1 exhibited a difference at the N-terminal region (the first 10 amino acids) compared with that of known phenol hydroxylase of Pseudomonas strains.

Keywords: Degradation; aromatic compounds; cresol; 4-hydroxybenzoic acid; syringic acid; vanillic acid; phenol hydroxylase; sequence; cloning

Potential use of cutinase in enzymatic scouring of cotton fiber cuticle by Ofir Degani; Shimon Gepstein; Carlos G. Dosoretz (pp. 277-289).

The present study characterized the ability of a bacterial cutinase to improve the wettability of raw cotton fabrics by specific hydrolysis of the cutin structure of the cuticle. The effect of cutinase was studied alone and in coreaction with pectin lyase. The changes in both the fabric and the reaction fluid were measured and compared to enzymatic hydrolysis with polygalacturonase, and to chemical hydrolysis with boiling NaOH. Water absorbancy, specific staining, fabric weight loss, and evaporative light-scattering reversephase high-performance liquid chromatography analysis of chloroform extract of the reaction fluid were measured to assess the enzymatic hydrolysis of the cuticle waxy layer. The pattern and extent of hydrolysis of the major cuticle constituents depended on the enzyme type and titers employed and paralleled the degree of wettability obtained. The combination of cutinase and pectin lyase resulted in a synergistic effect. The use of detergents improved enzymatic scouring. The major products released to the reaction medium by the cutinase treatment were identified by gas chromatography/mass spectrometry analysis as C:16 and C:18 saturated fatty acid chains.

Keywords: Cotton fiber; plant cuticle; cutinase; pectin lyase; polyalacturonase; bioscouring; textile industry

Engineering cytochrome P-450s by Suja Sukumaran; William Mark Atkins; Rishi Shanker (pp. 291-302).

Cytochrome P-450 isozymes represent a critical component of nature’s spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5α was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.

Keywords: Designer proteins; cytochrome P-450; P-450 101; chimeric P-450 2E1; homology modeling; camphor; detoxification; bacteria

Immobilization of α-amylase from Bacillus circulans GRS 313 on coconut fiber by Gargi Dey; Varima Nagpal; Rintu Banerjee (pp. 303-313).

A simple and inexpensive method for immobilizing α-amylase from Bacillus circulans GRS 313 on conconut fiber was developed. The immobilization conditions for highest efficiency were optimized with respect to immobilization pH of 5.5, 30°C, contact time of 4 h, and enzyme to support a ratio of 1:1 containing 0.12 mg/mL of protein. The catalytic properties of the immobilized enzyme were compared with that of the free enzyme. The activity of amylase adsorbed on coconut fiber was 38.7 U/g of fiber at its optimum pH of 5.7 and 48°C, compared with the maximum activity of 40.2 U/mL of free enzyme at the optimum pH of 4.9 and 48°C. The reutilization capacity of the immobilized enzyme was up to three cycles.

Keywords: Coconut fiber; adsorption; response surface methodology; amylase; Bacillus circulans GRS 313

Degradation of textile dyes mediated by plant peroxidases by T. S. Shaffiqu; J. Jegan Roy; R. Aswathi Nair; T. Emilia Abraham (pp. 315-326).

The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25–200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2–4 h and Brilliant Green 54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the halflife was found to be 60 h.

Keywords: Peroxidase; plant peroxidase; dye degradation; bioremediation; textile; dyes; azodyes; reactive dyes; Saccharum ; Ipomea

Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp by Shamla Ahammed; Parukuttyamma Prema (pp. 327-336).

The effect of carbon and nitrogen sources, lignocellulosic substrates, and metal ions on lignin peroxidase (LiP) activity of Aspergillus sp., which was isolated from a mangrove area, was studied. Glucose (1%) was found to be the best carbon source. Among the various lignocellulosic substrates used, coir pith at 3% concentration increased LiP activity twofold on the second day of incubation. Peptone and KNO₃ completely inhibited the enzyme synthesis while (NH₄)₂SO₄ at 12.5 mM produced maximum activity. Since seawater contained all the requisite metal ions, any added ions had a negative effect on activity. Cu²⁺ had the most inhibiting effect while K⁺ the least. When all the optimized conditions were provided, in nitrogen- and carbon-sufficient medium, a maximum LiP activity of 345 U/mL was obtained on the second day of incubation.

Keywords: Lignin degradation; white rot; soft rot; lignin peroxidase; Phanerochaete chrysosporium ; marine fungi; ligninase

Biodegradation of a medium-chain-length polyhydroxyalkanoate in tropical river water by Yen-Him Ho; Seng-Neon Gan; Irene K. P. Tan (pp. 337-347).

The medium-chain-length polyhydroxyalkanoate (PHA_MCL) produced by Pseudomonas putida PGA1 using saponified palm kernel oil as the carbon source could degrade readily in water taken from Kayu Ara River in Selangor, Malaysia. A weight loss of 71.3% of the PHA film occurred in 86 d. The pH of the river water medium fell from 7.5 (at d 0) to 4.7 (at d 86), and there was a net release of CO₂. In sterilized river water, the PHA film also lost weight and the pH of the water fell, but to lesser extents. The C8 monomer of the PHA was completely removed after 6 d of immersion in the river water, while the proportions of the other monomers (C10, C12, and C14) were reversed from that of the undegraded PHA. By contrast, the monomer composition of the PHA immersed in sterilized river water did not change significantly from that of the undegraded PHA. Scanning electron microscopy showed physical signs of degradation on the PHA film immersed in the river water, but the film immersed in sterilized river water was relatively unblemished. The results thus indicate that the PHA_MCL was degraded in tropical river water by biologic as well as nonbiologic means. A significant finding is that shorter-chain monomers were selectively removed throughout the entire PHA molecule, and this suggests enzymatic action.

Keywords: Biodegradation; medium-chain-length polyhydroxyalkanoate; tropical river water; saponified palm kernel oil

Modified enzymes for reactions in organic solvents by A. B. Salleh; M. Basri; M. Taib; H. Jasmani; R. N. Z. A. Rahman; M. B. A. Rahman; C. N. A. Razak (pp. 349-357).

Recent studies on biocatalysis in water—organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a w) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.

Keywords: Modified enzymes; organic solvents; polyethylene glycol; lipase

Screening of thermoacidophilic autotrophic bacteria for covellite solubilization by Valentina V. Umrania; Jitendra S. Joshi (pp. 359-366).

In an attempt to obtain suitable bacterial isolates for bioleaching of copper from chalcopyrite and covellite, soil samples taken from areas of the metal industry were screened using an enrichment procedure specially run at acidic pH and thermophilic temperature range, to overcome the limitations of mesophiles employed for the purpose besides having economic and environmental advantages. Of a total of 47 isolates, the most promising 3 having resistance to copper toxicity were evolved by subjecting them to gradually increasing concentrations of CuSO₄ by acclimatization runs conducted on an environmental shaker for 125 d at 65°C. The isolates, JVCu-8, JVCu-10, and JVCu-12, exhibited significantly enhanced bioleaching and copper tolerance ability at pH 3.5 and 60–70°C. The total solubilization of copper recorded was 87, 89.4, and 91.2% by JVCu-8, JVCu-10, and JVCu-12, respectively, and these isolates exhibited tolerance to CuSO₄ concentrations of 6.9, 7.2, and 7.2%, respectively. The isolates morphologically resembled Thiobacillus and Sulfolobus.

Keywords: Bioleaching; acidothermophiles; covellite and chalcopyrite solubilization; copper tolerant bacteria

Biosynthetic activity of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II by Yukti Bhatia; Saroj Mishra; Virendra S. Bisaria (pp. 367-379).

The hydrolysis and biosynthetic reactions of partially purified Pichia etchellsii β-glucosidase II from recombinant Escherichia coli pBG22:JM109 are described. With 167 mmol/L of initial glucose, the products of synthetic reactions, glucobiose and glucotriose, accumulated to 18 and 6 mmol/L, respectively. In transglycosylation reactions with 79 mmol/L of initial cellobiose, glucotriose and glucopentaose were obtained at 4.5 and 2 mmol/L, respectively. The effects of incubation time and substrate concentration were studied on the yield of synthesized oligosaccharides. In a reaction time of 24 h with 468 mmol/L of initial cellobiose, glucotriose and glucopentaose levels of 21.6 and 6.6 mmol/L, respectively, were obtained. The addition of dimethyl sulfoxide (DMSO) further increased the yields of the products by 10%. Detailed kinetic analysis indicated a significant (about twofold) increase in V _max/K M of synthetic reactions in the presence of DMSO. A study of other disaccharides in transglycosylation reactions indicated biosynthetic activity in the order of sophorose > gentiobiose > cellobiose.

Keywords: β-Glucosidase; Pichia etchellsii ; oligosaccharide synthesis

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum by Saurabh Chattopadhyay; A. K. Srivastava; V. S. Bisaria (pp. 381-393).

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20°C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH₄ ⁺:NO₃ ⁻ ratio (total nitrogen concentration of 60 mM) and PO₄ ³⁻ did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH₄ ⁺:NO₃ ⁻ ratio, and PO₄ ³⁻) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.

Keywords: Podophyllum hexandrum ; podophyllotoxin; Plackett-Burman design; response surface methodology; central composite design

Optimization of fermentation conditions for production of glycopeptide antibiotic vancomycin by Amycolatopsis orientalis by P. Naga Padma; A. Bhaskar Rao; J. S. Yadav; Gopal Reddy (pp. 395-405).

Glycopeptides produced by Streptomyces species are the drugs used against β-lactam drug-resistant staphylococcal infections, and vancomycin is important among them. Increased prevalence of resistant strains increased the usage of vancomycin worldwide and also promoted attempts for indigenous production. The optimum process conditions pH, temperature, inoculum size, agitation, and aeration for vancomycin production by Amycolatopsis orientalis were evaluated, statistically analyzed, and the response surface curves were constructed. The optimum process conditions were a pH of 7.6, a temperature of 29°C, an inoculum size of 4.5%, an agitation of 255 rpm, and an aeration of less than 1:10 medium-to-air ratio.

Keywords: Fermentation; antibiotic; glycopeptide; vancomycin; optimization

Culture conditions dictate protease and tannase production in submerged and solid-state cultures of Aspergillus niger Aa-20 by Cristóbal Noé Aguilar; Ernesto Favela-Torres; Gustavo Vinegra-González; Christopher Augur (pp. 407-414).

Undesirable protease production by Aspergillus niger Aa-20 in submerged culture and solid-state culture was evaluated using different concentrations of tannic acid as sole carbon source in a model system designed for tannase production. Protease production was found to be dependent on the culture system used (submerged culture or solid-state culture) and on the initial tannic acid concentration. Expression of protease activity in submerged culture was higher (up to 10 times) than activity obtained in solid-state culture, using identical culture medium composition. In submerged culture, the lowest final protease activity (0.13 IU) was obtained with the highest tannic acid concentration, while in solid-state culture protease activity was not affected by changes in initial substrate concentration. Absence of detectable proteolytic activity in solid-state culture is related to high production of tannase enzyme. Hence, the use of solid-state culture for fungal enzyme production may allow for higher and more stable enzyme titers present in culture extracts.

Keywords: Protease; solid-state fermentation; tannase; tannin acyl hydrolase; liquid fermentation; submerged fermentation; Aspergillus niger ; tannic acid

Mn²⁺ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions by Roni Cohen; Limor Persky; Zahit Hazan-Eitan; Oded Yarden; Yitzhak Hadar (pp. 415-429).

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn²⁺ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn²⁺ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of ¹⁴C-lignin mineralization was measured in the Mn²⁺-amended BSM, as determined during the course of 47 d of fermentation. Mn²⁺ amendment of the PM increased mineralization rate to that obtained in the Mn²⁺-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn²⁺ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn²⁺ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.

Keywords: Pleurotus ostreatus ; manganese peroxidase; versatile peroxidase; lignin degradation; solid-state fermentation

Effect of NaCl on in vitro propagation of sweet potato (Ipomoea batatas L.) by Archana Mukherjee (pp. 431-441).

in vitro propagation protocols offer a better option for production of quality planting materials in a clonal crop such as sweet potato, which is a food crop of versatile uses. Propagation through axillary shoot proliferation and organogenic and embryogenic regeneration were studied in different genotypes of sweet potato. The addition of NaCl enhanced the rate of multiplication as well as yielded hardy somatic embryos. Optimal doses of NaCl in each mode of propagation were different. The hardy somatic embryos produced in NaCl medium could be stored at 8°C with or without a protective alginate covering. High-frequency germination of stored hardy somatic embryos could facilitate the production of artificial seeds. Plantlets produced with the addition of NaCl in regeneration medium were established in vivo at a high frequency (95–100%). Yield and quality of storage roots developed from artificial seed-propagated plants were comparable with those of source plants.

Keywords: Sweet potato; organogenesis; somatic embryogenesis; artificial seeds; sodium chloride; storage roots; isozymes

Anaerobic-anoxic-aerobic sequential degradation of synthetic wastewaters by Saswati Chakraborty; H. Veeramani (pp. 443-451).

This study was conducted in a continuous three-stage system of anaerobic (R1)-anoxic(R2)-aerobic (R3) reactors with synthetic wastewater containing phenol (1000 mg/L), chemical oxygen demand (COD) (3000 mg/L), CN⁻ (30 mg/L), SCN⁻(400 mg/L), and NH ₄ ⁺ -N (600 mg/L) as principal pollutants and well-acclimated heterogeneous microbial cultures. The final effluent was partially returned to R2 with a recycle ratio of 1. Anaerobic stage served to detoxify the feed by removing up to 80% of cyanide. Complete SCN⁻ removal and denitrification could be achieved in the anoxic stage by utilizing phenol as an internal source of carbon. Nitrification efficiency of 93% was obtained in the aerobic reactor. The results demonstrated that the three-stage system can give the desired final treated effluent quality (0 mg/L of phenol, 0.2 mg/L of CN⁻, 210 mg/L of COD, and 20 mg/L of NH ₄ ⁺ -N) and that the NO ₃ ⁻ -N concentration can be lowered by a higher recycle ratio.

Keywords: Complex wastewater; acclimation; anaerobic detoxification; nitrification; denitrification; recycle ratio

Production of α-amylase with Aspergillus oryzae on spent brewing grain by solid substrate fermentation by B. Bogar; G. Szakacs; R. P. Tengerdy; J. C. Linden; A. Pandey (pp. 453-461).

Ten Aspergillus oryzae strains were screened in solid substrate fermentation for α-amylase production on spent brewing grain (SBG) and on corn fiber. SBG proved to be a better substrate for enzyme production than corn fiber. A Plackett-Burman experimental design was used to optimize the medium composition for the best strain. Solid substrate fermentation on optimized medium with A. oryzae NRRL 1808 (=ATCC 12892) strain in stationary 500-mL Erlenmeyer flask culture yielded 4519 U of α-amylase/g of dry matter substrate in 3 d. The whole solid substrate fermentation material (crude enzyme, in situ enzyme) may be considered a cheap biocatalytic material for animal feed rations and for bioalcohol production from starchy materials.

Keywords: Amylase; solid substrate fermentation; Aspergillus oryzae ; spent brewing grain; Plackett-Burman experimental design

Comparison of volatile compound production in fruit body and in mycelium of Pleurotus ostreatus identified by submerged and solid-state cultures by W. Kabbaj; S. Breheret; J. Guimberteau; T. Talou; J. -M. Olivier; M. Bensoussan; M. Sobal; S. Roussos (pp. 463-469).

Comparative analyses of the production of volatile compounds by Pleurotus ostreatus JMO.95 fruit body and its corresponding mycelium grown in liquid, on agar surface, and on solid support cultures were carried out by dynamic headspace concentration using gas chromatography/mass spectrometry and gas chromatography sniffing. The aroma produced by fruit body was owing essentially to the presence of octan-3-one and, to a lesser extent, to octan-3-ol. Other compounds, such as oct-1-en-3-ol, oct-1-en, 2-methylbutanol, and α-pinene were also present in low concentrations. Comparison of aromatic spectra of the fruit body with that of mycelia obtained under different culture conditions indicated that the main aromatic compounds present in the P. ostreatus fruit body and mycelium were produced in the same proportions on agar surface and on solid support culture, but not under submerged conditions.

Keywords: Pleurotus ostreatus ; mushroom fruit body; mycelium; oct-1-en-3-ol; aroma; headspace concentration; gas chromatography/mass spectrometry; gas chromatography sniffing

Reversible denaturation behavior of immobilized glucose oxidase by M. D. Gouda; M. S. Thakur; N. G. Karanth (pp. 471-480).

Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25±1°C. The soluble enzyme required a GdmCl concentration of 5M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.

Keywords: Glucose oxidase; guanidine hydrochloride; reversible denaturation; renaturation; stabilizing agents; immobilization

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.102-103, #1-6)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.102-103, #1-6)