Construction,expression and characterization of fusion enzyme from <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis> dextranase and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> amylase

An artificial fusion protein of Arthrobacter oxydans dextranase and Klebsiella pneumoniae α-amylase was constructed and expressed in Escherichia coli. Most of the expressed protein existed as an insoluble fraction, which was solubilized with urea. The purified fusion enzyme electrophoretically migrated as a single protein band; M = 137 kDa, and exhibited activities of both dextranase (10.8 U mg⁻¹) and amylase (7.1 U mg⁻¹), which were lower than that of reference dextranase (13.3 U mg⁻¹) and α-amylase (103 U mg⁻¹). The fusion enzyme displayed bifunctional enzyme activity at pH 5–7 at 37°C. These attributes potentially make the fusion enzyme more convenient for use in sugar processing than a two-enzyme system.     

Evaluation of inert and organic carriers for <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation

Growth and sporulation of Verticillium lecanii on inert and organic carriers (sugar-cane bagasse, corncob, rice straw, polyurethane foam and activated carbon) in a solid-state fermentation process was studied. Sugar-cane bagasse and polyurethane foam produced 10¹⁰ spores g⁻¹ dry carrier whereas corncob, rice straw, and activated carbon yielded, respectively 8 × 10⁹, 4 × 10⁹, and 3 × 10⁸ spores g⁻¹. Chitinase activity of the conidia was in the following order: sugar-cane bagasse (3.3 U mg⁻¹) > wheat bran (3.0 U mg⁻¹) > polyurethane foam (2.7 U mg⁻¹). There was no significant difference (2.5–2.7 U mg⁻¹) in the proteinase activity among the conidia from the three cultures. Scanning electron microscopy shows that aerial mycelium freely penetrated into the internal area of polyurethane foam. Sugar-cane bagasse provided enough area for vegetative hyphae to attach. Of the carriers analyzed, polyurethane foams and sugar-cane bagasse were the best carriers for V. lecanii growth and spore production.     

Cholesterol assimilation and biotransformation by Lactobacillus helveticus

Lactobacillus helveticus, grown at 37°C in MRS medium supplemented with 3 mM cholesterol, assimilated all the cholesterol in 42 h having 68 U mg⁻¹ of intracellular cholesterol oxidase activity. The strain transformed 1 g cholesterol to 0.05 g of androsta-1, 4-diene-3, 17-dione and 0.04 g of androst-4-ene-3, 17 dione within 48 h at 37°C with extracellular cholesterol oxidase activity at 12 U mg⁻¹ and intracellular oxidase at 0.5 U mg⁻¹.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Optimal activity and thermostability of xylose reductase from <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-170

Xylose reductase (XR) is the enzyme that catalyzes the first step of xylose metabolism. Although XRs from various yeasts have been characterized, little is known about this enzyme in Debaryomyces hansenii. In the present study, response surface analysis was used to determine the optimal conditions for D. hansenii UFV-170 XR activity. The influence of pH and temperature, ranging from 4.0 to 8.0 and from 25 to 55°C, respectively, was evaluated by a 2² central composite design face-centered. The F-test (ANOVA) and the Student’s t test were performed to evaluate the statistical significance of the model and the regression coefficients, respectively. The NADPH-dependent XR activity varied from 0.502 to 2.53 U mL⁻¹, corresponding to 0.07–0.352 U mg⁻¹, whereas the NADH-dependent one was almost negligible. The model predicted with satisfactory correlation (R ² = 0.940) maximum volumetric activity of 2.27 U mL⁻¹ and specific activity of 0.300 U mg⁻¹ at pH 5.3 and 39°C, which were fairly confirmed by additional tests performed under these conditions. The enzyme proved very stable at low temperature (4°C), keeping its activity almost entirely after 360 min, which corresponded to the half-time at 39°C. On the other hand, at temperatures ≥50°C it was lost almost completely after only 20 min.     

An improved nitrilase-mediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced <Emphasis Type="Italic">Nocardia globerula</Emphasis> NHB-2

Nitrilase of Nocardia globerula NHB-2 was induced by short-chain aliphatic nitriles (valeronitrile > isobutyronitrile > butyronitrile > propionitrile) and exhibited activity towards aromatic nitriles (benzonitrile > 3-cyanopyridine > 4-cyanopyridine > m-tolunitrile > p-tolunitrile). Hyperinduction of nitrilase (6.67 U mg_DCW⁻¹, 18.7 U mL⁻¹) was achieved in short incubation time (30 h, 30°C) through multiple feeding of isobutyronitrile in the growth medium. The nitrilase of this organism exhibits both substrate and product inhibition effects. In a fed batch reaction at 1 L scale using hyperinduced resting cells corresponding to 10 U mL⁻¹ nitrilase activity (1.5 mg_DCW mL⁻¹), a total of 123.11 g nicotinic acid was produced at a rate of 24 g h⁻¹ g_DCW⁻¹.     

Biochemical characterization of laccase from hairy root culture of <Emphasis Type="Italic">Brassica juncea</Emphasis> L. and role of redox mediators to enhance its potential for the decolorization of textile dyes

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg⁻¹ of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg⁻¹ specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0–6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and l-cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.     

Characterization of trehalose synthase from <Emphasis Type="Italic">Corynebacterium nitrilophilus</Emphasis> NRC

Trehalose synthase (TSII) from Corynebacterium nitrilophilus NRC was successively purified by ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-100 columns. The specific activity of the trehalose synthase was increased ~200-fold, from 0.14 U mg⁻¹ protein to 28.3 U mg⁻¹ protein. TSII was found to be a monomeric protein with a molecular weight of 67–69 kDa. Characterization of the enzyme exhibited optimum pH and temperature were 7.5 and 35°C, respectively. The purified enzyme was stable from pH 6.6 to 7.8 and able to prolong its thermal stability up to 35°C. The enzyme activity was inhibited strongly by Zn²⁺, Hg²⁺ and Cu²⁺ and moderately by Ba²⁺, Fe²⁺, Pb²⁺ and Ni²⁺. Other metal ions Ca²⁺, Mg²⁺, Co²⁺, Mn²⁺ and EDTA had almost no effect.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Characterization of a soluble oxidoreductase from the thermophilic bacterium Carboxydothermus ferrireducens

An NAD(P)H-dependent oxidoreductase has been purified approximately 40-fold from the soluble protein fraction of the dissimilatory iron-reducing, anaerobic, thermophilic bacterium Carboxydothermus ferrireducens. The enzyme, a flavoprotein, has broad-substrate specificity—reducing Fe³⁺, Cr⁶⁺, and AQDS with rates of 0.31, 0.33, and 3.3 U mg⁻¹ protein and calculated NADH oxidation turnover numbers of 0.25, 0.25, and 2.5 s⁻¹, respectively. Numerous quinones are reduced via a two-electron transfer from NAD(P)H to quinone, thus participating in managing oxidative stress by avoiding the formation of semiquinone radicals.     

Dielectric barrier discharge plasma as a novel approach for improving 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Dielectric barrier discharge plasma was used to generate a stable strain of Klebsiella pneumoniae (designated to as Kp-M2) with improved 1,3-propanediol production. The specific activities of glycerol dehydrogenase, glycerol dehydatase and 1,3-propanediol oxidoreductase in the crude cell extract increased from 0.11, 9.2 and 0.15 U mg⁻¹, respectively, for wild type to 0.67, 14.4 and 1.6 U mg⁻¹ for Kp-M2. The glycerol flux of Kp-M2 was redistributed with the flux to the reductive pathway being increased by 20% in batch fermentation. The final 1,3-propanediol concentrations achieved by Kp-M2 in batch and fed-batch fermentations were 19.9 and 76.7 g l⁻¹, respectively, which were higher than those of wild type (16.2 and 49.2 g l⁻¹). The results suggested that dielectric barrier discharge plasma could be used as an effective approach to improve 1,3-propanediol production in K. pneumoniae.     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the -factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg^–1 protein which was more than a recombinant P. pastoris GS115 (552 U mg^–1 protein) or KM71H (539 U mg^–1 protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg^–1 protein by P. pastoris GS115, 1176 U mg^–1 protein by P. pastoris KM71H and 1522 U mg^–1 protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 °C) than the wild-type PLC from B. cereus. Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co²⁺ and Mn²⁺ etc., also influenced the activity of the recombinant PLCs.     

Lignocellulose-degrading enzyme production by white-rot <Emphasis Type="Italic">Basidiomycetes</Emphasis> isolated from the forests of Georgia

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml⁻¹) and xylanase (195 U ml⁻¹) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l⁻¹), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570–620 U l⁻¹) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Purification and characterization of a cold active alkaline protease from <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp., isolated from Kashmir,India

A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4–37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg⁻¹). The maximum specific enzyme activity (398 U mg⁻¹) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg⁻¹. The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10–60°C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures.     

Overexpression of β-glucosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> for the production of highly purified aglycone isoflavones from soy flour

To produce aglycone isoflavones from soy flour, the β-glucosidase A gene (bglA) of Thermotoga maritima was overexpressed in Escherichia coli BL21-CodonPlus (DE3)-RIL. The K _m and V _max values of the purified BglA for pNPG were 0.43 mM and 323.6 U mg⁻¹, respectively, and those for salicin were 9.0 mM and 183.2 U mg⁻¹, respectively. The biochemical and kinetic characteristics of his-tagged BglA were found to be similar to those of BglA, except for the temperature stability and specific activity. Production of aglycone isoflavones from soy flour by BglA was examined by HPLC. For 3 h at 80°C, all the isoflavone glycosides approximated to the complete conversion into aglycone isoflavones, over seven times higher than that obtained from soy flour without BglA.     

Cholesterol biotransformation to androsta-1,4-diene-3,17-dione by growing cells of <Emphasis Type="Italic">Chryseobacterium gleum</Emphasis>

Cholesterol oxidase activity was studied during biotransformation of cholesterol to androsta-1,4-diene-3,17-dione (ADD) by Chryseobacterium gleum. Spent LB media, containing cholesterol (3 mM≈1 g l⁻¹) where the bacterium was grown for 24 h, at 30°C with constant shaking at 120 rpm, had the highest enzyme activity (167 U mg⁻¹). The growing cells produced 0.076 g ADD from 1 g cholesterol l⁻¹.     

Production and activities of chitinases and hydrophobins from Lecanicillium lecanii

The production of chitinases and hydrophobins from Lecanicillium lecanii was influenced by the cultivation method and type of carbon source. Crude enzyme obtained from solid-substrate culture presented activities of exochitinases (32 and 51 kDa), endochitinases (26 kDa), β-N-acetylhexosaminidases (61, 80, 96 and 111 kDa). Additionally, submerged cultures produced exochitinases (32 and 45 kDa), endochitinases (10 and 26 kDa) and β-N-acetylhexosaminidases (61, 96 and 111 kDa). β-N-acetylhexosaminidases activity determined in solid-substrate culture with added chitin was ca. threefold (7.58 ± 0.57 U mg⁻¹) higher than submerged culture (2.73 + 0.57 U mg⁻¹). Similarly, hydrophobins displayed higher activities in solid-substrate culture (627.3 ± 2 μg protein mL⁻¹) than the submerged one (57.4 ± 4.7 μg protein mL⁻¹). Molecular weight of hydrophobins produced in solid-substrate culture was 7.6 kDa and they displayed surface activity on Teflon.     

<Emphasis Type="SmallCaps">l</Emphasis>-tryptophan decarboxylase activity and tryptamine accumulation in callus cultures of <Emphasis Type="Italic">Vinca minor</Emphasis> L.

l-tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyses the formation of tryptamine from tryptophan, and therefore it plays a role in terpenoid indole alkaloids biosynthesis. In this study, TDC activity and tryptamine accumulation were monitored in callus cultures of important medicinal plant Vinca minor L. Callus cultures, established from leaf tissues, were incubated on Murashige and Skoog (MS) medium supplemented with 4.4 μM kinetin and different concentrations (0.44, 1.1, 2.2, 4.4 and 6.6 μM) of naphthaleneacetic acid (NAA), and grown either in the dark or under 16 h photoperiod. When the basal enzyme activity of TDC was determined in these cultures, it was 0.5–0.7 nmol tryptamine mg⁻¹ prot. min⁻¹. Moreover, this activity remained linear over time and over protein concentrations, and with optimum pH levels between 6.5 and 7.5, and an optimum temperature of 35°C. The Michaelis–Menten constant (K_m) for l-tryptophan was 1.3 mM. TDC cofactor, pyridoxal-5′-phosphate (1 mM), increased the enzyme activity. During later stages of callus culture growth cycle, an increase in TDC activity was observed, and this activity depended on culture conditions and age of callus cultures. In addition, TDC activity and tryptamine accumulation in callus cultures were strongly enhanced by light treatment.