Enzymes In Cell-free Extracts Research Articles

Synthesis of β(1 → 3) and β(1 → 6) galactooligosaccharides from lactose and whey using a recombinant β-galactosidase from Pantoea anthophila

Background: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by β-galactosidases. Results: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with β(1 → 6) and β(1 → 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. Conclusions: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Biological Control of Thielaviopsis paradoxa and Colletotrichum gloeosporioides by the Extracellular Enzymes of Wickerhamomyces anomalus

An alternative to chemical fungicides in post-harvest diseases are the use of biocontrol agents and their extracellular products against phytopathogens. Two relevant agents in post-harvest infections are Thielaviopsis paradoxa and Colletotrichum gloeosporioides, causing large economic losses in cacao, pineapple, and avocado during storage. In this work, we evaluated the effect of Wickerhamomyces anomalus, an effective biocontrol agent, against these filamentous fungi, focusing on the production of extracellular enzymes and their effect on fungal growth and germination. Moreover, we evaluated the use of inactivated fungal biomass as an inducer in complete (Potato Dextrose Agar and Yeast Peptone Dextrose) and minimal culture media. The antagonistic effect of W anomalus on the growth of both phytopathogens was also studied. The extracellular enzymes in YPD cultures, using T. paradoxa inactivated biomass as the best inducer, were capable of inhibiting the germination of both phytopathogens. In minimal media, only the production of a 30 kDa glucanase with activity against laminarin was observed. The enzyme was effective against the spore germination of T. paradoxa. In post-harvest crop protection tests, growth inhibition of T. paradoxa was observed using the cell-free enzyme extract, which is a promising system to protect cocoa fruits from T. paradoxa during post-harvest.

The special effect of the Aspergillus flavus and its enzymes on biological degradation of the intact polylactic acid (PLA) and PLA-Jute composite

The biodegradation of PLA and PLA-Jute (64/36) in an aqueous media with Aspergillus flavus CCUG 28296, as well as its cell-free enzyme extract, was investigated through their physical, molecular, and thermal characterization. Results indicated that the thicknesses of the fungal treated PLA and PLA-jute samples during seven months have reduced by 52% and 63%, respectively while for the enzyme-treated samples, 45% and 49% reduction in the thickness has occurred. Moreover, the gel permeation chromatography (GPC) revealed a substantial decrease (about 75%) in the weight average molecular weight (Mw) of PLA and PLA-Jute treated with fungus, which confirmed the effective performance of A. flavus on the biological degradation of PLA. The obtained results were further supported by differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) of the treated and control samples as well. Interestingly, the observed reduction in the Mw of PLA in PLA-Jute was 64% after the enzymatic treatment, while for the unblended PLA, it was just about 32%. These results pointed to the synergistic effect of jute on PLA degradation because of the promiscuous activity of the effective enzymes on jute degradation, which could accelerate the PLA decomposition.

Cell-assisted synthesis of conducting polymer - polypyrrole - for the improvement of electric charge transfer through fungal cell wall.

In this research we report the biological synthesis of electrically conducting polymer - Polypyrrole (Ppy). Cell-assisted enzymatic polymerization/oligomerization of Ppy was achieved using whole cell culture and cell-free crude enzyme extract from two white-rot fungal cultures. The selected fungal strains belong to Trametes spp., known laccase producers, commonly applied in bioremediation and bioelectrochemical fields. The biocatalytic reaction was initiated in situ through the copper-containing enzymes biosynthesized within the cell cultures under submerged aerobe cultivation conditions. The procedure was inspired by successful reports of laccase-catalyzed pyrrole polymerization. The usage of whole culture and/or crude enzyme extract has the advantage of overcoming enzyme purification and minimizing the liability of enzyme inactivation through improved stability of enzymes in their natural environment. Spectral and electrochemical techniques (UV-vis spectroscopy, infrared spectroscopy; cyclic voltammetry (CV)) and pH measurements provided insight into the evolution of pyrrole polymerization/oligomerization and the electrochemical features of the final product. Microscopy techniques (optical microscopy and scanning electron microscopy (SEM)) were primary tools for visualization of the formed Ppy particles. The relevance of our research is twofold: Ppy prepared in crude enzyme extract results in enzyme encapsulated within Ppy and/or Ppy-modified fungal cells can be formed when polymerization occurs in whole cell culture. The route of biocatalysis can be chosen according to the desired bioelectrochemical application. The reported study focuses on the improvement of charge transfer through the fungal cell membrane and/or cell wall by modification of the fungal cells with conducting polymer - polypyrrole.

Microbial cell-free extracts affect the biochemical characteristics and sensorial quality of sourdough bread

This study aimed to improve the sensorial quality of sourdough wheat bread by the addition of cell-free enzyme extracts (CFEs) from Lactobacillus sanfranciscensis (SF), Hafnia alvei (HF) and Debaryomyces hansenii (DH). CFEs were suitable sources of peptidases, glutamate dehydrogenase and cystathionine γ-lyase. The concentration of free amino acids (FAA) in the sourdoughs containing CFEs was higher than the control sourdough, produced without addition of CFEs. The community-level catabolic profiles showed that the highest number of carbohydrates, polymers and carboxylic acids were consumed in the SF sourdough. Breads produced with CFEs were characterized by higher specific volume than the control. The use of CFEs impacted on the profile of volatile organic compounds. Overall, positive correlations were found between some key-aroma compounds and enzyme activities/precursor FAA. The SF bread, characterized by highest level of alcohols, received the highest score for aroma and sweetness in the sensory analysis.

Multiple microbial cell-free extracts improve the microbiological, biochemical and sensory features of ewes’ milk cheese

This study used cell-free enzyme (CFE) extracts from Lactobacillus casei, Hafnia alvei, Debaryomyces hansenii and Saccharomyces cerevisiae to condition or accelerate Pecorino-type cheese ripening. Compositional, microbiological, and biochemical analyses were performed, and volatile and sensory profiles were obtained. Lactobacilli and cocci increased during ripening, especially in cheeses containing CFE from L. casei, H. alvei and D. hansenii (LHD-C) and L. casei, H. alvei and S. cerevisiae (LHS-C). Compared to control cheese (CC), several enzymatic activities were higher (P < 0.05) in CFE-supplemented cheeses. Compared to the CC (1907 mg kg−1 of cheese), the free amino acid level increased (P < 0.05) in CFE-supplemented cheeses, ranging from approximately 2575 (LHS-C) to 5720 (LHD-C) mg kg−1 of cheese after 60 days of CFE-supplemented ripening. As shown by GC/MS analysis, the levels of several volatile organic compounds were significantly (P < 0.05) lower in CC than in CFE-supplemented cheeses. All cheeses manufactured by adding multiple CFEs exhibited higher scores (P < 0.05) for internal structure, acid taste and juiciness than CC samples. This study shows the possibility of producing ewes’ milk cheese with standardized characteristics and improved flavor intensity in a relatively short time.

Digestion of Tannin by Bacteria Enterobacter cloacae from the Gut of Indian Mole Cricket (Gryllotalpa krishnani)

Insects are the most successful animal on earth; the gut microbes present might play an important role in food digestion as well as the interaction with hosts. In this study insect Gryllotalpa krishnani has been used to isolate the symbiotic associated organisms which play role in host metabolism, promote efficient digestion and to protect the host from the harmful microbes. In the present study, microorganisms were isolated from the gut of G. krishnani and characterized for tannase enzyme activity. It was observed that 5 isolates (TAH 6, TAH 13, TAH 36, TAH 38 and TAH 41) out of total 120 tested were able to show significant growth on tannic acid plate. Among these five potential isolates, strain TAH 41 exhibited maximum tannase activity in tannase plate assay and was selected for the further study. The bacterial strain TAH41 was analyzed for biochemical analysis, 16S rDNA sequencing and the result confirmed the strain as Enterobacter cloacae. HPLC analysis showed the formation two peaks representing gallic acid and glucose as a by-product FT-IR analysis also confirmed the same. The polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis showed the molecular mass of tannase enzyme in cell free extract was ~45 kDa, the analysis suggested this tannase enzyme to be one of the smallest of the bacterial source, which could be attributed to the formation of di or triA¢Â€Â‘galloyl glucose. The present study was the first report E. cloacae with tannase activity from G. krishnani gut. E. cloacae which may endow the insect with some ecological advantages by enabling them to overcome the anti-nutritional effects of plant tannins.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus

β-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000U of β-galactosidase activity per liter of culture corresponding to approximately 170mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205gL−1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

Kinetic properties of pyruvate ferredoxin oxidoreductase of intestinal sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9

Intestinal sulfate-reducing bacteria reduce sulfate ions to hydrogen sulfide causing inflammatory bowel diseases of humans and animals. The bacteria consume lactate as electron donor which is oxidized to acetate via pyruvate in process of the dissimilatory sulfate reduction. Pyruvate-ferredoxin oxidoreductase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. have never been well-characterized and have not been yet studied. In this paper we present for the first time the specific activity of pyruvate-ferredoxin oxidoreductase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains. Microbiological, biochemical, biophysical and statistical methods were used in this work. The optimal temperature (+35°C) and pH 8.5 for enzyme reaction were determined. The spectral analysis of the puri- fied pyruvate-ferredoxin oxidoreductase from the cell-free extracts was demonstrated. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period) and maximum velocity of the pyruvate-ferredoxin oxidoreductase reaction (V ) were defined. Michaelis constants (Km) of the enzyme reaction were calculated for both intestinal bacterial strains. The studies of the kinetic enzyme properties in the intestinal sulfate-reducing bacteria strains in detail can be prospects for clarifying the etiological role of these bacteria in the development of inflammatory bowel diseases.

Activity and kinetic properties of phosphotransacetylase from intestinal sulfate-reducing bacteria

Phosphotransacetylase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. has never been well-characterized and has not been studied yet. In this paper, the specific activity of phosphotransacetylase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains were presented at the first time. The microbiological, biochemical, biophysical and statistical methods in this work were used. The optimal temperature and pH for enzyme reaction was determined. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period, τ) and maximum velocity of the phosphotransacetylase reaction (Vmax) were defined. Michaelis constants (Km) of the enzyme reaction (3.36 ± 0.35 mM for D. piger Vib-7, 5.97 ± 0.62 mM for Desulfomicrobium sp. Rod-9) were calculated. The studies of the phosphotransacetylase in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in intestinal sulfate-reducing bacteria, their production of acetate in detail can be perspective for clarification of their etiological role in the development of the humans and animals bowel diseases. These studies might help in predicting the development of diseases of the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.

Kinetic properties of adenosine triphosphate sulfurylase of intestinal sulfate-reducing bacteria.

The investigation of specific activity of ATP sulfurylase and kinetic properties of the enzyme in cell-free extracts of intestinal bacterial strains Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 is presented. The microbiological, biochemical, biophysical and statistical methods were used in the work. The optimal temperature (35°C) and pH 8.0-8.5 for enzyme reaction were determined. An analysis of kinetic properties of ATP sulfurylase has been carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period, τ) and maximum velocity of the ATP sulfurylase reaction (Vmax) have been defined. Michaelis constants (Km(Sulfate), Km(ATP), Km(APS), and Km(Pyrophosphate)) of the enzyme reaction were demonstrated for both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains.

Biodegradation of dimethyl phthalate ester using free cells, entrapped cells of Variovorax sp. BS1 and cell free enzyme extracts: A comparative study

Whole cells of Variovorax sp. BS1 had the ability to biodegrade up to 600 mg L−1 (3.08 mM) of dimethyl phthalate (DMP) at the end of 30 h of incubation at 30 °C. The total organic carbon analyses suggested mineralization of DMP using isolated bacterial strain. The cell-free extracts of Variovorax sp. BS1 contained mixture of enzymes required for the complete biodegradation of DMP, diethyl phthalate (DEP) and dibutyl phthalate (DBP). Transient intermediates or end products were not detected by HPLC and LC-MS analyses during or after biodegradation of DMP by Variovorax sp. BS1. Addition of Zn+2 to assay mixture containing DMP and cell free extract resulted in the accumulation of detectable quantity of metabolite, ortho-phthalate thereby suggesting that DMP biodegradation proceeded via de-esterification reactions. Cells of Variovorax sp. BS1 entrapped in calcium alginate beads could be re-used for atleast ten sequential cycles in plain water with diminutive loss of DMP biodegradation activity. Results suggested that whole cells, alginate entrapped cells of Variovorax sp BS1 and enzymes present in the cell free extracts may be used for efficient biodegradation of dimethyl phthalate.

Catalase Activity of Sulfate-Reducing Bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 Isolated from Human Large Intestine

Catalase activity of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 isolated from the human large intestine was studied. The high activity of the enzyme in cell-free extracts of both bacterial strains was determined (1745.21±154.67 and 873.11±72.23 U×mg-1 protein for D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively). The effect of different temperature and pH as well as H2O2 concentration and time of incubation on the catalase activity in the cell-free extracts of D. piger Vib-7 and Desulfomicrobium sp. Rod-9 were examined. The maximum catalase activity for both bacterial strains at +30ºC temperature was determined. The highest activity of the studied enzyme in the cell-free extracts of D. piger Vib-7 at pH 7.5 and Desulfomicrobium sp. Rod-9 at pH 7.0 was measured. Based on experimental data, the analysis of the kinetic properties of the catalase by the studied bacteria was carried out. Increasing of hydrogen peroxide concentrations from 0.5 to 5.0 mM causes a monotonic rise of studied enzyme activity and the activity was maintained on an unchanged level (plateau) under substrate concentrations over 5.0 mM. The catalase activity, initial (instantaneous) reaction rate (V0) and maximum rate of the catalase reaction (Vmax) were significantly higher in D. piger Vib-7 than in Desulfomicrobium sp. Rod-9 cells. Michaelis constants (Km) of the catalase reaction were 8.01±0.77 and 10.33±0.98 mM for D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively. DOI: http://dx.doi.org/10.3329/mh.v3i1.19776 Microbes and Health, June 2014. 3(1): 15-20

The effect of alkanes on the physiology and metabolism of the entomopathogenic fungus, Isaria fumosorosea

The influence of different alkanes on spore morphology, glyoxlate pathway enzyme activities, total lipid contents and fatty acid composition of Isaria fumosorosea were investigated under laboratory conditions. Fungal spores grown on different alkanes showed higher germination and mycelial growth when compared to control. A strong induction of glyoxlate cycle enzymes in cell-free extracts was observed for cells grown on different alkanes when compared to glucose and control. Higher activities of glyoxlate cycle enzymes were observed for cells grown on alkanes when compared to other treatments. Even numbered fatty acids accounted for the majority of fatty acid production with a significant increase in relative amounts of linoleic acid and palmatic acid observed for conidia grown on alkanes. These results indicate that addition of alkanes to culture media can be a tool to pre-induce metabolic adaptations that can facilitate successful infection of insect host by entomopathogenic fungi.

Enhanced artemisinin production from engineered yeast precursors upon biotransformation

Production of artemisinin in genetically modified microorganisms is an attractive option to enable sufficient supply of the effective antimalarial agent. Although a sundry of artemisinin precursors are available from engineered bacteria or yeast, no artemisinin has been manufactured by engineering any microbial platforms due to inaccessibility to unidentified steps. To this end, it is essential to consider how to convert artemisinin precursors to artemisinin, either biochemically or chemically. To establish a novel procedure of artemisinin production, we incubate the mixture of artemisinin precursors from engineered Sacchromyces cerevisiae with the cell-free enzyme extract of Artemisia annua. For the single gene-expressing strain INVScI (pYES-ADS), amorpha-4,11-diene accumulation within 48 h or 14 days led to higher artemisinin content than the control. In the multiple gene-expressing strain YPH501 (pYES-ADS:: pESC-CYP71AV1-DBR2), artemisinin accumulation from the 14-day-induced yeast precursor mixture was nearly equivalent between the single gene-transferred strain and the multiple gene-transferred strain. Alternatively, biotransformation of 48-hour-induced yeast amorpha-4,11-diene mixture by the cold-acclimated A. annua cell-free extract that possesses the abundant enzymes relevant to artemisinin biosynthesis gave rise to considerable elevation of artemisinin content up to 0.647% in maximum, accounting to 15-folds increase as the A. annua cell-free extract without cold-acclimation (0.045%), thereby providing a practical protocol for artemisinin overproduction through the interplay of engineered microbial artemisinin precursors with upregulated plant enzymes.

Biosynthesis of a cholesterol-derived brassinosteroid, 28-norcastasterone, in Arabidopsis thaliana

A metabolic study revealed that 28-norcastasterone in Arabidopsis is synthesized from cholesterol via the late C-6 oxidation pathway. On the other hand, the early C-6 oxidation pathway was found to be interrupted because cholestanol is converted to 6-oxocholestanol, but further metabolism to 28-norcathasterone was not observed. The 6-oxoBRs were found to have been produced from the respective 6-deoxoBRs administered to the enzyme solution, thus indicating that these 6-oxoBRs are supplied from the late C-6 oxidation pathway. Heterologously expressed CYP85A1 and CYP85A2 in yeast catalysed this C-6 oxidation, with CYP85A2 being much more efficient than CYP85A1. Abnormal growth of det2 and dwf4 was restored via the application of 28-norcastasterone and closer precursors. Furthermore, det2 and dwf4 could not convert cholesterol to cholestanol and cholestanol to 6-deoxo-28-norcathasterone, respectively. It is, therefore, most likely that the same enzyme system is operant in the synthesis of both 28-norcastasterone and castasterone. In the presence of S-adenosyl-L-methionine, the cell-free enzyme extract catalysed the C-24 methylation of 28-norcastasterone to castasterone, although the conversion rates of 28-norteasterone to teasterone and 28-nortyphasterol to typhasterol were much lower; this suggests that 28-norcastasterone is the primary precursor for the generation of C28-BRs from C27-BRs.

Isolation and characterization of tetrachloroethylene- and cis-1,2-dichloroethylene-dechlorinating propionibacteria

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹⁴C] PCE and [1,2-¹⁴C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.

Process development for the production of prebiotic galacto‐oligosaccharides from lactose using β‐galactosidase fromLactobacillussp.

Galacto-oligosaccharides (GOS) are formed from lactose in discontinuous mode of conversion using beta-galactosidase from Lactobacillus sp. (beta-gal). The discontinuous process was optimized for technical application with regard to GOS yield, enzyme preparation, reaction temperature and substrate source. It proved to be advantageous to directly apply the crude cell-free enzyme extract for the conversion, since similar GOS yields and composition were obtained as when using the pure enzyme preparation, but expensive purification could be avoided. Reaction temperature was lowered to 17 degrees C to limit microbial contamination when using technical substrates. Thereby GOS yield decreased from 30% to 28% of total sugars and enzyme demand increased 2.7-fold. Whey permeate was compared to buffered lactose solution as a substrate source. The initial reaction rate was found to be 1.8 times higher for the whey permeate substrate; however, GOS yield was slightly lower (approximately 25% of total sugar at 17 degrees C) mainly due to smaller amounts of allolactose[beta-D-Galp-(1-->6)-D-Glc] and the trisaccharide beta-D-Galp-(1-->6)-D-Lac formed.

Brassinosteroids are inherently biosynthesized in the primary roots of maize, Zea mays L.

GC-MS analysis revealed that primary roots of maize contain 6-deoxocathasterone, 6-deoxoteasterone and 6-deoxotyphasterol. These brassinosteroids, and the previously identified campesterol, campestanol, 6-deoxocastasterone and castasterone, in the roots are members of a biosynthetic pathway to castasterone, namely the late C-6 oxidation pathway, suggesting that its biosynthetic pathway is operative in the roots. To verify this, a cell-free enzyme extract was prepared from maize roots, and enzymatic conversions from campesterol to castasterone through the aforementioned sterols and brassinosteroids were examined. The presence for the biosynthetic sequences, campesterol → 24-methylcholest-4-en-3β-ol → 24-methylcholest-4-en-3-one → 24-methylcholest-5 α-cholestan-3-one → campestanol and 6-deoxoteasterone → 6-deoxo-3-dehydroteasterone → 6-deoxotyphasterol → 6-deoxocastasterone → castasterone were demonstrated. These results indicate that maize roots contain a complete set of enzymes involved in the late C-6 oxidation pathway, thereby demonstrating that endogenous brassinosteroids are biosynthesized in the roots.

Hexavalent Chromium Reduction by Immobilized Cells and the Cell-Free Extract of Bacillus sp. ES 29

Bacillus sp. ES 29 (ATCC: BAA-696) is an efficient chromate reducing bacterium. We evaluated hexavalent chromium (Cr[VI]) reduction by immobilized intact cells and the cell-free enzyme extracts of Bacillus sp. ES 29 in a bioreactor system. Influences of different flow rates (3 to 14 mL h−1), Cr(VI) concentration (2 to 8 mg L−1), and immobilization support materials (Celite, amberlite, and Ca-alginate) on Cr(VI) reduction were examined. Both immobilized intact cells and the cell-free extract of Bacillus sp. ES 29 displayed substantial Cr(VI) reduction. Increasing flow rates from 3 to 6 mL h−1 did not affect the rate of Cr(VI) reduction, but above 6 mL h−1, the Cr(VI) reducing capacity of the immobilized intact cells and cell-free extract of Bacillus sp. ES 29 decreased. With both intact cells and the cell-free extracts, the rate of Cr(VI) reduction was inversely related to the concentration. Intact cells immobilized to Celite displayed the highest rate (k = 0.443 at 3 mL h−1) of Cr(VI) reduction. For the immobilized cell-free extract, maximal reduction (k = 0.689 at 3 mL h−1) was observed with Ca-alginate. Using initial Cr(VI) concentrations of 2 to 8 mg L−1 at flow rates of 3 to 6 mL h−1 both immobilized intact cells and the cell-free extracts reduced 84 to 98% of the influent Cr(VI). Results indicate that immobilized cells and the cell-free extracts of Bacillus sp. ES 29 could be used for large-scale removal of Cr(VI) from contaminated water and waste streams in containment systems.