Simultaneous determination of hydrolysis and mutarotation rates during the enzymatic hydrolysis of lactose

An experiment is described in which a custom-made glucose electrode is used to directly monitor the enzymatic hydrolysis of lactose to glucose. The transient profile of beta- d-glucose can be used to simultaneously determine the rate constants for mutarotation and for enzymatic hydrolysis by applying a dynamic nonlinear regression routine. Due to differences in the mutarotation rate constants between lactose and glucose, the beta- d-glucose concentration "overshoots" equilibrium under certain conditions, which can be modeled mathematically. This overshoot can be observed reliably and used to quantify the differences in mutarotational equilibria between glucose and lactose. These observations may be important for the analysis of dairy products and commercial lactase preparations and illustrate an unusual kinetic phenomenon caused by intramolecular forces. This approach may also be important for the accurate determination of a variety of oligosaccharides such as glycogen, which tend to be composed primarily of one stereoisomer.     

Stimulation of nodulation and plant growth of chickpea (Cicer arietinum L.) by Pseudomonas spp. antagonistic to fungal pathogens

Two strains of Pseudomonas MRS23 and CRP55b showed antagonistic activity towards the pathogenic fungi Aspergillus sp., Fusarium oxysporum f. sp. ciceri, Pythium aphanidermatum and Rhizoctonia solani under culture conditions. Larger growth inhibition zones were obtained on nutrient agar (NA) and King's B media in comparison to potato dextrose agar and pigment production media. Both the strains produced siderophore in agar plates as well as in liquid cultures. Fungal inhibition zones were reduced in size and abolished in iron-supplemented NA medium by Pseudomonas strains MRS23 and CRP55b, respectively, indicating that some other metabolites along with siderophores are involved in growth inhibition of fungi by strain MRS23, whereas CRP55b produced only siderophores. Only Pseudomonas strain MRS23 was found to produce hydrocyanic acid (HCN). Seed bacterization with Pseudomonas strains of two chickpea (Cicer arietinum L.) cultivars, H8618 and C235, showed root-stunting effects at 5 days, whereas this inhibitory effect was overcome at 10 days of seedling growth in cv. H8618. Coinoculation of chickpea with Pseudomonas strains MRS23 and CRP55b, and Mesorhizobium sp. Cicer strain Ca181 resulted in the formation of 68.2-115.4% more nodules at 80 and 100 days after planting as compared to single inoculation with the Mesorhizobium strain under sterile conditions. The shoot dry weight ratios of coinoculated treatments at different stages of plant growth varied from 1.18 to 1.35 times that of Mesorhizobium-inoculated and 3.25 to 4.06 times those in uninoculated controls. The plant N contents were also increased significantly on coinoculation. Coinoculation effects of HCN-producing strain MRS23 were significantly lower than those of non-HCN-producing strain CRP55b in terms of shoot dry weight and shoot N. The results demonstrated the potential benefits of using rhizosphere bacteria as coinocula in nodule promotion and plant growth in chickpea.     

Cloning and characterization of a plant defensin VaD1 from azuki bean

A recombinant mungbean defensin VrD1 was previously shown to exhibit antifungal and bruchid-resistant activity. To study the function and regulation of VrD1, genomic DNAs of plant defensins were isolated from Vigna radiata VC6089A and azuki bean Vigna angularis Kao Hsiung No. 6. The azuki bean defensin genomic DNA VaD1 was sequenced and converted to VaD1 cDNA. VaD1 defensin was purified from Vigna angularis Kao Hsiung No. 6 to apparent homogeneity. The complete amino acid sequence of the purified VaD1 was determined and was found to be exactly the same as the sequence deduced from VaD1 cDNA. VaD1 is a basic protein containing 46 amino acids with four conserved disulfide bonds and shares high sequence homology (78.3%) with VrD1. VaD1 inhibited the growth of Fusarium oxysporum, Fusarium oxysporum f. sp. pisi, Staphylococcus epidermidis, and Salmonella typhimurium. VaD1 also inhibited in vitro protein synthesis and bruchid larval development, but was less active than the recombinant VrD1.     

大米降压肽酶法制备工艺及其性质研究

该文利用碱性蛋白酶水解大米蛋白制备大米降压肽，确定碱性蛋白酶水解终点为水解度18.17%。采用9种大孔吸附树脂吸附大米降压肽，结果显示NKA型树脂对大米降压肽的吸附效果较好，大米降压肽中灰分含量由脱盐前的16.64%减少到脱盐后的2.32%。对大米降压肽的相对分子质量、溶解性进行分析，大米降压肽的相对分子质量在138～1461之间；在pH 3～11的范围内，大米降压肽的溶解度在97.6%左右。     

Performance of chickpea (Cicer arietinum L.) to bio-fertilizer and nitrogen application in arid condition

Abstract

Field experiments were conducted during 2013–2014 at Tashkent, Uzbekistan to evaluate the performance of chickpea variety “Jakhongir” with the variable proportion of nitrogen (N) and bio-fertilizer inoculation in the moderate saline (5.6?±?0.6?dSm^?1) soil condition. The studied treatments were No control (non-fertilized), N1 mineral-N (50?kg?N?ha^?1), N2, mineral-N (75?kg?N?ha^?1), N3, mineral-N (100?kg?N?ha^?1) equivalent 0%, 50%, 75%, and 100% from recommended rate for chickpea, Rhizobium inoculation (Bio)?+?No control, Rhizobium inoculation (Bio)?+?N1, Rhizobium inoculation (Bio)?+?N2, and Rhizobium inoculation (Bio)?+?N3. Seed inoculation with Rhizobium was significantly superior over no inoculation treatments at all rate of N fertilization. The middle rate of N fertilization 75?kg?N?ha^?1 combined with biofertilizer inoculation had of superior effect on chickpea, producing 73.2% more yield (1.68?Mg ha^?1), oil, protein, and sugar content performed 16.4%; 15.0%, and 17.9% higher value, respectively, in comparison to control.     

Evaluation of plant growth promoting activities of microbial strains and their effect on growth and yield of chickpea (Cicer arietinum L.) in India

The aims of our study were to enhance growth, yield and disease control of chickpea by various combinations of microbial strains (Mesorhizobium, Azotobacter chroococcum, Pseudomonas aeruginosa and Trichoderma harzianum). Pseudomonas and Trichoderma showed positive IAA (indole-3-acetic acid) production, phosphate solubilisation and antagonistic activities against Fusarium oxysporum and Rhizoctonia solani as compared to other strains. In two year investigations, tetra-inoculants have shown significant growth attributes, yield and phytopathogen growth inhibition followed by tri-inoculants than control. Therefore, tetra-inoculants (Mesorhizobium-Azotobacter-Pseudomonas-Trichoderma) may be used as efficient biofertilizer and bio-control agent for chickpea production (Cicer arietinum L.) in eastern Uttar Pradesh.     

Chemical hybridizing agents for chickpea (Cicer arietinum L.): leads from QSAR analysis of ethyl oxanilates and pyridones

In the self-pollinated crops such as chickpea, induction of male sterility by deployment of chemical hybridizing agents (CHAs) facilitating "two-line" approach holds immense potential in heterosis breeding. A total of 40 test CHAs comprising 20 ethyl oxanilates and 20 pyridones were screened as potential CHAs on chickpea (variety BG 1088) at 500, 800, and 1000 ppm. Three test compounds mostly having either F (4)/Br (5)/CF(3) (19) at the para position of the aryl ring from a pool of 20 ethyl oxanilates were identified as the most potent CHAs causing >or=99% induction of pollen sterility and >90% total flower sterility at 1000-ppm test concentration. Among pyridone derivatives, N-(4-chlorophenyl)-5-carbethoxy-4,6-dimethyl, 1,2-dihydropyrid-2-one (26) was found to be the most active. Quantitative structure activity relationship (QSAR) analysis has revealed a direct involvement of Swain-Lupton field constant, F(p), with the target bioactivity which implied that field rather than resonance effect (R) had a positive effect on the activity. The real guiding principle for selectivity was found out to be the hydrophobic parameter pi value. The QSAR models indicated that increased steric bulk at the 4-position on the phenyl ring is associated with enhanced activity. The CHAs appeared to act by mimicking UDP-glucose, the key substrate in the synthesis of callose, or lead to an imbalance in acid-base equilibrium in pollen mother cells resulting in dissolution of callose wall by premature callase secretion.     

Effect of inoculum rate on the performance of chickpea (Cicer arietinum L.) Rhizobium strains in the field

Summary The influence of three inoculum rates on the performance of three chickpea (Cicer arietinum L.) Rhizobium strains was examined in the field on a Mollisol soil. Increasing amounts of inoculum improved the performance of the strains. A normal dose (10⁴ cells per seed) applied at different intervals gave non-significant increases in nodulation, nitrogenase activity (acetylene reduction assay), nitrogen uptake and grain yield. A ten-fold increase in inoculum increased nodule number, shoot dry weight, nitrogenase activity (ARA) and grain yield, but increases over the control were significant only for nodule dry weight and nitrogen uptake by shoot and grain. The highest level of inoculum (100 × normal) significantly increased nodule dry weight, grain yield, total nitrogenase activity (ARA) and nitrogen uptake by shoot and grain. Strain TAL 620 was more effective than the other two. Combined nitrogen (60 kg N ha^–1) suppressed nodulation and nitrogenase activity (ARA).Research paper No. 4345 from the Experiment Station, G. B. P. U. A. & T., Pantnagar, Nainital, U. P.     

Influence of Vermicompost Fertilizer and Water Deficit Stress on Morpho-Physiological Features of Chickpea (Cicer arietinum L. cv. karaj)

One goal in the face of deficit water conditions is to increase growth and yield. Agro-industrial production frequently causes environmental pollution by using chemical fertilizers. In recent decades, bio-fertilizers such as vermicompost have been used as a safe alternative to chemical fertilizer. The present study considered the response of the chickpea to different combinations of vermicompost and water deficit stress in a greenhouse environment. Plant response was determined by measuring a range of morpho-physiologic parameters. The treatments were addition of 0%, 10%, 20%, and 30% of vermicompost to soil, and water deficit stress at the following levels: non-stress (100% of field capacity), moderate water stress (75% of field capacity), and severe water stress (25% of field capacity). The results showed that vermicompost had a significant effect on all traits under stress and non-stress conditions. The vermicompost treatments under non-stress conditions significantly increased plant height, number of pods, leaf area, stem and leaf dry weight, pod dry weight, chlorophyll a, carotenoid, total chlorophyll content, CO₂ assimilation rate, internal CO₂ concentration, and water-use efficiency over that of the control condition. The addition of 30% vermicompost under moderate and severe water stress conditions significantly increased plant height, number of pods, leaf area, leaf dry weight, carotenoids, and water-use efficiency over that of the control level. This study confirmed that vermicompost improved the morphological features, soil biological activity, and quality of the chickpea, but did not positively influence the physiological features under moderate and severe water deficit stress.     

Topography of the Cotyledon Surfaces and Adjoining Seed Coat of Chickpea (Cicer arietinum L.) Genotypes Differing in Milling Performance

Milling of pulse seeds generally refers to dehulling (decortication or seed coat removal) and splitting (division of the two cotyledons) to produce split seeds, known as dhal. Reduction of whole seeds or dhal to flour, in comparison, is generally termed “grinding” for pulses. Many pulses are consumed as dhal, including desi chickpea (Cicer arietinum L.), faba bean (Vicia faba L.), lentil (Lens culinaris L.), field pea (Pisum sativum L.), pigeon pea (Cajanus cajan L.), mungbean (Vigna radiata L.), black gram (Vigna mungo L.), and hyacinth bean (Lablab purpureus L.). Hence, ease‐of‐milling is an important quality attribute of pulse species that are commonly dehulled or split prior to consumption. Seed structure and the surface topography at the junctions between seed coat and cotyledons have the potential to influence the varietal differences observed in the ease of milling. The epicuticular wax surfaces of six genotypes of chickpea differing in ease of milling were examined by scanning electron microscopy to evaluate whether the physical attributes of seed coat and cotyledon surfaces could be involved in adhesion. Differing epicuticular wax patterns were observed for each of the six genotypes. The possible roles these patterns play in genotypic differences in ease of milling are discussed.     

Stimulatory effect of phosphate-solubilizing fungal strains (Aspergillus awamori and Penicillium citrinum) on the yield of chickpea (Cicer arietinum L. cv. GPF2)

The effect of six phosphate-solubilizing fungi (PSF, two strains of Aspergillus awamori, and four of Penicillium citrinum) isolated from rhizosphere of various crops, was observed on the growth and seed production of chickpea plants (Cicer arietinum L. cv. GPF2) in pot experiments. The phosphate (P) solubilizing activity of PSF in liquid varied from 38 to 760 μg ml^?1 for tricalcium phosphate (TCP) and 28–248 μg ml^?1 for mussoorie rock phosphate (MRP). All PSF isolates were biocompatible and produced growth-promoting hormone, Indole acetic acid (IAA), varying in concentration from 2.5 to 9.8 μg ml^?1. Of the various pot experiments carried out in green house, maximum stimulatory effect on chickpea plants growth was observed by inoculation of two A. awamori strains. This treatment resulted in 7–12% increase in shoot height, nearly three-fold increase in seed number and two-fold increase in seeds weight as compared to the control (un-inoculated) plants. Inoculation of four strains of P. citrinum exhibited lesser stimulatory effect. It showed 7% increase in shoot height, two-fold increase in seed number and 87% increase in seeds weight as compared to the control plants. However, a consortium of all the six fungal isolates showed no stimulatory effect on chickpea plants growth.     

Impact of single and co-inoculations with Rhizobial and PGPR isolates on chickpea (Cicer arietinum) in cereal-growing zone soil

Strains isolated from chickpea (Cicer arietinum L.) rhizospheric soil from selected sites in Algeria were screened for their plant-growth-promoting potential, for indole acetic acid production and P solubilization ability. Then, we selected native rhizobial strains with high nitrogen-fixing potential. On the basis of their efficiency under controlled conditions, two plant-growth-promoting rhizobacteria (PGPR) isolates and three nodulating bacteria were selected. Then, the effect of single PGPR isolates inoculation was compared to their combination with rhizobial inoculants on plant growth, on native cereal-growing soils under greenhouse conditions. No effects were observed on chickpea yield by using rhizobial inoculation alone, nor by PGPR-rhizobial co-inoculation on two soils presenting weak and no nodulation pattern in natural conditions. Only PGPR inoculation improved growth of plants on soil with no nodulation pattern. These findings emphasized inoculation on native soils at a little scale before large assays on field because no one could predict inocula behavior with native soil microflora.     

Improving nodulation,growth and yield of Cicer arietinum L. through bacterial ACC-deaminase induced changes in root architecture

Bacteria containing ACC-deaminase in the vicinity of roots may influence plant growth by modifying root architecture through their potential to regulate ethylene synthesis in plant roots. Approximately 138 isolates capable of utilizing ACC as the sole source of N were isolated from the rhizosphere soil of chickpea (Cicer arietinum L.) plants. Under axenic conditions, some rhizobacterial isolates were highly effective in increasing root length (up to 2.08 fold), number (up to 3.7 fold) and length (up to 3.9 fold) of lateral roots, and root biomass (up to 83%) of chickpea as compared to uninoculated control. Serratia proteamaculans strain J119 was found to be the most effective plant growth promoting rhizobacterium (PGPR) in improving root and shoot growth, nodulation and grain yield of chickpea as compared to respective controls in growth pouches, pot and field trials. A highly significant direct correlation (r = 0.99) was observed between number of lateral roots under axenic conditions (jar trial) and number of nodules per plant in pot and field trials. Interestingly, S. proteamaculans J119 also exhibited highest ACC-deaminase activity in addition to root colonization compared to other tested strains. The results of this study demonstrated that changes in root growth and architecture (particularly lateral roots) as a result of inoculation with PGPR containing ACC-deaminase are crucial for improving growth, yield and nodulation of chickpea under field conditions.     

Comparison between discontinuous and continuous lactose conversion processes for the production of prebiotic galacto-oligosaccharides using beta-galactosidase from Lactobacillus reuteri

Galacto-oligosaccharide (GOS) formation from lactose in discontinuous and continuous modes of conversion was investigated using beta-galactosidase (beta-gal) from Lactobacillus reuteri. A continuous stirred tank reactor (CSTR) with an external crossflow membrane was set up, and continuous GOS production was analyzed and compared to the batchwise formed GOS product. Marked differences were detected for the two reactor setups. Above 65% lactose conversion, the GOS yield was lower for the CSTR due to a lower content of tri- and tetrasaccharides in the reaction mixture. In the CSTR, beta-gal from L. reuteri showed up to 2-fold higher specificity toward the formation of beta-(1-->6)-linked GOS, with beta-D-Galp-(1-->6)-D-Glc and beta-D-Galp-(1-->6)-D-Gal being the main GOS components formed under these conditions. This could be used to synthesize more defined GOS products.     

Investigation of enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle (Collichthys niveatus) and evaluation of its functional properties

This study was carried out to investigate the enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle of the marine fish species Collichthys niveatus. About 160 fish samples were tested, and the analyzed fish species was found to be a lean fish with low fat (1.77 ± 0.01%) and high protein (16.76 ± 1.21%). Fish muscle of C. niveatus was carefully collected and hydrolyzed with four commercial enzymes: Alcalase, Neutrase, Protamex, and Flavourzyme under the conditions recommended by the manufacturers. Among the tested proteases, Neutrase catalyzed the hydrolysis process most effectively since the hydrolysate generated by Neutrase has the highest content of sweet and umami taste amino acids (SUA). The effect of hydrolysis conditions was further optimized using response surface methodology (RSM), and the optimum values for temperature, pH, and enzyme/substrate ratio (E/S ratio) were found to be 40.7 °C, 7.68, and 0.84%, respectively. Finally, the amino acid composition of the hydrolysate was analyzed by AccQ·Tag derivatization and HPLC-PDA determination. Major amino acids of the muscle of C. niveatus were threonine, glutamic acid, phenyalanine, tryptophan, and lysine, accounting for respectively 10.92%, 10.85%, 10.79%, 9.86%, and 9.76% of total amino acid content. The total content of essential amino acids was 970.7 ng·mL(-1), while that of nonessential amino acids was 709.1 ng·mL(-1). The results suggest that the fish muscle and its protein hydrolysate from C. niveatus provide a versatile supply of the benefits and can be incorporated as supplements in health-care foods.     

Growth,nutrient uptake and yield parameters of chickpea (Cicer arietinum L.) enhance by Rhizobium and Azotobacter inoculations in saline soil

Abstract

A field experiment was conducted during two consecutive growing seasons (2013 and 2014) to evaluate the effects of inoculations with Rhizobium and Azotobacter on the growth and yield of two chickpea (Cicer arietinum L.) varieties under saline (5.8 dS m^?1) arid condition. The single treatment of either Rhizobium or Azotobacter exhibited to promote the growth of chickpea to some level, however, co-inoculation produced more effects and increased the shoot dry weight (30.3 and 26.4%), root dry weight (17.5 and 26.3%), nodule number (79.1 and 43.8 piece per plant), nitrogen content in roots (9.62 and 10.9%), in shoots (12.6 and 8.3%) and seed protein (7.1 and 4.3%) in both Flip06-102 and Uzbekistan-32 chickpea varieties compared to the control. Our studies showed that the highest yield response of 429 (27.9%) and 538 (23.9%) kg?ha^?1 over the control was revealed by the co-inoculation with Rhizobium and Azotobacter inoculants in Flip 06-102 and Uzbekistan-32, respectively. A new introduced Flip 06-102 chickpea variety was more salt tolerant and had higher root nodulation than the local Uzbekistan-32 chickpea variety. Nitrogen (N), phosphorus (P), and potassium (K) contents in the shoots and roots were significantly (p?Rhizobium plus Azotobacter could be applied to improve the vegetative growth and yield of chickpea and to alleviate the effects of salt stress.     

Fractionation and enzymatic hydrolysis of soluble protein present in waste liquors from soy processing

A waste effluent from a soymeal concentrates plant was centrifuged and ultrafiltered by successive processing in membranes of 10, 30, and 50 kDa, with further concentration of the resulting stream using a 5 kDa membrane. The separated fractions (5-10, 10-30, 30-50, and >50 kDa) were subjected to chemical, nutritional, and functional characterization. Resuspension of the retentates in salt-containing systems improved the protein solubility, the emulsifying capacity, and the gelation capacity, whereas the emulsion stability and the foam capacity and stability decreased with respect to the values obtained using distilled water, and the oil absorption capacity was not affected. The digestibility of the fraction >50 kDa was comparable to that of casein. The fractions of higher molecular mass (30-50 and >50 kDa) were subjected to enzymatic hydrolysis with a commercial protease, to give products with improved emulsifying activity and stability, particularly for hydrolysates with a degree of hydrolysis between 20 and 30%.     

Effect of processing methods on the calcium, phosphorus, and phytic acid contents and nutritive utilization of chickpea (Cicer arietinum L.).

The effect of chickpea (Cicer arietinum L.) processing methods on the nutritive utilization of calcium and phosphorus and on phytic acid, a seed component that affects mineral utilization, was studied. Chemical and biological methods were used for nutritional determinations in growing rats. The digestive utilization of calcium from raw chickpea was adequate for growing rats; however, processing resulted in a slight decrease. The metabolic utilization of chickpea calcium was low because of the low rates of net absorption. This was reflected in the decreased calcium levels in longissimus dorsi muscle in the absence of mobilization of calcium from the femur. Soaking in acid solution followed by cooking decreased phytic acid content, suggesting that processing made part of the phytic acid phosphorus available. The absorbed phosphorus was greater than the nonphytic phosphorus supplied by the diet. The digestive utilization of phosphorus was similar in processed and raw chickpeas, despite the loss of soluble anion as a result of processing. These results may indicate the contribution of phosphorus in the form of inositol hexaphosphate-phosphorus.     

Molecular cloning and characterization of the gene encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4

The gene bgaP encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the BgaP gene revealed an open reading frame of 2031 bp encoding for a protein of 677 amino acid residues. The BgaP was heterologously expressed in E. coli and purified followed by Ni2+ affinity chromatography. The molecular mass of the native enzyme was approximately 156 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the BgaP indicated molecular masses of 78 and 77.311 kDa, respectively, suggesting that the BgaP is a dimer. The purified BgaP had an isoelectric point of 4.8 and exhibited maximal activity at 20 degrees C and pH 6.8 under the assay conditions used. The enzyme is particularly thermolabile, losing all activity in only 10 min at 45 degrees C. It was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG); the Km values with ONPG and lactose were calculated to be 5.4 and 20.4 mM at 5 degrees C, respectively. The catalytic efficiencies of BagP for lactose at 5 and 20 degrees C had 14 and 47 times more than that of E. coli beta-galactosidase at 20 degrees C, respectively. Therefore, cold-active beta-galactosidase from the psychrotrophic and halotolerant Planococcus sp. L4 could conceivably be developed to fulfill the practical requirements to enable its use for lactose removal in milk and dairy products at low temperature or a reporter enzyme for psychrophilic genetic systems.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.