A novel acid phosphatase from cactus (Opuntia megacantha Salm-Dyck) cladodes: Purification and biochemical characterization of the enzyme.

Acid phosphatase (ACP) plays an important role in regulating phosphate nutrition in plants. Herein, for the first time, a novel ACP from Opuntia megacantha Salm-Dyck cladodes was purified to homogeneity and biochemically characterized. Specific activity of 8.78 U/mg was obtained with 11.29-fold purification and 15% yield. ACP was purified as monomer with molecular weight of 44 kDa as determined by SDS-PAGE under denaturing and nondenaturing conditions. Optimum pH and temperature for ACP activity was 5.5 and 60 °C, respectively. Thermodynamic parameters (Ea, ΔH, ΔG and ΔS) were also determined. ACP activity was stimulated by Ca2+, strongly inhibited by Cu2+ and Fe3+, and moderately inhibited by Mg2+ and Zn2+. Br-, CN-, F-, I- and N3- weakly inhibited ACP activity, where more than 70% of enzyme activity was remained at 5 mM. In addition, effect of ß-ME, Cys, DTT, EDTA, H2O2, PMSF, SDS and TX-100 on ACP activity was investigated. km, Vmax, kcat and kcat/km of ACP for p-NPP were found to be 0.09 mM, 2.75 U/mL, 9.60 s-1 and 106.67 s-1 mM-1, respectively. The biochemical properties of ACP from Opuntia megacantha Salm-Dyck cladodes provide novel features with other plant ACPs and basic knowledge of ACP in Opuntia species.

Effects of the Consumption of Prickly Pear Cacti (Opuntia spp.) and its Products on Blood Glucose Levels and Insulin: A Systematic Review.

Background and Objectives: There is confusion as to which component of the Opuntia spp. cacti has demonstrated anti-hyperglycemic effects or anti-diabetic properties. It is important to clarify these health benefits due to the increasing need for prevention and treatment of chronic diseases. The aim of this review is to identify the effects of Opuntia spp. cacti consumption on biomedical measures; glucose and insulin with consideration of its' components; fruit, leaf and combined or unidentified Opuntia spp. products. Materials and Methods: Prior to commencing the searches, this systematic review was registered with PROSPERO (CRD42018108765). Following the PRISMA 2009 guidelines, six electronic databases (Food Science and Technology Abstracts (EBSCO), Medline, Scopus, CINAHL, Web of Science and Cochrane) were searched for articles investigating the effect of Opuntia spp. consumption on glucose and insulin in humans. Results: Initially, 335 articles were sourced and filtered by exclusion criteria (human interventions, control trials and articles published in English) resulting in 20 relevant articles. The included studies were characterized by such plant components as fruit (n = 4), cladode (n = 12), and other Opuntia spp. products (n = 4), further separated by clinical populations ('healthy', hyperlipidemic, hypercholesterolemic, Type 2 Diabetes Mellitus). The findings of this review indicate variations in effects between cacti components and products. Cladode and select Opuntia spp. products predominately demonstrated significant reductions in serum glucose and insulin, indicating potential as a functional food candidate. Prickly Pear fruit was predominately reported to have no significant effects on glucose or insulin. The quality of evidence appeared to vary based on the type of Opuntia spp. product used. Studies that used specifically the fruit or cladode had high risk of bias, whereas studies which used combined Opuntia spp. products had a lower risk of bias. Numerous mechanisms of action were proposed where positive findings were reported, with emphasis on dualistic glucose-dependent and independent actions, however, mechanisms require further elucidation. Conclusion: Currently, there is a lack of evidence to support the recommendation of using Opuntia spp. fruit products as an alternative or complementary therapy in the reduction of risk or management of Type 2 Diabetes Mellitus. The Cladode does however show promise in potential glucose-lowering effects which warrant further investigation.

Molecular and computational approaches to characterize thermostable laccase gene from two xerophytic plant species.

Laccases are blue multicopper oxidases that carry out single electron transfers in the oxidation of phenols to quinones. In plants, they confer structural stability to the cell wall. Thermostable laccases were identified in xerophytes Cereus pterogonus and Opuntia vulgaris that could be used in biotechnology and industrial processes. Polyclonal anti-laccase antibodies were generated against purified laccase enzyme isoforms capable of 98-99% inhibition of the catalytic activity. Antibodies raised against lower molecular weight isoforms inhibited 70% of the catalytic activity of higher molecular forms. Only 20% inhibition was noted when assayed in reverse. A partial gene sequence of thermostable xerophytic laccase comprising 712 and 880 bp was identified employing cDNA as template. The nucleotide sequence was submitted to GenBank. The gene sequence was in silico translated into protein sequence and a 3-D structure was predicted using I-Tasser and Genesilico online servers that justified the experimental observations. Anti-laccase antibodies and nucleotide gene sequence of this thermostable plant laccase can be utilized for predicting laccase antigenic sequences and for cloning and expression of the thermostable eukaryotic laccase.

Purification and characterization of an amylase from Opuntiaficus-indica seeds.

BACKGROUND: In Tunisia, prickly pear fruit grow spontaneously; it is consumed as fresh fruit, juice or jam. When the fruit is used for juice production, the seeds are discarded and go to waste. Our study aimed to extract biomolecules from seeds by producing value-added products from the fruits. RESULTS: An amylase from Opuntia ficus-indica seeds was extracted and purified to homogeneity. An increase in specific activity of 113-fold was observed. The apparent molecular mass of the enzyme is 64 kDa. The optimum pH and temperature for enzyme activity were pH 5 and 60 °C, respectively. Under these conditions, the specific activity is 245.5 U mg(-1) . The enzyme was activated by Co(2+) and Mg(2+) (relative activity 117% and 113% respectively) at lower ion concentrations. It was strongly inhibited by Mn(2+) and Fe(2+) . Cu(2+) inhibited totally the activity of this enzyme, but Ca(2+) has an inhibitory effect which increases with ion concentration. CONCLUSION: The extracted enzyme belongs to the exo type of amylases and is classified as a ß-cyclodextrin glycosyltransferase since it generates mainly ß-cyclodextrin from starch. It exhibits high thermal stability and a broad range of pH stability, making it a promising prospect for industrial and food applications.

Isolation, purification, and characterization of two thermostable endo-1,4-ß-D-glucanase forms from Opuntia vulgaris.

Four endoglucanase temperature isoforms (T (30), T (50), T (70), and T (90)) were identified and purified from the cladodes of the xerophytic plant Opuntia vulgaris. These isoforms exhibited optimum catalytic activity at 30 °C, 50 °C, 70 °C, and 90 °C and yielded an apparent molecular mass of 150, 20, 74, and 45 kDa, respectively, on gel filtration chromatography. These isoforms were purified 24-, 25-, 29-, and 27-fold with a yield of 15%, 12%, 17%, and 19% and having a specific activity of 120, 125, 144, and 136 U/mg, respectively. The thermostable T (70) and T (90) isoforms exhibited optimum activity at pH 4.5 and 7 and also yielded a molecular weight of 66 and 36 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T (70) had a K (m) of 43 mM and a V (max) of 12.5 µmol min(-1) µg(-1) of protein, and the T (90) isoform had a K (m) of 40 mM, with an apparent V (max) of 10 µmol min(-1) µg(-1) of protein. Western blot, immunodiffusion, and in vitro inhibition assays established the reactivity of the T (90) isoform with polyclonal anti-T (90) antibody raised in rabbit. Cross-reactivity of this antibody with the T (70) endoglucanase isoform was also noted.

Purification and characterization of a novel thermostable xylose isomerase from Opuntia vulgaris mill.

Thermophilic xylose isomerase from the xerophytic eukaryote Opuntia vulgaris can serve as a good alternate source of enzyme for use in the production of high fructose corn syrup. The existence of two temperature stable isoforms having optimal activity at temperatures 70 °C (T(70)) and 90 °C (T(90)), respectively, is reported here. These isoforms were purified to homogeneity using column chromatography and SDS-polyacrylamide gel electrophoretic techniques. Only the T(90) isoform was subjected to full biochemical characterization thereafter. The purified T(90) isoform was capable of converting glucose to fructose with high efficiency under the assay conditions. The enzyme at pH 7.5 exhibited a preference to yield the forward isomerization reaction. The melting temperature of the native enzyme was determined to be 90 °C employing differential scanning colorimetery. Thermostability of the enzyme protein was established through temperature-related denaturation kinetic studies. It is suggested that the thermostability and the wide pH activity of this eukaryotic enzyme will make it an advantageous and dependable alternate source of catalytic activity for protected use in the high fructose corn syrup sweetener industry.

Thermophilic laccase from xerophyte species Opuntia vulgaris.

Two laccase temperature isoforms capable of oxidizing phenolic compounds to quinones were isolated and purified to homogeneity from the cladodes of the xerophyte species Opuntia vulgaris. These catalytically active proteins exhibit apparent molecular masses of 137 and 90 kDa. Under reducing conditions, both isoforms yielded a subunit molecular mass of 43 kDa, suggesting that the enzyme is a multimer of the 43 kDa subunit. The 137 kDa isoform when heated at 80°C for 3 min generated three polypeptide bands on activity stained polyacrylamide gels exhibiting 137, 90 and 43 kDa molecular forms. All isoforms of the enzyme exhibited an optimum pH of 10 when 2,6-dimethoxyphenol was used as a substrate. The optimum temperature of the 137 kDa enzyme form was noted to be 80°C and that of the 90 kDa enzyme form was 70°C. Denaturation kinetics of both the laccase isoforms carried out at their respective optimum temperatures for 30 min exhibited enzyme activity in excess of their t(1/2) values throughout the assay period. The K(m) for the 137 kDa form was determined to be 2.2 ± 0.3 mm and the V(max) was 2.8 ± 0.2 IU/mL. These high temperature stable laccase isoforms having alkaline pH optima can find significant industrial use.

A process to produce penicillin G acylase by surface-adhesion fermentation using Mucor griseocyanus to obtain 6-aminopenicillanic acid by penicillin G hydrolysis.

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V (max), and the Michaelis constant, K (M), were determined using the Michaelis-Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0-10 mM). K(M) and V(max) were determined to be 3.0 x 10(-3) M and 4.4 x 10(-3) mM/min, respectively.

Characterization of a highly stable trypsin-like proteinase inhibitor from the seeds of Opuntia streptacantha (O. streptacantha Lemaire).

A trypsin inhibitor from Opuntia streptacantha Lemaire (Prickly pear) seeds was purified and characterized. Of several proteases tested, this inhibitor showed specificity to trypsin-like enzymes. The major inhibitor present in these seeds showed distinctive characteristics, most notably a low molecular weight of 4.19 kDa, as determined by MALDI TOF, and an unusually high thermal stability, retaining most of the activity after heating the sample 1h to 120 degrees C with a pressure of 1 kg/cm(2). Its complete amino acid sequence was obtained through mass spectrometry, this establishing presence a blocked N-terminal region. When comparing its sequence in the MEROPS database for peptidases and peptidase inhibitors, it showed 34.48% identity with a serine-proteinase inhibitor from the I15 family.

Biochemical and nutritional characterization of three prickly pear species with different ripening behavior.

Biochemical and nutritional changes were studied during the ripening process of three Opuntia morphospecies with different ripening behavior: Naranjona (O. ficus-indica), Blanca Cristalina (Opuntia sp.), and Esmeralda (Opuntia sp.) of early, early-intermediate, and intermediate-late ripening, respectively. In loss of fresh weight, Naranjona showed the highest values, while in Blanca Cristalina and Esmeralda, a discrete weight loss was found. No significant differences were found among morphospecies in soluble solids, total titratable acidity and pH during the postharvest days. Blanca Cristalina and Esmeralda showed an increase in the content of carotenoids, while these diminished in Naranjona. The cell wall enzymes evaluated showed particular behaviors during the ripening of each morphospecies suggesting a fine biochemical control and not a clear relationship between fruit softening and enzyme activity. This study provides basic information on prickly pear ripening, in order to understand this process for its control and for improving shelf life.

Hydrolytic activity and ultrastructural changes in fruit skins from two prickly pear (Opuntia sp.) varieties during storage.

The activity of four cell wall hydrolases, pectinmethylesterase (PME), polygalacturonase (PG), cellulase, and beta-galactosidase (beta-Gal), was measured in fruit skins of two prickly pear varieties, Naranjona and Charola, during storage at 18 degrees C and 85-95% relative humidity (RH). In Naranjona (Opuntia ficus indica), of short postharvest life (ca. 2 weeks), PG, cellulase, and beta-Gal increased their activity more than twice, whereas PME activity tended to increase only slightly during storage. In Charola (Opuntia sp.), of long postharvest life (ca. 2 months), only beta-Gal increased its activity (77%), showing a high PG activity from the beginning of storage. Transmission electron microscopy observations showed middle lamella dissolution at the end of storage for both varieties. Naranjona showed a higher cell wall enzymatic activity than Charola, in agreement with their storability differences. Our results suggest that PG and cellulase in Naranjona and PG and beta-Gal in Charola are the main enzymes responsible for cell wall hydrolytic and ultrastructural changes in skins of stored prickly pears.