Factors affecting invertase activity in soils

Summary The rate of reducing sugars released through invertase activity exhibited a buffer pH optimum of 5.0. Generally, the decline in invertase activity in its pH-profile near the optimal pH range was due to a reversible reaction that involved ionization or deionization of the functional groups in the active centre of the protein, but under highly acidic or alkaline conditions (pH<4 to >9) the reduced activity appears to be due to irreversible inactivation of the enzyme. The dependence of the reaction on the amount of enzyme present was linear up to 3 g of soil. By varying the substrate concentration, it was found that the reaction rate of this enzyme approached zero-order kinetics when 145mM of sucrose solution was added to soils. Application of three linear transformations of the Michaelis-Menten equation indicated that the apparent K_m constants varied among the soils studied, but the results obtained by the three plots were similar. By using the Lineweaver-Burk plot, the K_m values in five soils ranged from 16.3 to 42.1 (avg.=24.5) mM and V_max values ranged from 1.98 to 7.37 mg of reducing sugars released/g of soil/24 h. The optimum temperature for invertase activity in soils was observed at 50°C and denaturation of the enzyme began at 55°C. The activation energy (E_a) and enthalpy of activation (H^*) values for invertase activity, expressed in kJ/mole, ranged from 6.1 to 43.1 and 3.5 to 40.5, respectively. The Q₁₀ values for the invertase reaction in soils with a temperature range to 10 to 50°C ranged from 1.08 to 1.96. Under standerd conditions, the accumulation of reducing sugars was linear with time up to 48 h. Among the various pretreatments that affected invertase activity in soils, toluene, TCA, and PMA inhibited the enzyme by an average of 19, 54, and 11%, respectively. Steam-sterilization essentially destroyed soil invertase.     

葛根芩连汤对泄泻肠道湿热证小鼠肠道双糖酶活性的影响

目的探究葛根芩连汤对泄泻肠道湿热证小鼠肠道双糖酶活性的影响。方法采用“高糖高脂+高温高湿+白酒复合灌胃冰水”的方法制备泄泻肠道湿热证小鼠模型,并运用葛根芩连汤进行干预治疗。分别在干预的第0、2、4、6天无菌采集各组小鼠肠黏膜,运用DNS法测定蔗糖酶活性,ONPG法测定乳糖酶活性,观察葛根芩连汤对泄泻肠道湿热证小鼠肠道双糖酶活性的影响。结果经肠道湿热泄泻造模后,小鼠肠道前段和后段黏膜的乳糖酶和蔗糖酶活性均显著下降,与正常组小鼠相比差异有统计学意义(均P<0.01)。随着治疗时间的延长,治疗组小鼠肠道乳糖酶和蔗糖酶活性与正常组相比有显著的提高(均P<0.05),治疗6 d后,肠道前段黏膜蔗糖酶和乳糖酶活性恢复至正常组水平(P>0.05)。肠道后段乳糖酶活性在第4天时最高,后段黏膜蔗糖酶活性在第6天时最高,与正常组和自愈组相比差异有统计学意义(均P<0.05)。结论泄泻肠道湿热证造模使小鼠肠道黏膜蔗糖酶、乳糖酶等双糖酶的活性显著下降,葛根芩连汤能调节泄泻肠道湿热证小鼠肠道黏膜乳糖酶和蔗糖酶的活性,从而发挥治疗泄泻肠道湿热证的疗效。     

Phenotypic flexibility of digestive morphology and physiology of the South American omnivorous rodent Akodon azarae (Rodentia: Sigmodontinae)

We have determined the occurrence of responses at different levels (morphological, physiological and biochemical) in the omnivorous rodent Akodon azarae upon cold acclimation (15 degrees C). A short-term enhancement in food consumption appeared to account for the maintenance of both mass and body composition. At the morphological level, the main response was an increase in the dimensions of small intestine, which constitutes the section of the gut where absorption and secretion take place. An increase in sucrase specific activity was only found in small intestine. Sucrose independent maltase activity was very low since 99.8% of total maltase activity was due to sucrase-isomaltase (SI) complex. Protease specific activities were not affected. The fact that resting metabolic rates determined at 15 and 23 degrees C were similar in cold acclimated animals suggests a change in lower critical temperature. In conclusion, our results show that A. azarae exhibits different strategies to support cold environment that could lead to an enhancement in digestion and absorption efficiency. Furthermore, this work suggests that low temperature is an independent cue of other environmental factors to trigger the strategies allowing the maintenance of body condition in A. azarae.     

Effects of diet quality on phenotypic flexibility of organ size and digestive function in Mongolian gerbils (Meriones unguiculatus)

In the context of evolution and ecology, there is a trade-off between the benefits of processing food through a digestive system with specific phenotypic attributes and the cost of maintaining and carrying the digestive system. In this study, we tested the hypothesis that digestive modulations at several levels can match each other to meet the energy and nutrient demands of Mongolian gerbils, a small granivorous rodent species, by acclimating them to a high-quality diet diluted with alfalfa powder. Mongolian gerbils on the diluted diet maintained metabolizable energy intake by an integrated processing response (IPR), which included increases in dry matter intake, gut capacity and rate of digesta passage after 2-weeks of acclimation. Down-regulation of hydrolytic enzyme activity in the intestinal brush-border membrane supported the adaptive modulation hypothesis. The absence of up-modulation of summed enzyme hydrolytic capacity on the diluted diet indicated that greater mass of small intestine on a high-fibre diet is not a direct indicator of digestive or absorptive capacity. Changes in mass of vital organs and carcass suggested that the amount of energy allocated to various organs and hence physiological functions was regulated in response to diet shift.     

Sucrose hydrolases from the midgut of the sugarcane stalk borer Diatraea saccharalis

A beta-fructosidase (EC 3.2.1.26) was isolated from the midgut of larval sugar cane stalk borer Diatraea saccharalis by mild-denaturing electrophoresis and further purified to near homogeneity by gel filtration. beta-Fructosidase hydrolysed sucrose, raffinose and the fructosyl-trisaccharide isokestose, but it had no activity against maltose, melibiose and synthetic substrates for alpha-glucosidases. Two other sucrose hydrolases, one resembling a alpha-glucosidase (EC 3.2.1.20) and the other one active specifically against sucrose (sucrase) were detected in the larval midgut of D. saccharalis. All three sucrose hydrolases were associated with the midgut epithelium of larval D. saccharalis. Relative molecular mass (M(r)) of the beta-fructosidase was estimated around 45,000 (by gel filtration). The other two sucrose hydrolases had M(r) of 54,000 (alpha-glucosidase) and 59,000 (sucrase). The pH optima of the sucrose hydrolases were 5-10 for both alpha-glucosidase and sucrase and 7-8 for beta-fructosidase. Considering V(max)/K(m) ratios, beta-fructosidase preferentially cleaves isokestose rather than raffinose and sucrose. In order to evaluate the possible contribution of microorganisms isolated from the midgut to the pool of sucrose hydrolases, washed midgut epithelia were homogenised and plated onto appropriate media. Seven bacterial and one yeast species were isolated. None of the sucrose hydrolases extracted from the microorganisms corresponded to the enzymes isolated from midgut tissue homogenates. This result suggests that the major sucrose hydrolases found in the midgut of larval D. saccharalis were probably produced by the insect themselves not by the gut microflora.     

Characterization of a novel low molecular weight sucrase from filamentous fungus Termitomyces clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 °C and pH 5.0. K_m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity.     

Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239 bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).     

Method of measuring invertase activity in soils

Summary Invertase (-D-fructofuranoside fructohydrolase, EC [Enzyme Commission] 3.2.1.26) is the enzyme that catalyzes the hydrolysis of sucrose and yields glucose and fructose. The activity of this enzyme was monitored by systematically developing a sensitive and rapid method to detect reducing sugars with the precision of 1.4 to 6.1% C.V. The method involves the colorimetric determination of reducing sugars which react with 3,5-dinitrosalicylic acid when soil is incubated with buffered sucrose solution and toluene at 37°C for 24 h. The detection limit for the method described is 100 g of reducing sugar per ml of soil extract. The color intensity remained constant up to 24 h. Comparative studies showed that the method described was in good agreement to other invertase assay procedures reported in the literature.Studies on the stability and distribution of invertase in soils by using the method described showed that air-drying of field-moist soil samples resulted in decreased activity ranging from 15.3 to 23.7% (avg.=19.8%). Statistical analyses indicated that invertase activity was significantly correlated with total N (r=0.78^***) and organic C (r=0.70^***) in the topsoil of 19 diverse samples. There was no significant correlation between invertase activity and soil pH, cation exchange capacity, percentage of clay and percentage of sand. The activity of this enzyme was concentrated in surface soils and decreased with profile depth. Regression analyses showed that invertase activity was significantly correlated with organic carbon content of three soil profiles examined.     

Time- and dose-dependency of intestinal lactase activity in adult rat on starch intake

Although it is generally accepted that lactase (β-d-galactosidase, EC 3.2.1.23) activity is not influenced by intake of saccharides containing α-linkages, an effect of these carbohydrates on lactase activity was never thoroughly investigated. Activity of lactase and sucrase (sucrose α-d-glucohydrolase, EC 3.2.1.48) was determined in proximal, middle and distal thirds of the jejunoilem of female, 12-week-old rats, fed for 2 weeks a low-starch (5 cal%), high-fat (73%) diet, and in rats, that after this introduction period were fed for 1,2 and 3 days, an isocaloric middle-starch (40%), middle-fat (36%) diet or an isocaloric high-starch (70%), low-fat (7%) diet. During the entire experimental period, the body weight changes, food intake and the amount of protein per segment were practically the same in all three dietary groups. In all intestinal segments, increased intake of starch was followed by an increase of lactase and sucroase activity (both expressed as per tissue protein or per intestinal segment) within the first day. The increase continued during the second day and leveled off during the third day. A highly significant linear correlation was found between the search content of the diets and the lactase activity in all three segments. A highly significant correlation was also established in all three segments between sucrase and lactase activities. These studies thus demonstrated a dose- and time-dependency between the intake of starch (a carbohydrate containing only α-linkages) and the activity of lactase, a neutral β-galactosidase in adult rats.