ENZYME ACTION IN ECHINODONTIUM TINCTORIUM ELLIS AND EVERHART

In Echinodontium tinctorium the presence of the following enzymes was demonstrated: esterase, maltase, lactase, sucrase, raffinase, diastase, inulase, cellulase, hemicellulase, urease, rennet, and catalase.     

Simultaneous production of inulase and lactase in batch and continuous cultures of Kluyveromyces fragilis

Under different induction conditions, the industrial yeast Kluyveromyces fragilis is an excellent producer of the enzymes inulase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26) and lactase (β-d-galactoside galactohydrolase, EC 3.2.1.23), producing 27 and 1.6 U mg^?1dry cell weight, respectively. In order to improve overall enzyme yields, conditions for the simultaneous production of both enzymes in a one-stage fermentation have been examined. Techniques employed include carbon-limited batch and continuous culture, single and mixed carbon substrates, and the use of a mutant semi-constitutive for inulase production. Synthesis of both enzymes suffered strongly from carbon catabolite repression in batch cultures grown on single and mixed inducing substrates. Only glycerol and dl-malate did not repress either enzyme. The non-metabolizable analogues of lactose, isopropyl-β-d-thiogalactoside and methyl-β-d-thiogalactoside induced lactase in glycerol grown batch cultures, but were ineffective in sucrose grown continuous cultures. They also depressed the normally high levels of inulase in such continuous cultures. The highest simultaneous inulase and lactase activities in the wild-type yeast were obtained in continuous culture on an equal mixture of d-fructose and d-galactose; 25 and 0.78 U mg^?1dry cell weight, respectively. In this fermentation the combined yield per unit carbon substrate of the two enzymes was 141%, compared to a reference value of 100% for the highest yield of each enzyme in separate fermentations. On the same mixture of d-fructose and d-galactose, the mutant produced ～60 and 0.70 U mg^?1dry cell weight, respectively. The combined enzyme yield per unit of carbon substrate was 172%.     

Organ culture of suckling rat intestine: Comparative study of various hormones on brush border enzymes

Summary Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10⁻⁶ M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10⁻⁸ M) and dbcAMP (10⁻³ M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity. This work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and a grant 79.7.1243 from the Délégation Générale a la Recherche Scientifique et Technique. P. M. S. is a recipient of a grant from Fondation de la Recherche Médicale (France).     

Hydrolytic Enzyme Production by Rhizobium

Cellulase and hemicellulase activity was detected in temperate (infective and noninfective) and tropical strains (infective) of Rhizobium. Hydrolytic enzymes were initially detected by a cup-plate assay. The presence of cellulase and hemicellulase was confirmed by viscometric assay. Implications of the presence of these enzymes in Rhizobium are discussed.     

Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes

The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of $\text{5-}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones.     

Carbohydrases in the digestive tract of the African bony-tongue Heterotis niloticus (Pisces: Osteoglossidae)

The presence of amylase and maltase in the stomach, intestine and pyloric caeca of Heterotis niloticus is demonstrated. Amylase activity was highest in the fore-gut, followed by the pyloric caeca, while the lowest activity was in the hind-gut. Optimum pH for intestinal amylase was 8.45. Sucrase, lactase and cellulase were not detected.     

Origin of intestinal disaccharidases of Ascaris suum

Disaccharidases from the gut of Ascaris suum were investigated to determine whether they were synthesized by the worm or whether they were host enzymes adsorbed to the worms' intestinal cells. Alpha-d-glucoside glucohydrolase (maltase) (EC 3.2.1.20), Beta-d-fructofuranoside fructohydrolase (invertase) (EC 3.2.1.26) and 1-glucohydrolase (trehalase) (EC 3.2.1.28) from Ascaris were studied in both a membrane (brush border)-bound and solubilized form with regard to temperature stability and pH optima. Data collected were compared to similar data on hog intestinal enzymes. Worm maltase and trehalase were relatively heat labile, whereas the hog enzymes were more stable to heat inactivation. Worm invertase was heat stable in comparison to the hog enzyme. The pH optima for Ascaris maltase and invertase were different from those of hog disaccharidases, whereas the pH optimum for trehalase from both parasite and host were similar. Tissue homogenates of second-stage larvae contained measurable maltase, but not sucrase, or trehalase activity. Results suggested that Ascaris intestinal disaccharidases represent three distinct enzymes of parasite rather than host origin.     

Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog.

Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.     

The effect of dexamethasone on the development of rat intestinal brush border enzymes in organ culture

The effect of dexamethasone on the evolution pattern of brush border enzymes was examined in the rat jejunum cultured in vitro at different postnatal stages (4 to 21 days). Enzymic activities were analyzed in purified brush border membranes isolated from noncultured intestine and from explants cultured for 24 and 48 hr. The data obtained from this study indicated that dexamethasone exhibits two types of effects on the cultured intestinal tissue: (1) a nonspecific but protective effect against the drastic drop of all enzyme activities as well as against a loss of villus cells observed in control cultures, and (2) a direct and specific effect on precocious induction of sucrase and on stimulation of maltase activity. The SDS-polyacrylamide gel patterns of brush border membrane proteins showed that in the 6-day-old intestine, appearance of sucrase as well as stimulation of maltase activities elicited by dexamethasone were accompanied by a simultaneous appearance or enhancement of the corresponding protein bands. Furthermore, the radioactivity peaks on gels due to the incorporation of ¹⁴C-valine and of ¹⁴C-fucose indicated that dexamethasone induces the synthesis of new proteins or at least the glycosylation of preexisting proteins which may lead to the formation of active maltase and sucrase molecules.     

Furofuran lignans as a new series of antidiabetic agents exerting α-glucosidase inhibition and radical scarvenging: Semisynthesis,kinetic study and molecular modeling

A new series of furofuran lignans containing catechol moiety were prepared from the reactions between lignans and a variety of phenolics. All 22 products obtained were evaluated against three different α-glucosidases (maltase, sucrase and Baker’s yeast glucosidase) and DPPH radical. Of furofuran lignans evaluated, β-14, having two catechol moieties and one acetoxy group, was the most potent inhibitor against Baker’s yeast, maltase, and sucrase with IC₅₀ values of 5.3, 25.7, and 12.9 µM, respectively. Of interest, its inhibitory potency toward Baker’s yeast was 28 times greater than standard drug, acarbose and its DPPH radical scavenging (SC₅₀ 11.2 µM) was 130 times higher than commercial antioxidant BHT. Subsequent investigation on mechanism underlying the inhibitory effect of β-14 revealed that it blocked Baker’s yeast and sucrase functions by mixed-type inhibition while it exerted non-competitive inhibition toward maltase. Molecular dynamics simulation of the most potent furofuran lignans (4, α-8b, α-14, and β-14) with the homology rat intestinal maltase at the binding site revealed that the hydrogen bond interactions from catechol, acetoxy, and quinone moieties of furofuran lignans were the key interaction to bind tightly to α-glucosidase. The results indicated that β-14 possessed promising antidiabetic activity through simultaneously inhibiting α-glucosidases and free radicals.     

Food composition and digestive enzymes in the gut of pond-cultured Clarias isheriensis (Sydenham 1980), (Siluriformes: Clariidae)

Food composition, diestive enzyme distribution and activity in the gut of pond-cultured Clarias isheriensis were studied It had an omnivorous diet but fed mainly on plancon (particularly Cyanophyceae) and detritus. Qualitative determinations of digestive enzymes in the gut showed that it is capable of digesting carbohydrates, proteins and lipids in its diet. Carbohydrases (amylase, cellulase, maltase, salicinase, sucrase and trehalase) were detected and their activities restricted to the stomach, duodenum and ileum. The incidence of cellulase activity could be responsible for the capacity of this fish species to digest large quantities of Cyanophyceae present in the pond. Lower activity of proteases (chymotrypsin, pepsin and trypsin) and lipases were recorded. Enzyme activity was not recorded in either oesophagus or rectum. The relative distribution and activity of the various digestive enzymes were possibly induced by the nutritional requirements of this catfish species.     

Production of β-xylosidase from <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.     

Biological comparative study between Wolbachia-infected Aedes aegypti mosquito and Wolbachia-uninfected strain,Jeddah city,Saudi Arabia

In this study, samples of Wolbachia-infected Aedes aegypti mosquitoes were collected from Al-Safa district in Jeddah city, Saudi Arabia. The presence of Wolbachia bacteria in mosquitoes was confirmed by PCR technique and they were reared and propagated in the laboratory. Comparative studies were conducted between Wolbachia-infected A. Aegypti and the Wolbachia-uninfected laboratory strain in terms of their ability to withstand drought, resist two types of insecticides and the activities of pesticide detoxification enzymes. The Wolbachia-infected A. aegypti strain proved less able to withstand the drought period, as the egg-hatching rate of the Wolbachia-uninfected strain was greater than that of the Wolbachia-infected strain after one, two and three months of dry periods. Compared to the Wolbachia-uninfected strain, the Wolbachia-infected strain demonstrated a relatively greater resistance to tested pesticides, namely Baton 100EC and Fendure 25EC which may be attributed to the higher levels of the detoxification enzymes glutathione-S-transferase and catalase and the lower levels of esterase and acetylcholine esterase.     

Roles of extracellular lactose hydrolysis in cellulase production by Trichoderma reesei Rut C30 using lactose as inducing substrate

Lactose, an inexpensive, soluble substrate, offers reasonably good induction for cellulase production by Trichoderma reesei. The fungus does not uptake lactose directly. Lactose is hydrolyzed to extracellular glucose and galactose for subsequent ingestion. The roles of this extracellular hydrolysis step were investigated in this study. Batch and continuous cultures were grown on the following substrates: lactose, lactose–glycerol mixtures, glucose, galactose, and glucose–galactose mixtures. Cell growth, substrate consumption, lactose hydrolysis, and lactase and cellulase production were followed and modeled. Cells grew much faster on glucose than on galactose, but with comparable cell yields. Glucose (at >0.3 g/L) repressed the galactose consumption. Cellulase synthesis was growth-independent while lactase synthesis was growth-dependent, except at D < ∼0.065 h⁻¹ where a basal level lactase production was observed. For cellulase production the optimal D was 0.055–0.065 h⁻¹ where the enzyme activity and productivity were both near maxima. The model suggested that lactase synthesis was subject to weak galactose repression. As the galactose concentration increased at high D (>0.1 h⁻¹), lactase synthesis became repressed. The insufficient lactase synthesis limited the lactose hydrolysis rate. Extracellular lactose hydrolysis was concluded to be the rate-limiting step for growth of T. reesei Rut C30 on lactose.     

Impact of Enterococcus faecium on specific activity of disaccharidases in small intestine of gnotobiotic pigs

The aim of the present work was to study the changes in the activity of disaccharidase enzymes (lactase. maltase, saccharase) in the small intestine of gnotobiotic pigs aged 0–35 days and inoculated with Enterococcus faecium. The continual decrease of lactase activity was observed from the 14^th day of age up to the end of the experiment. The most significant decrease of specific lactase activity in the duodenum (2.1 μmol/mg protein/hour) was noted from the 21^st to the 28^th day of age. On the other hand, the specific saccharase activity increased moderately during the post weaning period and maltase activity maintained a constant level. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Pancreatic and Pancreatic-Like Microbial Proteases Accelerate Gut Maturation in Neonatal Rats

ObjectivesPostnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats.MethodsSuckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14–16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability.ResultsGavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner.ConclusionFeeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals.     

Genetic Modification of Carbon Catabolite Repression in Trichoderma reesei for Improved Protein Production

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Δcre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.The filamentous fungus Trichoderma reesei (Hypocrea jecorina) produces large amounts of extracellular enzymes. The majority of the secreted proteins are various plant polymer-degrading enzymes; the most abundant of these enzymes are the cellobiohydrolases and endoglucanases that act synergistically to break down cellulose. This fungus has been used as a production host for various industrial enzymes, including products tailored for textile, feed, food, and pulp and paper applications (6, 10). It has been reported that protein production levels in the industrial T. reesei process exceed 100 g/liter (7).The major cellulase and hemicellulase genes are regulated in a coordinate manner by the carbon source available (2, 9, 14). Cellulose and other plant materials and other substances (for example, lactose) induce the expression of cellulase and hemicellulase genes, while glucose acts as a repressing carbon source. Several genes coding for regulators of cellulase and hemicellulase expression have been isolated. These include CREI mediating carbon catabolite repression, the repressor ACEI, the activator ACEII, the CCAAT binding complex Hap2/3/5 (reviewed in references 2, 17, and 27) and the activator XYRI (29). The CREI protein has sequence similarity with other fungal proteins mediating glucose repression, such as Aspergillus nidulans CREA (8) and Saccharomyces cerevisiae MIG1 and RGR1 (22). In T. reesei, glucose repression has been shown to occur upon binding of CREI to specific sequences in the cbh1 promoter (13). A mutant cre1 gene (cre1-1) encoding a truncated form of CREI has been isolated from the hypercellulolytic T. reesei strain Rut-C30, which is capable of cellulase and hemicellulase production on glucose-containing media. Further evidence for the function of CREI in glucose repression was obtained by complementation of the cre1-1 mutation of Rut-C30 by the wild-type cre1 gene, which restored the glucose-repressed phenotype of the strain (15).In this paper, we wanted to address three questions. (i) What is the effect of cre1 mutations in the wild-type background? (ii) Is cre1-1 a null mutation? (iii) Can enzyme production be further improved by cre1 deletion in an industrial production strain improved greatly by mutagenesis and screening programs? Therefore, we introduced cre1-1 allele and cre1 deletion to the wild-type strain QM6a and the cre1 deletion into the industrial strain VTT-D-80133 and studied the effects of these mutations on enzyme production.     

A Novel Compound from the Mushroom Cryptoporus volvatus Inhibits Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) In Vitro

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a serious contagious disease in the swine industry. At present, there are no effective control strategies against PRRSV. Thus, there is an urgent need for new treatment regimens that have efficacious antiviral activity to compensate for vaccines. The anti-infective effect of Cryptoporus volvatus has previously been demonstrated in Tradational Chinese Medicine. In this report, we expected to identify a new anti-PRRSV agent in the aqueous extract of C. volvatus, by employing a combination of modern chromatographic purification techniques and indirect immunofluorescence assay (IFA). Our results showed that C. volvatus extracts from every separation step differed in their inhibitory potency on PRRSV. One anti-PRRSV component designated as C_M-H-L-5 was isolated from water-soluble fraction of C. volvatus. The inhibition induced by C_M-H-L-5 occurred in a dose-dependent manner. C_M-H-L-5 appeared to be a low-molecular-weight polyol fragment with amide groups and carboxylic acid groups. Collectively, our findings imply that C_M-H-L-5 from the aqueous extract of C. volvatus has the potential to be used for anti-PRRSV therapy.