Digestive protease,lipase and amylase activities in stage i larvae of the american lobster,Homarus americanus

• 1.1. Digestive protease, lipase, and amylase of Stage I larvae of the American lobster Homarus americanus are characterized.
• 2.2. A sensitive method for detection of crustacean lipase was developed using an latroscan which combines thin-layer chromatography and flame ionization detection to quantify free fatty acids generated by lipase digestion.
• 3.3. pH optima of the three enzymes occurred at or near the pH of gastric fluid.
• 4.4. A time course study demonstrated slight increases in protease and amylase activities during the first larval stage, regardless of whether the lobsters were fed or not, whereas lipase activity was constant.

     

A new bacterial strain of Antarctica,Alteromonas sp. that produces a heteropolymer slime

A bacterial strain NF₃ was isolated from a sludge sample from Inlet Admiralty Bay (Antarctica) and has been identified as belonging to the genus Alteromonas. The isolate was a Gram-negative rod with a single polar flagellum arrangement. The strain was catalase and oxidase positive and capable of gelatin hydrolysis. The G + C content was 42 mol%. The fatty acid composition and the protein profile has confirmed its identification as Alteromonas sp. During cell growth, the strain produced an exopolymer composed of 76–86% protein and 8–14% sugars depending on the medium used.     

Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe

This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Deltaalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the D-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 micro g of the competitive Alr inhibitor D-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of D-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.     

Meta-analysis of phosphorus utilization by growing pigs: effect of dietary phosphorus,calcium and exogenous phytase

Optimizing phosphorus (P) utilization in pigs requires improving our capacity to predict the amount of P absorbed and retained, with the main modulating factors taken into account, as well as precisely determining the P requirements of the animals. Given the large amount of published data on P utilization in pigs, a meta-analysis was performed to quantify the impact of the different dietary P forms, calcium (Ca) and exogenous phytases on the digestive and metabolic utilization criteria for dietary P in growing pigs. Accordingly, the amount of phytate P (PP) leading to digestible P (g/kg) was estimated to be 21%, compared with 73% for non-phytate P (NPP) from plant ingredients and 80% for NPP from mineral and animal ingredients (P < 0.001). The increase in total digestible dietary P following the addition of microbial phytase (PhytM) from Aspergillus niger (P < 0.001) was curvilinear and about two times higher than the increase following the addition of plant phytase, which leads to a linear response (P < 0.001). The response of digestible P to PhytM also depends on the amount of substrate, PP (PhytM² × PP, P < 0.001). The digestibility of dietary P decreased with dietary Ca concentration (P < 0.01) independently of phytase but increased with body weight (BW, P < 0.05). Although total digestible dietary P increased linearly with total NPP concentration (P < 0.001), retained P (g/kg), average daily gain (ADG, g/day) and average daily feed intake (ADFI, g/day) increased curvilinearly (P < 0.001). Interestingly, whereas dietary Ca negatively affected P digestibility, the effect of dietary Ca on retained P, ADG and ADFI depended on total dietary NPP (NPP × Ca, P < 0.01, P < 0.05 and P < 0.01, respectively). Increasing dietary Ca reduced retained P, ADG and ADFI at low NPP levels, but at higher NPP concentrations it had no effect on ADG and ADFI despite a positive effect on retained P. Although the curvilinear effect of PhytM on digestible P increased with PP (P < 0.001), this effect was lessened by total NPP for ADG and ADFI (PhytM × NPP and PhytM² × NPP, P < 0.05) and depended on both total NPP and Ca for retained P (PhytM² × NPP × Ca, P < 0.01). This meta-analysis improves our understanding of P utilization, with major modulating factors taken into account. The information generated will be useful for the development of robust models to formulate environmentally friendly diets for growing pigs.     

The influence of dietary microbial phytase and calcium on the accumulation of cadmium in different organs of pigs.

A total of 72 barrows (initial body weight 16.7 kg) was used, to evaluate the influence of microbial phytase supplementation alone or in combination with calcium to barley soybean meal diets on the accumulation of cadmium (Cd) in kidney, liver, muscle, brain and bone. The control group received the basal diet with 6 g Ca and a low native Cd concentration of 0.03 mg/kg dry matter (DM). In the experimental groups 2, 3, 4 and 5 dietary cadmium concentration was elevated to 0.78 mg/kg DM. The diet of group 3 was supplemented with 800 U microbial phytase/kg, the diet of group 4 with 6 g Ca/kg. The diet of group 5 contained both supplements. The addition of microbial phytase caused an increase of Cd retention in kidney and liver at 30 and 50 kg body weight. This effect was counteracted by the contemporary addition of calcium. A supplementation of Ca alone showed no effect on the Cd accumulation in kidney and liver. In muscle, brain and bone no effects of phytase and calcium on the accumuLation of Cd could be found.     

How conformational changes can affect catalysis,inhibition and drug resistance of enzymes with induced-fit binding mechanism such as the HIV-1 protease

A central question is how the conformational changes of proteins affect their function and the inhibition of this function by drug molecules. Many enzymes change from an open to a closed conformation upon binding of substrate or inhibitor molecules. These conformational changes have been suggested to follow an induced-fit mechanism in which the molecules first bind in the open conformation in those cases where binding in the closed conformation appears to be sterically obstructed such as for the HIV-1 protease. In this article, we present a general model for the catalysis and inhibition of enzymes with induced-fit binding mechanism. We derive general expressions that specify how the overall catalytic rate of the enzymes depends on the rates for binding, for the conformational changes, and for the chemical reaction. Based on these expressions, we analyze the effect of mutations that mainly shift the conformational equilibrium on catalysis and inhibition. If the overall catalytic rate is limited by product unbinding, we find that mutations that destabilize the closed conformation relative to the open conformation increase the catalytic rate in the presence of inhibitors by a factor exp(ΔΔG_C/RT) where ΔΔG_C is the mutation-induced shift of the free-energy difference between the conformations. This increase in the catalytic rate due to changes in the conformational equilibrium is independent of the inhibitor molecule and, thus, may help to understand how non-active-site mutations can contribute to the multi-drug-resistance that has been observed for the HIV-1 protease. A comparison to experimental data for the non-active-site mutation L90M of the HIV-1 protease indicates that the mutation slightly destabilizes the closed conformation of the enzyme. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Influence of microbial phytase and dietary calcium on the accumulation of lead in different organs of pigs

The objective of this study was to investigate the influence of microbial phytase and calcium supplementation to diets for growing pigs on the retention of lead in the kidney, liver, muscle, brain, and bone (phalanx 1). The experiments were carried out with barrows over the body weight range from 17 to 50 kg. The average lead concentration of the diets was 1.45 mg/kg dry matter. Diets were prepared with or without a supplement of 800 units of microbial phytase. The calcium concentration in the diets was 6.53 or 13.4 g/kg dry matter. The addition of microbial phytase showed an increase of lead concentration in bone. By increasing the calcium concentration to 13.4 g/kg dry matter, it was possible to avoid the phytase-induced increase of lead retention in bone.     

Acid production by Metarhizium anisopliae: Effects on virulence against mosquitoes and on detection of in vitro amylase,protease, and lipase activity

The imperfect fungus Metarhizium anisopliae infects and kills larvae of many insect species, including the mosquito Culex pipiens. Mutants of M. anisopliae selected for enhanced production of amylase have been found to have simultaneously acquired hypervirulence against C. pipiens larvae. In the present work, wild-type and some mutant strains of M. anisopliae were found to excrete an acid or acids which alter the pH of fungal cultures below that permissible for amylase activity. The amylase-enhanced hypervirulence mutants did not excrete acid. Detection of protease and lipase activities was complicated by the acid excretion. When this was taken into account, mutants having altered lipase or protease production were found to have no alteration in virulence against mosquito larvae. The link between acid excretion, amylase activity, and hypervirulence is discussed.     

Molecular cloning,nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101

Summary Alkaliphilic Bacillus sp. no. AH-101 produces an extremely thermostable alkaline serine protease that has a high optimum pH (pH 12–13) and shows keratinolytic activity. The gene encoding this protease was cloned in Escherichia coli and expressed in B. subtilis. The cloned protease was identical to the AH-101 protease in its optimum pH and thermostability at high alkaline pH. An open reading frame of 1083 bases, identified as the protease gene, was preceded by a putative Shine-Dalgarno sequence (AAAGGAGG) with a spacing of 11 bases. The deduced amino acid sequence revealed a pre-pro-peptide of 93 residues followed by the mature protease comprising 268 residues. AH-101 protease showed slightly higher homology to alkaline proteases from alkaliphilic bacilli (61.2% and 65.3%) than to those from neutrophilic bacilli (54.9–56.7%). Also AH-101 protease and other proteases from alkaliphilic bacilli shared common amino acid changes and a four amino acid deletion when compared to the proteases from neutrophilic bacilli. AH-101 protease, however, was distinct among the proteases from alkaliphilic bacilli in showing the lowest homology to the others.Correspondence to: H. Takami     

Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells.

Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found.     

Pseudomonas sp. ST4 produces variety of active compounds to interfere fungal sexual mating and hyphal growth

Sexual mating of compatible sporida is essential for Sporisorium scitamineum to form dikaryotic mycelia and then cause infection on sugarcane. Our previous work identified a Pseudomonas sp. ST4 from a soil sample, which showed a promising biocontrol potential by inhibiting the mating of S. scitamineum sporida and hyphal growth. In this study, we set to isolate the active compounds from Pseudomonas sp. ST4 through solid fermentation. High-performance liquid chromatography (HPLC) separation coupling with bioassay showed that Pseudomonas sp. ST4 produced a range of antimicrobial compounds. Two of the major components were purified following acetate extraction, silica gel and HPLC separation. Nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC-MS) analysis identified these active compounds are 4-hydroxybenzaldehyde and indole-3-carbaldehyde respectively. Further analysis showed that the former compound only inhibited the hyphal growth of the fungus at a concentration of 3 mM, while the latter interfered the fungal sexual mating at a concentration of 0.6 mM and affected hyphal growth at a concentration of 2 mM. Treatment of corn plants with 3 mM indole-3-carbaldehyde significantly inhibited corn smut infection, with a control rate up to 94%. Further analysis of the structure and activity relationship revealed that indole has a much stronger inhibitory activity against the fungal sexual mating than indole-3-carbaldehyde. The results from this study provide new agents for control and prevention of the sugarcane smut disease, and the active compounds could also be used to probe the molecular mechanisms of fungal sexual mating.     

Volume infusion produces abdominal distension, lung compression, and chest wall stiffening in pigs.

The effect of severe generalized edema on respiratory system mechanics is not well described. We measured airway pressure, gastric pressure, and four vertical pleural pressures in 13 anesthetized paralyzed pigs ventilated in the upright position. Pressure-volume relationships of the respiratory system, chest wall, and lung were measured on deflation from total lung capacity to residual volume and during tidal breathing both before (control) and 50 min after one of two interventions. In one series of experiments, a volume equal to 15-20% of the pig's body weight was infused intravenously. In a second series, a balloon was placed in the peritoneal space to distend the abdomen to the same gastric pressures as achieved in the first series. Measurements were compared before and after either abdominal balloon inflation or volume infusion. Volume infusion increased the pleural pressure in dependent lung regions, decreased both total lung capacity (34%) and functional residual capacity (62%) (both P less than 0.05), and markedly shifted the respiratory system and chest wall pressure-volume curves to the right, but it only moderately affected the lung deflation curve. Tidal compliances of the respiratory system, chest wall, and lung decreased 36, 31, and 49%, respectively (all P less than 0.05). The effect of abdominal balloon inflation on respiratory system mechanics was similar to that of volume infusion. We conclude that infusing large volumes of fluid markedly alters chest wall mechanics, mainly by causing abdominal distension that prohibits descent of the diaphragm.     

Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas

Translation products of a reticulocyte lysate reaction, programmed with poly(A)-rich RNAs from the male mouse submaxillary gland, were subjected to affinity chromatography on a tubulin-Sepharose column. Analysis of the bound proteins in sodium dodecylsulfate/polyacrylamide gels revealed two polypeptides of Mr 27 000 and 45 000, that were shown to bind to tubulin in a specific manner. These polypeptides were absent from the translation products coded by poly(A)-rich RNAs from the female mouse. They were eluted from the tubulin-Sepharose resin under conditions similar to those employed for the dissociation of immune complexes. The Mr-27 000 and Mr-45 000 proteins were identified by immunoprecipitation with specific antisera as the precursors of the gamma subunit of the nerve growth factor (NGF) and renin respectively. These two precursors as well as a third, unidentified polypeptide of Mr 38 000, probably unrelated to the beta subunit of NGF, bound also to microtubules. The mature form of renin, purified from the submaximillary gland, also displayed an affinity for the microtubules. In contrast, the mature form of the gamma subunit of NGF did not bind to the microtubules. The possible involvement of the microtubules (tubulin) in the biosynthesis of these two secretory proteins is discussed.     

Quantification of protease, amylase and lipase in the gut of Schizodactylus monstrosus during the postembryonic developmental stage. Effect of starvation, stress and topical application of juvenile hormone analogue

A quantitative picture is presented on the activities and distribution of three major digestive enzymes in the postembryonic and adult stages of Schizodactylus monstrosus. The activity of enzymes in adult insects of both sexes has also been studied under starvation stress and after the topical application of juvenile hormone analogue. Results show that amylase activity was much higher in the early stages of development while in later stages protease and lipase activity was more significant. Protease activity was highest in the adult male, while the activity of all the enzymes reached its maximum in pregnant females. Starvation stress in adult individuals led to a slight decline of enzyme activity which was insignificant statistically. Topical application of JHa in high doses caused a steady increase in the activity of all the enzymes of which the increase of protease was statistically significant. The probable reasons for the quantitative variation have been discussed.     

Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.     

Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101

A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.     

The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33

In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently.