Screening of cellulose degradation bacteria from Min pigs and optimization of its cellulase production

BACKGROUND: Cellulose as a potential feed resource hinders its utilization because of its complex structure, and cellulase is the key to its biological effective utilization. Animal endogenous probiotics are more susceptible to colonization in the intestinal tract, and their digestive enzymes are more conducive to the digestion and absorption of feed in young animals. Min pigs are potential sources of cellulase probiotics because of the high proportion of dietary fiber in their feed. In this study, the cellulolytic bacteria in the feces of Min pigs were isolated and screened. The characteristics of enzymes and cellulase production were studied, which provided a theoretical basis for the rational utilization of cellulase and high-fiber food in animal production. RESULTS: In our study, 10 strains of cellulase producing strains were isolated from Min pig manure, among which the M2 strain had the best enzyme producing ability and was identified as Bacillus velezensis. The optimum production conditions of cellulase from strain M2 were: 2% inoculum, the temperature of 35°C, the pH of 5.0, and the liquid loading volume of 50 mL. The optimum temperature, pH and time for the reaction of cellulase produced by strain M2 were 55°C, 4.5 and 5 min, respectively. CONCLUSIONS: Min pigs can be used as a source of cellulase producing strains. The M2 strain isolated from feces was identified as Bacillus velezensis. The cellulase from M2 strain had a good activity and the potential to be used as feed additive for piglets.

Diversity of culturable moderately halophilic bacteria producing extracellular hydrolytic enzymes from marine sediments / Diversidade de bactérias cultiváveis moderadamente halofílicas que produzem enzimas hidrolíticas extracelulares de sedimentos marinhos

A total of 114 moderately halophilic bacteria were isolated from marine sediment environments. The isolates are belonged to 23 species based on the 16S rRNA sequence analysis. 63, 52, 47, 57, 74, 15 and 4 isolates are able to produce protease, amylase, lipase, pectinase, pulluanase, xylanase, cellulase, respectively. Combined hydrolytic enzyme activity analysis show that 15 strains present 1 hydrolytic activity, 32 strains present 2 hydrolytic activities, 21 strains present 3 hydrolytic activities, 26 strains present 4 hydrolytic activities, 11 strains present 5 hydrolytic activities and 2 strains present 6 hydrolytic activities. Hydrolase activities are widely distributed in a variety of species. The highest rates for production of protease, amylase, lipase, pectinase, pullanase, xylanase and cellulase were observed in species of B. baekryungensis, Hallobacillus sp., B. pumilus, B. megaterium or P. chungwhensis, B. amyloliquefaciens, B. pumilus, B. baekryungensis, respectively. However, the higher activities of protease, pectinase and pulluanase are frequently produced by the species of Halomonas sp. B. amyloliquefaciens or P. chungwhensis, and Vibrio sp. respectively. This investigation show that the diversity of halophilic bacteria from marine sediments could serve as a potential source of hydrolytic enzymes for industrial applications. (AU)

Um total de 114 bactérias moderadamente halofílicas foram isoladas de ambientes de sedimentos marinhos. Os isolados pertencem a 23 espécies com base na análise da sequência 16S rRNA. 63, 52, 47, 57, 74, 15 e 4 isolados são capazes de produzir protease, amilase, lipase, pectinase, pululanase, xilanase, celulase, respectivamente. A análise da atividade enzimática hidrolítica combinada mostra que 15 cepas apresentam 1 atividade hidrolítica, 32 cepas apresentam 2 atividades hidrolíticas, 21 cepas apresentam 3 atividades hidrolíticas, 26 cepas apresentam 4 atividades hidrolíticas, 11 cepas apresentam 5 atividades hidrolíticas e 2 cepas apresentam 6 atividades hidrolíticas. Atividades de hidrolase são amplamente distribuídas em uma variedade de espécies. As maiores taxas de produção de protease, amilase, lipase, pectinase, pululanase, xilanase e celulase foram observadas em espécies de B. baekryungensis, Hallobacillus sp., B. pumilus, B. megaterium ou P. chungwhensis, B. amyloliquefaciens, B. pumilus, B. baekryungensis, respectivamente. No entanto, as atividades mais elevadas de protease, pectinase e pululanase são freqüentemente produzidas pelas espécies de Halomonas sp. B. amyloliquefaciens ou P. chungwhensis e Vibrio sp. respectivamente. Esta investigação mostra que a diversidade de bactérias halofílicas de sedimentos marinhos pode servir como uma fonte potencial de enzimas hidrolíticas para aplicações industriais. (AU)

Evaluation of resistance to natural poisoning by Amorimia septentrionalis in goats which had received sodium monofluoroacetate degrading bacteria / Avaliação da resistência à intoxicação natural por Amorimia septentrionalis em caprinos que receberam bactérias degradadoras de monofluoroacetato de sódio

Amorimia septentrionalis is an important sodium monofluoroacetate (MFA) containing plant that causes sudden death in ruminants in northeastern Brazil. MFA degrading bacteria are being used in the prevention against poisoning by this plant. The aim of this study was to evaluate if goats which had per os received MFA degrading bacteria remained resistant when exposed to natural poisoning by A. septentrionalis. Eighteen goats were randomly distributed into three groups: the goats of Group 1 previously received, during 40 days, a solution containing the bacteria Ralstonia sp. and Burkholderia sp., those goats in the Group 2 received the bacteria Paenibacillus sp. and Cupriavidus sp. and goats from Group 3 did not receive any bacteria. After the administration period, during 60 days, the animals of all groups were released to graze on a one hectare paddock, with significant amount of A. septentrionalis. They were observed daily for the spontaneous consumption of A. septentrionalis leaves and the occurrence of clinical signs of poisoning or sudden death. Goats from all groups consumed significant amounts of A. septentrionalis during the experimental period. Goats that did not receive MFA-degrading bacteria (Group 3) became sick and died from the 25th to the 27th day of the experiment, whereas the goats of the groups that received MFA-degrading bacteria showed only clinical sings when A. septentrionalis regrowth after the 55th day of the experiment. The days elapsed from field observation to death of Group 3 goats (25.5±0.9 days) were significantly lower (p<0.05) than Group 1 (58.6±1.3 days) and Group 2 (57.8±1.5 days). Thus, it can be concluded that administration of MFA degrading bacteria increases the resistance to natural poisoning by A. septentrionalis.(AU)

Amorimia septentrionalis que contém monofluoroacetato de sódio (MFA) é responsável pela ocorrência de mortes súbitas em ruminantes no nordeste do Brasil. Bactérias degradadoras desse composto estão sendo utilizadas na prevenção contra a intoxicação por essa planta. O objetivo deste estudo foi avaliar se caprinos que receberam, via oral, bactérias degradadoras de MFA permaneciam resistentes quando expostos a intoxicação natural por A. septentrionalis. Dezoito caprinos foram divididos em três grupos, os caprinos do Grupo 1 receberam anteriormente, durante 40 dias, uma solução contendo as bactérias Ralstonia sp. e Burkholderia sp., os do Grupo 2 receberam, também por 40 dias as bactérias Paenibacillus sp. e Cupriavidus sp. e os do Grupo 3 não receberam nenhuma bactéria. Após o período de administração, durante 60 dias, os animais de todos os grupos foram soltos para pastar em um piquete de um hectare, que apresentava uma quantidade significativa da planta. Diariamente eles foram observados quanto ao consumo espontâneo das folhas de A. septentrionalis e quanto à presença de sinais clínicos de intoxicação ou morte. Os caprinos de todos os grupos consumiram quantidades significantes da planta durante o período experimental. Os caprinos que não receberam as bactérias degradantes de MFA (Grupo 3) adoeceram e morreram entre o 25º e o 27º dia de experimento, enquanto que os que receberam as bactérias degradantes de MFA (Grupo 1 e 2) só apresentaram sinais clínicos no 55º dia de experimento, o que coincidiu com a rebrota da planta. Os dias transcorridos desde a observação a campo até a morte dos caprinos do Grupo 3 (25,5±0,9 dias) foram significativamente menores (p<0,05) que os do Grupo 1 (58,6±1,3 dias) e do Grupo 2 (57,8±1,5 dias). Com isso pode-se concluir que a administração de bactérias degradadoras de MFA aumenta à resistência a intoxicação natural por A. septentrionalis.(AU)

A thermophilic microorganism from Deception Island, Antarctica with a thermostable glutamate dehydrogenase activity

BACKGROUND: The Antarctic continent is a source of extreme microorganisms. Millions of years of isolation have produced unique biodiversity with adaptive responses to its extreme environment. Although the Antarctic climate is mainly cold, the presence of several geothermal sites, including thermal springs, fumaroles, hot soils and hydrothermal vents, provides ideal environments for the development of thermophilic and hyperthermophilic microorganisms. Their enzymes, called thermoenzymes, are the focus of interest in both academic and industrial research, mainly due to their high thermal activity and stability. Glutamate dehydrogenase, is an enzyme that plays a key role in the metabolism of carbon and nitrogen catalyzing reversibly the oxidative deamination of glutamate to alpha-ketoglutarate and ammonium. It belongs to the family of oxidoreductases, is widely distributed and it has been highly regarded for use as biosensors, particularly for their specificity and ability to operate in photochemical and electrochemical systems. However, the use of enzymes as biosensors is relatively problematic due to their instability to high temperatures, organic solvents and denaturing agents. The purpose of this study is to present the partial characterization of a thermophilic microorganism isolated from Deception Island, Antarctica, that displays glutamate dehydrogenase activity. RESULTS: In this work, we report the isolation of a thermophilic microorganism called PID15 from samples of Deception Island collected during the Antarctic Scientific Expedition ECA 46. This microorganism is a thermophile that grows optimally at 50 °C and pH 8.0. Scanning electron microscopy shows rod cells of 2.0 to 8.0 µm of length. Phylogenetic analysis of 16S rRNA gene revealed that this microorganism is closely related to Bacillus gelatini. This microorganism contains a thermostable glutamate dehydrogenase with optimal activity at pH 8.0 and temperatures for its activity from 37 to 50 °C, range of temperature of interest for biotechnological applications. This glutamate dehydrogenase is a highly thermostable enzyme. CONCLUSION: This is the first report of a microorganism from Antarctica containing a thermostable glutamate dehydrogenase that maintains its activity in a broad range of temperatures making it of potential interest for biotechnological applications.

Isolation and characterization of a novel endo-beta-1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

Enzymatic potential of heterotrophic bacteria from a neutral copper mine drainage

Abstract Copper mine drainages are restricted environments that have been overlooked as sources of new biocatalysts for bioremediation and organic syntheses. Therefore, this study aimed to determine the enzymatic activities (esterase, epoxide hydrolase and monooxygenase) of 56 heterotrophic bacteria isolated from a neutral copper mine drainage (Sossego Mine, Canaã dos Carajás, Brazil). Hydrolase and monooxygenase activities were detected in 75% and 20% of the evaluated bacteria, respectively. Bacterial strains with good oxidative performance were also evaluated for biotransformation of organic sulfides. Fourteen strains with good enzymatic activity were identified by 16S rRNA gene sequencing, revealing the presence of three genera: Bacillus, Pseudomonas and Stenotrophomonas. The bacterial strains B. megaterium (SO5-4 and SO6-2) and Pseudomonas sp. (SO5-9) efficiently oxidized three different organic sulfides to their corresponding sulfoxides. In conclusion, this study revealed that neutral copper mine drainages are a promising source of biocatalysts for ester hydrolysis and sulfide oxidation/bioremediation. Furthermore, this is a novel biotechnological overview of the heterotrophic bacteria from a copper mine drainage, and this report may support further microbiological monitoring of this type of mine environment.

Cellulase activity and dissolved organic carbon release from lignocellulose macrophyte-derived in four trophic conditions

Abstract Considering the importance of lignocellulose macrophyte-derived for the energy flux in aquatic ecosystems and the nutrient concentrations as a function of force which influences the decomposition process, this study aims to relate the enzymatic activity and lignocellulose hydrolysis in different trophic statuses. Water samples and two macrophyte species were collected from the littoral zone of a subtropical Brazilian Reservoir. A lignocellulosic matrix was obtained using aqueous extraction of dried plant material (≈40 °C). Incubations for decomposition of the lignocellulosic matrix were prepared using lignocelluloses, inoculums and filtered water simulating different trophic statuses with the same N:P ratio. The particulate organic carbon and dissolved organic carbon (POC and DOC, respectively) were quantified, the cellulase enzymatic activity was measured by releasing reducing sugars and immobilized carbon was analyzed by filtration. During the cellulose degradation indicated by the cellulase activity, the dissolved organic carbon daily rate and enzyme activity increased. It was related to a fast hydrolysable fraction of cellulose that contributed to short-term carbon immobilization (ca. 10 days). After approximately 20 days, the dissolved organic carbon and enzyme activity were inversely correlated suggesting that the respiration of microorganisms was responsible for carbon mineralization. Cellulose was an important resource in low nutrient conditions (oligotrophic). However, the detritus quality played a major role in the lignocelluloses degradation (i.e., enzyme activity) and carbon release.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.

Sourcing Starter Cultures for Parkia biglobosa Fermentation Part II: Potential of Bacillus subtilis Strains.

Aim: To select good strains of Bacillus subtilis for use as starter culture in the fermentation of Parkia biglobosa. Study Design: Fifteen (15) strains of Bacillus subtilis group obtained from commercial samples were used in starter-culture fermentation of Parkia biglobosa seeds to produce ‘iru’. Place and Duration of Study: Food Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pahumthani, Thailand, between March to May 2010. Methodology: The quality of the starter culture-fermented products were compared on the bases of sensory evaluation, degree of hydrolysis (DH), level of ammonia nitrogen (NH3-N), pH and enzymatic activities. The 15 strains were also screened for haemolytic activity. Results: On the basis of the sensory scores of 5 parameters (color, odor, consistency, texture and over-all liking), particularly the over-all liking, 5 strains were rated the best (in descending order): BC4333 > 8B > 2B > 7A > 5A, amongst the 15 tested. There were good correlations between pH and DH (r= 0.926), DH and NH3-N (r=0.962) and between pH and NH3-N (r=0.945). The strain BC4333 produced the very soft variant of ‘iru’ (‘iru-pete’), without the addition of ‘kuuru’ (local potash). The quantity of extracellular enzymes (protease, amylase, pectinase, phytase and lipase) produced during fermentation varied significantly. None of the 5 strains was haemolytic on sheep blood agar. Conclusion: The 5 strains of Bacillus subtilis (BC4333, 8B, 2B, 7A, 5A) that showed potentials of being used as starter cultures for industrial production of ‘iru’, were nonhemolytic on blood agar.

Isolation and characterization of novel thermophilic lipase-secreting bacteria

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Fermenation Products of β-Mannanase Producing Bacteria Inhibit Selected Poultry Borne Pathogens.

Aim: The study evaluated the inhibitory effect of fermentation products of β-mannanaseproducing bacteria on selected poultry borne pathogens. Study Design: The first experiment, bacterial isolates previously confirmed positive for mannanase by plate assay technique were further screened for mannanase production in submerged state fermentation. In the second experiment, inhibitory effect of fermentation products of mannanase-producing bacteria on selected poultry pathogens was evaluated. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: Bacterial isolates from agricultural wastes previously confirmed positive for mannanase activity by plate assay were further screened for their potential performance under submerged state fermentation and enzyme activity determined by dinitrosalicylic acid method. The inhibitory action of β-mannanase-producing bacteria was determined by supplementation of supernatant and plating method. Results: Isolate 1A showed highest mannanase activity (13.430 U/ml), displayed broad inhibition to selected poultry borne pathogens; Klebsiella oxytoca, Shigella alkalescens, Escherichia coli, Salmonella typhii, Staphylococcus aureus and Streptococcus sp. Apart from isolate 1A, fermentation products of other isolates generated from the mannolytic action of β-mannanase on mannan containing substrate displayed different percentage inhibition on selected poultry borne pathogens. Conclusion: The results suggested that fermentation products from β-mannanaseproducing bacteria might possess antibacterial properties which could be applied in poultry farms.

Endo-and exoglucanase activities in bacteria from mangrove sediment

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.

Bioprospection of cellulolytic and lipolytic South Atlantic deep-sea bacteria

Background: Cellulases and lipases have broad industrial application, which calls for an urgent exploration of microorganisms from extreme environments as valuable source of commercial enzyme. In this context, the present work describes the bioprospection and identification of deep-sea bacteria that produce cellulases and lipases, as well their optimal temperature of activity. Results: The first step of this study was the screening of cellulolytic and lipolytic deep-sea bacteria from sediment and water column, which was conducted with substrates linked with 4-Methylumbelliferyl. Among the 161 strains evaluated, 40 were cellulolytic, 23 were lipolytic and 5 exhibited both activities. Cellulolytic and lipolytic bacteria are more common in sediment than at the water column. Based on the ability to produce cellulases and lipases three isolates were selected and identified (16S rRNA sequencing) as Bacillus stratosphericus, B. aerophilus and B. pumilus. Lipases of strain B. aerophilus LAMA 582 exhibited activity at a wide temperature range (4º to 37ºC) and include psychrophilic behaviour. Strain Bacillus stratosphericus LAMA 585 can growth in a rich (Luria Bertani) and minimal (Marine Minimal) medium, and does not need an inducer to produce its mesophilic cellulases and lipases. Conclusions: Deep-sea sediments have great potential for bioprospection of cellulase and lipase-producing bacteria. The strains LAMA 582 and LAMA 585 with their special features, exhibit a great potential to application at many biotechnology process.

Biotechnological application of bacterial alkaline thermostable enzymes in bio-detergent industry

This is an investigation concerned on the production of alkaline thermostable microbial enzymes for application in biodetergent technology. Bacillus licheniformis- B42 and Geobacillus stearothermophilus -B78 were selected and identified among one hundred and fifty-three thermophilic bacterial isolates with respect to their ability to produce alpha-amylases, cellulases, proteases and lipases grown on some agro-industrial wastes at 55 degree C and at pH 9 for application in biodetergent technology. Productivity of four alkaline thermostable enzymes by both selected strains using slaughter house wastes [SHW] as best substrate for proteases and lipases and potato peel [PP] as best substrate for alpha-amylases and cellulases. The enzymatic level more affected by incubation temperature, pH, SHW and PP concentrations, inoculum size, incubation period, carbon, nitrogen, metal inducer and vitamins sources, under shaking conditions. Four alkaline thermostable enzymes were produced under all optimal nutritional and environmental conditions and purification by column chromatography on Sephadex G200 and G100, respectively were performed. Purification of four produced alkaline thermostable enzymes steps resulted in raising the purification fold to 17.04,15.24, 411.9 and 27.33 times in comparable with crude enzymes for alpha-amylase. cellulase, protease and lipase, respectively. The wash performing analysis of the four enzymes revealed that, it could effectively remove a variety of stains such as blood, apple, chocolate, mango, strawberry, salad and pomegranate by treatment at 55 degree C for 15 min when alkaliphilic-thermostable crude/purified enzymes were added separately or in combination with or without detergent [Rabso] as an Egyptian local detergent product. The crude enzymes of these two bacterial strains proved to be potentially candidates for the application in the detergent technology

Honey as a natural preservative of milk.

The anti-bacterial property and preservative nature of honey has been studied by evaluating the role of hydrogen peroxide in these properties, against bacterial strains isolated and identified from pasteurized milk samples. The antibacterial property of honey examined by agar incorporation assay and turbidometry, indicated a concentration dependent inhibition of bacterial growth in all catalase negative strains in comparison with catalase positive strains, highlighting a probable role of hydrogen peroxide. Samples of commercial milk stored at 40C in presence of honey were shown to inhibit opportunistic bacterial growth better compared to samples stored without honey. Due to the bactericidal property of hydrogen peroxide and its preservative nature, honey which is chiefly a combination of various sugars and hydrogen peroxide, can be used a preservative of milk samples.

Preliminary report of NAD[+]-dependent amino acid dehydrogenase producing bacteria isolated from soil

Amino acid dehydrogenases [L-amino acid: oxidoreductase deaminating; EC 1.4.1.X] are members of the wider superfamily of oxidoreductases that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either NAD[+], NADP[+] or FAD. These enzymes have been received much attention as biocatalysts for use in biosensors or diagnostic kits to screen amino acid metabolism disorders such as phenylketonuria [PKU], maple syrup urine disease [MSUD], homocystinuria [HCY] and hyperprolinemia. This study was aimed to isolation and screening of novel amino acid dehydrogenases from soil bacteria. The enzyme producing bacteria were selected among L-methionine and L-phenylalanine utilizers isolated from soil by thin layer chromatography, activity staining and confirmed by enzyme assay. Bacterial strains were identified by phenotypic and biochemical characteristics. The steady-state kinetic studies of enzymes were also performed. In total of 230 tested strains, four of them were recognized as amino acid dehydrogenase producers that belong to species of Pseudomonas, Citrobacter and Proteus. They exhibited the desired NAD[+]-dependent dehydrogenase activities toward L-isoleucine, L-methionine, L-cysteine, L-serine and L-glutamine in oxidative deamination reaction. The specific activity of L-isoleucine dehydrogenase, L-methionine dehydrogenase and L-glutamine dehydrogenase for oxidative deamination of L-isoleucine, L-methionine and L-glutamine were 1.59, 1.2 and 0.73 U/mg, respectively. The Kcat /Km [s-1.mM -1] values in these strains were as follows: L-isoleucine, 113.6, L-methionine, 62.05 and L-glutamine, 95.83. This is the first report of occurrence a specific isoleucine dehydrogenase, glutamine dehydrogenase and methionine dehydrogenase in bacteria

Effect of denaturants on the structure and activity of 3-hydroxybenzoate-6-hydroxylase.

The effect of denaturants such as urea, sodium dodecylsulphate (SDS), guanidinium hydrochloride (Gu.HCl) on the structure of enzyme 3-hydroxybenzoate-6-hydroxylase was studied using intrinsic fluorescence and far and near-UV-CD spectroscopic techniques. Also, activity profiles of the enzyme, as a function of increasing concentrations of denaturants were studied. The far-UV CD spectrum of the enzyme did not show appreciable alterations in the presence of urea, SDS or Gu.HCl, thereby suggesting that the protein does not undergo gross conformational changes in its alpha-helical secondary structure. The treatment of enzyme with 2 M urea resulted in almost complete loss of catalytic activity, accompanied by the reduction of emission fluorescence of enzyme. Similarly, treatment with 0.01% SDS also caused almost complete loss of activity and quenching of enzyme fluorescence as well as a red shift in the emission peak. In addition, reduction in the intensity of near-UV-CD spectrum, especially at 280 nm was observed. About 70% of the activity was lost by treatment with 20 mM Gu.HCl, accompanied by quenching of intrinsic fluorescence of the enzyme. The change in intrinsic fluorescence of the enzyme in the presence of 5 mM-100 mM Gu.HCI could be correlated to progressive loss of catalytic activity. Thus, intrinsic fluorescence (due to tryptophan residues) could be used as an effective probe to provide an insight into the relation between the activity and subtle conformational changes of the enzyme. The results suggested that denaturants caused very slight conformational changes in the enzyme that perturbed the microenvironment of aromatic amino acid residues such as tryptophan accompanied by reduction or loss of catalytic activity.

Phylogenetic tree construction of butyrylcholinesterase sequences in life forms.

Butyrylcholinesterase is an enzyme with few known physiological functions. It is related to acetylcholine that was shown to be expressed in a variety of life forms. We performed a search using the human butyrylcholinesterase gene (HGNC:983;MIM:177400), and found the sequence in a broad spectrum including plants, bacteria and animals. Therefore butyrylcholinesterase appears to have evolved early in evolution, and to have been conserved.