Yeasts associated with Manteca

Manteca is a traditional milk product of southern Italy produced from whey deriving from Caciocavallo Podolico cheese-making. This study was undertaken to obtain more information about the microbiological properties of this product and particularly about the presence, metabolic activities, and technological significance of the different yeast species naturally occurring in Manteca. High numbers of yeasts were counted after 7 days ripening (10(4)-10(5) cfu g(-1)) and then decreased to 10(2) at the end. A total of 179 isolates were identified and studied for their phenotypic and genotypic characteristics. The most frequently encountered species were Trichosporon asahii (45), Candida parapsilosis (33), Rhodotorula mucilaginosa (32), Candida inconspicua (29). Some of these yeasts showed lipolytic activity (32 strains) and proteolytic activity (29 strains), NaCl resistance up to 10% and growth up to 45 degrees C (42 strains). Biogenic amines were formed by proteolytic strains, in particular phenylethylamine, putrescine and spermidine. Spermidine was produced by all the yeasts tested in this work, but only Trichosporon produced a great quantity of this compound. Histamine was not detectable. Caseinolytic activity was common to almost all strains, corresponding to the ability to efficiently split off amino-terminal amino acids. The highest and most constant activity expressed by all species was X-prolyl-dipeptidyl aminopeptidase. The findings suggest that the presence of yeasts may play a significant role in justifying interactions with lactic acid bacteria, and consequently with their metabolic activity in the definition of the peculiar characteristics of Manteca cheese.     

Involvement of Microbial Alkyl Hydroxybenzenes in the Regulation of Autolytic Degradation of Yeast Cells

A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d₂ (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under the concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d₁, which belong to alkylhydroxybenzenes and fulfill functions of chemical chaperones. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.     

Synthesis and in vitro antibacterial activity of novel 5'-O-analog derivatives of zidovudine as potential prodrugs

An efficient, short synthesis of four potential prodrugs of 3'-azido-3'-deoxythymidine (AZT) and their antibacterial activity are reported. The 5'-OH group of AZT was functionalized with oxalyl chloride obtaining an acyl chloride derivative (AZT-Ox), which by further transformation with leucine, isoleucine and valine amino acids led to the corresponding AZT analogs, namely AZT-Leu, AZT-iLeu and AZT-Val. A carboxyl acid derivative (AZT-Ac) was also obtained by hydrolysis of AZT-Ox. These compounds, which exhibit anti HIV activity, have killed collection and clinical strains of some opportunistic infectious agents in AIDS-related complex. Thus, the clinical strains, K. oxytoca, S. typhi and K. pneumoniae, and collection strain K. pneumoniae ATCC 10031 showed sensitivity to antibiotics. The activity order for the studied compounds against the most sensitive strain (K. pneumoniae ATCC 10031) was AZT-Leu > AZT-iLeu > AZT-Val > AZT-Ac > AZT. On the other hand, the activity order for the second most sensitive strain (K. oxytoca) was AZT-Leu > AZT-Val = AZT-Ac > AZT-iLeu > AZT. The most effective antibacterial drug AZT-Leu, M.I.C.=0.125 microgmL(-1)) was 16 times more active than AZT (AZT, M.I.C.=2 microg mL(-1)) against K.     

Autolysis of Lactococci: Detection of Lytic Enzymes by Polyacrylamide Gel Electrophoresis and Characterization in Buffer Systems

Lactococcal strains were screened for bacteriolytic activity against Micrococcus luteus cells, lactococcal cells, and cell walls. Thirty strains were screened for bacteriolytic activity against cells and cell walls incorporated into agar medium. Enzymes from all strains hydrolyzed the substrates; however, the activity against Micrococcus cells was much higher than against Lactococcus cells or cell walls. Electrophoretic profiles of bacteriolytic activities of culture supernatants, sodium dodecyl sulfate-treated cell extracts, cell wall fractions, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing M. luteus cells or lactococcal cell walls as the substrate. The 22 strains tested contained two to five lytic bands in the culture supernatant, ranging in size between 32 and 53 kDa. The cell extracts, the sodium dodecyl sulfate-treated cell extracts, and the cell wall fractions revealed two lytic bands of 47 and 53 kDa. Effects of external factors on autolysis of some strains were determined in buffer systems. Optimal autolysis was observed in the exponential growth phase at pH 6.0 to 7.5 and at a temperature of 30(deg)C. Two of three strains tested seemed to contain a glycosidase, and all three strains contained an N-acetylmuramyl-l-alanine amidase or an endopeptidase.     

Purification of β-Glucosidase from Saccharomyces lactis Strains Y-14 and Y-1057A

Two strains of Saccharomyces lactis (Y-14 and Y-1057A), medium (B(m)) and low (B(1)) constitutive producers of beta-glucosidase, were grown in enriched medium. beta-Glucosidase was extracted by autolysis and purified by ammonium sulfate precipitation, gel filtration, and calcium phosphate gel adsorption-elution. The kinetics of release, purification, and stability of beta-glucosidase from strains Y-14 and Y-1057A were compared with the enzyme from strain Y-123. The ability of glycerol, sorbitol, and mannitol to stabilize the beta-glucosidases is presented. A lower molecular weight, labile form of the Y-14 enzyme is demonstrated. Differences in the initial specific activites of beta-glucosidase among the three strains are discussed.     

Evaluation of different phenol hydroxylase-possessing phenol-degrading pseudomonads by kinetic parameters

Phenol-degrading pseudomonads possessing different phenol hydroxylases (PH) were evaluated by the values of apparent half-saturation constant for phenol-oxygenating activity (K ( S )), maximum specific growth rate (mu (max)), lag-time length (lambda), inhibition constant (K ( I )) and growth yield factor (Y ( X/S )). Strains of the same PH type showed similar kinetic parameters: single-component PH (sPH) harbouring strains had higher values of K ( S ) and lower values of mu (max) than the strains having multicomponent PH (mPH). However, the values of K ( I ) and the dependencies of the lag-time length on initial phenol concentration were strain-specific. The elevated ratio between specific activities of catechol 1,2-dioxygenase (C12O) and muconate cycloisomerase in sPH-strains caused irreversible accumulation of a high amount of exogenous cis,cis-muconate (CCM) which resulted in decreased Y ( X/S ) values. Co-presence of sPH and mPH genes did not give the strains PC16 and P69 any extra advantage and according to determined kinetic parameters only one PH was active during phenol degradation. At the same time simultaneous functioning of catechol ortho and meta cleavage pathways (strain PC20) resulted in higher mu (max) and Y ( X/S ) values. Evaluation of strains showed that the type of PH determined the efficiency of phenol degradation, whereas the tolerance to elevated phenol concentrations was strain-specific.     

Limited autolysis reduces the Ca2+ requirement of a smooth muscle Ca2+-activated protease

Chicken gizzard smooth muscle contains large amounts of Ca2+-activated protease activity. Approximately 15 mg of purified enzyme can be obtained from 1 kg of fresh muscle. The enzyme consists of two subunits (Mr = 80,000 and 30,000) present in a 1:1 molar ratio. In the presence of CaCl2, the 80,000/30,000-dalton heterodimer (form I) is rapidly converted by limited autolysis to a 76,000/18,000-dalton species (form II). Both the 80,000- and 30,000-dalton subunits are degraded simultaneously. Moreover, the Ca2+ dependence for autolysis (K0.5 = 300 microM) is identical for both subunits. Neither the time course nor the Ca2+ dependence of the autolytic conversion reaction is altered by 10- and 20-fold molar excesses of substrate. Limited autolysis markedly reduces the Ca2+ requirement for substrate degradation. Using N-[ethyl-2-3H]maleimide-labeled 27,000-dalton cardiac myosin light chains as substrate, the Ca2+ requirement of form I was found to be quite high (K0.5 = 150 microM). Under similar conditions, the Ca2+ requirement of form II was 30-fold lower (K0.5 = 5 microM). Limited autolysis did not alter the specific activity of the enzyme. Our results demonstrate that smooth muscle contains an abundant amount of Ca2+-activated protease. Moreover, autolysis of this enzyme may play an important regulatory role by converting the native form to a species that is fully active at physiological levels of intracellular calcium ion.     

Clonal population structure of Pseudomonas stutzeri, a species with exceptional genetic diversity

Genetic diversity and genetic relationships among 42 Pseudomonas stutzeri strains belonging to several genomovars and isolated from different sources were investigated in an examination of 20 metabolic enzymes by multilocus enzyme electrophoresis analysis. Forty-two distinct allele profiles were identified, indicating that all multilocus genotypes were represented by a single strain. All 20 loci were exceptionally polymorphic, with an average of 15.9 alleles per locus. To the best of our knowledge, this P. stutzeri sample exhibited the highest mean genetic diversity (H = 0.876) found to date in all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles was identified. The index of association (I(A)) for the P. stutzeri strains analyzed was 1.10. The I(A) values were always significantly different from zero for all subgroups studied, including clinical and environmental isolates and strains classified as genomovar 1. These results suggest that the population structure of P. stutzeri is strongly clonal, indicating that there is no significant level of assortative recombination that might destroy linkage disequilibrium.     

Control of Aspergillus growth and aflatoxin production using antioxidants at different conditions of water activity and pH

AIMS: The effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone (THB) and propyl paraben (PP) (at concentrations of 1, 10 and 20 mmol l(-1)) on germination, growth and aflatoxin B1 production by Aspergillus section Flavi was evaluated. METHODS AND RESULTS: Studies on the percentage of spore germination, elongation rate, growth rate and aflatoxin B1 production were carried out in vitro in relation to water activity (aw) at 0.982, 0.937, 0.809 and 0.747 values. At 0.809 and 0.747aw values none of the isolates was able to germinate. Overall, PP and BHA were the antioxidants most effective at inhibiting germination of both species. In the presence of the lowest concentration of BHA and PP (1 mmol l(-1)) the conidial germination percentage ranged from 2 to 19% after 15 h of incubation at the highest water activity tested. BHA and PP at 10-20 mmol l(-1) completely inhibited conidial germination. The antioxidants more efficient in controlling Aspergillus elongation rate were PP, BHT and BHA. All strains were much more sensitive to all antioxidants tested on the percentage of spore germination and growth rate at 0.937aw. The antioxidants PP and BHA completely inhibited aflatoxin B1 production by all strains when added at 1 mmol l(-1). Decreased aflatoxin B1 levels in comparison with the control, were observed with BHT at 1, 10 and 20 mmol(-1) with the strain T20 at 0.982aw. In contrast, stimulation was observed with the antioxidant THB at 10 and 20 mmol l(-1) at 0.937aw with the strains T20 and T23. The effect of BHA and PP at 1 mmol l(-1) on lag phase and growth rate was maintained in the pH range between 6 and 8. At all pH values the inhibitory effect of BHA was higher than PP. No aflatoxin B1 was detected at all pH values. CONCLUSIONS: The data show that BHA and PP could be considered as effective fungitoxicants for A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained show promise for controlling growth and aflatoxin B1 in stored maize. Futher studies should be carried out to examine the potential for antioxidants, such as BHA and PP to effectively control both growth and aflatoxin production.     

Formation of resting forms of Arthrobacter globiformis in autolysing cell suspensions

Under conditions of spontaneous or induced autolysis of thick cell suspensions, Arthrobacter globiformis strains produced cells exhibiting features typical of resting microbial forms. The number of viable resting cells was greater under conditions of induced rather than spontaneous autolysis. The thermoresistance of the resting cells of A. globiformis strains isolated from 2- to 3 million-year-old permafrost was higher than that of the collection A. globiformis strain.     

Shrimp processing by-products protein hydrolysates: Evaluation of antioxidant activity and application in biomass and proteases production

Protein hydrolysates were prepared from shrimp processing by-products (SPBP) using five proteolytic enzymes: trypsin, Alcalase®, crude enzyme extract from sardinelle (Sardinella aurita) viscera and enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1. The obtained hydrolysates exhibited different degrees of antioxidant activities evaluated through three main tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity, reducing power and β-carotene bleaching assays. Hydrolysates were also tested as nitrogen source for microbial growth and proteases production by Escherichia coli, Saccharomyces cerevisiae, B. mojavensis A21 and B. subtilis A26. The reached results showed that the SPBP protein hydrolysates (SPBPPHs) could be a promising alternative to currently available commercial nitrogen sources of other origins.     

Bacillus naphthovorans sp. nov. from oil-contaminated tropical marine sediments and its role in naphthalene biodegradation

A Bacillus sp., designated as strain MN-003, was isolated as the dominant cultivatable naphthalene-degrading organism from oil-contaminated tropical marine sediments. Strain MN-003 is strictly aerobic, rod-shaped, Gram-positive, catalase positive, oxidase negative, and forms endospores. Strain MN-003 grew at salinities ranging from 0.28 to 7.00% and temperatures ranging from 15 to 41 degrees C. Phylogenetic analyses reveal that strain MN-003 is most similar to Bacillus sp. VAN14, with a 16S rRNA sequence identity of 97.9%. Based on taxonomic and 16S rRNA data, strain MN-003 was named Bacillus naphthovorans sp. nov. When grown with naphthalene as sole carbon source, strain MN-003 had a maximal specific growth rate (mu(max)) of 0.32 +/- 0.03 h(-1), and a half-saturation constant (K(S)) of 22.3 +/-4.2 microM. A batch study of the tropical marine sediments enriched with naphthalene showed that cells of the Bacillus genus grew to become dominant members of the microbial community. The bacilli comprised 39.5 +/- 6.5% of the microbial fraction after 20 days of enrichment.     

乳清分离蛋白酶解物的抗氧化活性研究

研究了乳清分离蛋白(WPI)酶解物对DPPH.、超氧阴离子自由基和羟基自由基的清除效果,同时用还原法研究了其抗氧化活性。结果表明,WPI酶解物在体外具有较强的抗氧化能力。木瓜蛋白酶酶解物和胰蛋白酶酶解物对DPPH.、超氧阴离子自由基、羟基自由基的清除能力和还原能力强于胰凝乳蛋白酶酶解物和胃蛋白酶酶解物。     

Rational redesign of glucose oxidase for improved catalytic function and stability

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.     

Effect of regulators on bacterial autolysis

The aim of this work was to study the effect of autolysis regulators (the fraction of microbial teichoic acids) on the rate of autolysis and the activity of bacterial extracellular lytic enzymes. The regulators of autolysis isolated from 23 cultures belonging to 10 microbial species regulated the rate of autolysis in Bacillus, E. coli and Streptococcus lactis. The regulators either activated or inhibited autolysis depending on the substrate (of a bacterium to be subjected to autolysis). The quantitative dependence of the autolysis rate on the regulator concentration was specific for each pair 'regulator--substrate'. The regulatory properties of the fraction of teichoic acids varied depending on the age of a culture from which they had been isolated. The regulators of autolysis, with an exception of the preparation from E. coli, inhibited the activity of B. subtilis extracellular lytic enzymes in the course of their action on E. coli cells. The possibility for using the regulators of autolysis in microbiological processes is discussed.     

Formation of resting forms of arthrobacter globiformis in autolyzing cell suspensions

Under conditions of the spontaneous or induced autolysis of thick cell suspensions,Arthrobacter globiformis strains produced cells exhibiting features typical of resting microbial forms. The number of viable resting cells was greater under conditions of induced rather than spontaneous autolysis. The thermoresistance of the resting cells of A.globiformis strains isolated from 2-to 3 million-year-old permafrost was higher than that of the collectionA. globiformis strain.     

Involvement of microbial alkyl hydroxybenzenes in the regulation of autolytic degradation of yeast cells

A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d2 (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d1, which belong to alkylhydroxybenzenes and fulfil functions of chemical chaperons. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.     

Diversity in antifungal activity of strains of <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> from the Brazilian Amazon

Chromobacterium violaceum is a free-living Gram-negative bacterium found in soil and aquatic habitats; abundantly present in the Brazilian Amazon, it is an important example of exploitable microbial diversity of the tropics. In this study, 24 strains from the Brazilian Amazon and ATCC 12472(T) were investigated for biocontrol potential of seven fungi pathogenic to soybean [Glycine max (L.) Merril] seed. Both cells and the supernatants of two Brazilian strains, 07-1 and 27-1, together with ATCC 12472(T) were strongly antagonistic to six out of the seven fungi. The antifungal activity of the Brazilian strains to Fusarium sp., Phomopsis sp. and Cercospora kikuchi was consistently stronger than that of ATCC 12472(T). In addition, the two Brazilian strains, but not ATCC 12472(T), were effective against Corynespora sp., and all three strains and their supernatants were equally effective against Aspergillus sp. and Colletotrichum sp. None of the strains had antifungal activity against Botroyodiplodia sp. Three potential mechanisms related to the antibiosis were investigated: violacein toxicity, cyanide production and chitinolytic activity; however, it was not possible to associate any of them with the antifungal activity. The results highlight the biotechnological potential still to be explored within the poorly characterized microbial biodiversity of the tropics.     

Kinetics of factor Xa inhibition by recombinant tick anticoagulant peptide: both active site and exosite interactions are required for a slow- and tight-binding inhibition mechanism

Recombinant tick anticoagulant peptide (rTAP) is a competitive slow- and tight-binding inhibitor of factor Xa (FXa) with a reported equilibrium dissociation constant (K(I)) of approximately 0.2 nM. The inhibitory characteristics and the high selectivity of rTAP for FXa are believed to arise from the ability of the inhibitor to specifically interact with the residues of both the active site as well as those remote from the active site pocket of the protease. To localize the rTAP-interactive sites on FXa, the kinetics of inhibition of wild-type and 18 different mutants of recombinant FXa by the inhibitor were studied by either a discontinuous assay method employing the tight-binding quadratic equation or a continuous assay method employing the slow-binding kinetic approach. It was discovered that K(I) values for the interaction of rTAP with four FXa mutants (Tyr(99) --> Thr, Phe(174) --> Asn, Arg(143) --> Ala, and a Na(+)-binding loop mutant in which residues 220-225 of FXa were replaced with the corresponding residues of thrombin) were elevated by 2-3 orders of magnitude for each mutant. Further studies revealed that the characteristic slow type of inhibition by rTAP was also eliminated for the mutants. These findings suggest that the interaction of rTAP with the P2-binding pocket, the autolysis loop, and the Na(+)-binding loop is primarily responsible for its high specificity of FXa inhibition by a slow- and tight-binding mechanism.     

地锦草内生细菌多样性、拮抗及促生特性测定

【背景】植物内生细菌既能抑制病原菌对植物的侵染,也具有促生作用。分离具有拮抗和促生活性的内生细菌可为开发微生物菌肥提供理论依据。【目的】筛选内生细菌中的优势拮抗、促生菌种资源。【方法】以地锦草为材料,采用4种分离培养基分离该植物内生细菌,通过形态特征以及16S r RNA基因序列分析,鉴定内生细菌的分类归属。采用平板对峙法,测定内生细菌对棉花立枯丝核菌(Rhizoctonia solani)、小麦赤霉病菌(Fusarium graminearum)、玉米小斑病菌(Bipolaris maydis)的拮抗活性。通过固氮、解磷、产吲哚乙酸(Indole Acetic Acid,IAA)、产铁载体能力等指标初步检测地锦草内生细菌的促生活性。【结果】共分离到133株内生细菌,分属于4门5纲8目13科25属,其中变形菌门(Proteobacteria)为优势门(52.63%),优势属为芽孢杆菌属(Bacillus),占15.79%。发现有8株菌相似性小于98.65%,可能为潜在新物种。拮抗活性结果表明,22株菌有不同程度的抑菌作用,其中菌株DHL56、DHN17、DHP3、DHP8对这3种病原菌...