Tom W. Young

Bacillus subtilis ORF yybQ encodes a manganese-dependent inorganic pyrophosphatase with distinctive properties : the first of a new class of soluble pyrophosphatase ?

The N-terminal 15 amino acids of the major protein associated with inorganic pyrophosphatase activity in Bacillus subtilis WB600 are identical to those of B. subtilis ORF yybQ. This ORF was amplified from B. subtilis WB600 DNA by PCR and cloned into an overexpression vector in Escherichia coli. Induction of overexpression produced a soluble protein of 34,000 Da by SDS-PAGE and by matrix-assisted laser desorption and ionization mass spectrometry. The overexpressed protein had a high specific activity for the hydrolysis of magnesium pyrophosphate, and was specifically and reversibly activated by Mn2+ ions. These properties are identical to those of inorganic pyrophosphatase purified from B. subtilis WB600. No significant similarity was found between the derived sequence of the B. subtilis yybQ-encoded protein and published sequences of identified inorganic pyrophosphatases of Eukarya, Bacteria or Archaea domains. However, there is significant similarity to three putative proteins of unknown function from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from Streptococcus gordonii. The genomes of B. subtilis, M. jannaschii and A. fulgidus do not contain sequences similar to those of hitherto known soluble inorganic pyrophosphatases. The present findings, together with a survey of the properties of inorganic pyrophosphatases from 38 different sources, suggest that the B. subtilis yybQ-encoded protein is the first fully characterized member of a new class of inorganic pyrophosphatase.

The sxa2‐dependent inactivation of the P‐factor mating pheromone in the fission yeast Schizosaccharomyces pombe

Haploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P‐factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2‐dependent carboxypeptidase that inactivates P‐factor by removal of the C‐terminal leucine residue.

The extracellular acid protease gene of Yarrowia lipolytica: sequence and pH-regulated transcription

The gene encoding an acid extracellular protease (AXP) from Yarrowia lipolytica (Candida olea) 148 was cloned and the complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AXP consists of 353 amino acids with an M, of 37427. The gene also encodes a putative 17 amino acid hydrophobic prepeptide and a 27 amino acid propeptide containing no potential N-glycosylation sites. The mature extracellular enzyme is produced by cleavage between Phe and Ala. AXP is a member of the aspartyl family of proteases. AXP shows homology to proteases of several fungal genera and to human progastricin. The coding sequence is preceded by a potential regulatory region of 1982 bp. Transcription of both AXP and alkaline extracellular protease genes of Y. lipolytica 148 is regulated by the pH of culture.

pH-regulated expression of the acid and alkaline extracellular proteases of Yarrowia lipolytica.

The pH-regulated expression of the acid (AXP) and alkaline (AEP) extracellular proteases of the yeast Yarrowia lipolytica 148 was analysed. Expression in batch and continuous cultures was determined at the mRNA level by Northern blotting, and at the enzyme level by enzyme assays and Western blotting. Culture pH regulated AEP and AXP expression predominantly at the level of mRNA content. Highest levels of AEP mRNA were detected at pH 6.5 whereas highest levels of AXP mRNA were detected at pH 5.5. At pH values either side of these maxima AEP and AXP expression were progressively down-regulated. For both enzymes, the variation in mRNA levels with culture pH occurred progressively rather than by discrete steps. AXP expression did not occur above pH 7.0. Some degree of AEP expression occurred at all pH values tested in two unrelated strains of Y. lipolytica.

Process intensification by direct product sequestration from batch fermentations: application of a fluidised bed, multi-bed external loop contactor

A critical comparison has been made of the relative efficacy of the primary purification of an extracellular acid protease produced by the yeast Yarrowia lipolytica. The performance of conventional, discrete sequences of fermentation, broth clarification and fixed bed, anion exchange chromatography has been compared with fluidised bed adsorption directly interfaced with post-term fermentation broth and fluidised bed adsorption directly integrated with productive fermentations (so-called direct product sequestration; DPS). Advantages of the latter, in terms of the improved yield and molecular quality of the protease end product are discussed in terms of the design, assembly and operation of component parts of DPS devices and their generic application to other extracellular bioproducts of microbial fermentations. Copyright 1999 John Wiley & Sons, Inc.

Ultrastructure of Septa in Dimargaris cristalligena R. K. Benjamin

Summary: The fine structure of the septum in the vegetative hypha of the mycoparasite Dimargaris cristalligena is described. The cross-wall, continuous with the inner electron-lucent layer of the hyphal wall, bears a central pore with a flared margin occupied by a biumbonate electron-dense plug. A globose body, surrounded by a zone of ribosome-free cytoplasm, is situated next to the septal pore on each side of the cross-wall. In the neck of the haustorium, a globose body is normally observed only on that side of the septal pore next to the appressorium. Globose bodies appear structureless; they are electron-dense when fixed in potassium permanganate and of variable electron density in aldehyde-osmium fixations. Cytoplasmic continuity is maintained through the septal pore by the plasmalemma. In the sporangiophore and sporiferous branchlets, the septal plug bears an upper globose pro-tuberance and a lower obconic one. The hyphal septum in Tiegherniomyces californicus is ultrastructurally similar to that of D. cristalligena. The possible functional and taxonomic significance of such septa is indicated.

Purification of N-methylputrescine oxidase from Nicotiana rustica

Abstract The yield of N-methylputrescine oxidase extracted from Nicotiana rustica roots grown in organ culture was improved by supplementing Whites medium with 1.15 mM (NH4)2SO4. The enzyme was purified from these roots by salt fractionation, anion exchange, affinity and hydrophobic interaction chromatography. The purified protein had Mr 54 000 by SDS-PAGE.

Secretion and pH‐dependent self‐processing of the pro‐form of the Yarrowia lipolytica acid extracellular protease

The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro‐enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N‐linked carbohydrate moieties. Using pulse‐chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro‐form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly‐catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side‐chain specificity of mature AXP towards the oxidized B‐chain of insulin.

R-factor-mediated resistance to tetracycline in Escherichia coli K12: An R-factor with a mutation to temperature-sensitive tetracycline resistance

Abstract R-factor-mediated resistance to tetracycline was studied in Escherichia coli K12 carrying an fi + F-like R-factor which has a mutation resulting in temperature-sensitive resistance to the drug. The rate of uptake of tetracycline in this mutant is sensitive to temperature, but the rate of efflux is not. These results are consistent with the view that resistance to tetracycline is expressed at the level of the influx rather than of the efflux of tetracycline.

Beer quality and flavor

There are two distinct aspects to beer quality. The first is its relative nature, kind, or character and the second, its degree of excellence. In the former case, we are, for example, talking of beer types in terms of the method of production, place of origin, or style. Examples would be top fermented, double decoction, Pilsner, and Burton ale. This aspect would also include many quality parameters that are perceived even before the beer is consumed. For example, color, clarity, degree of carbonation, and presence (or not) of foam. The brewer might also refer to beer types as lager, ale, or dark quality. In recent years, there has been an increased market share for low and no alcohol beer types. Clearly the quality referred to in these ways is largely determined by the brewing practice. The same applies to other beer parameters such as pH and alcohol content. Traditionally, beers arose as characteristic styles depending on the water supply, available raw mate rials, method of temperature control in brewhouse, fermentation, and storage. Today, it is technologically possible for a Single brewer to produce a whole variety of beer qualities from the same equipment. Quality assurance procedures are used to set overall specifications and often techniques are available for fine tuning them. Table 6–1 gives some examples.