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 34000 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 yybQencoded protein is the first fully characterized member of a new class of inorganic pyrop hosp hatase.I

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The principles of extrusion cooking are summarised. In small scale trials good extracts were obtained from extruded barley when it was mashed with industrial enzymes, using a programmed temperature cycle. Extruded barley, wheat and maize and wheat flour yielded acceptable levels of extract when mashed with lager malt (70%) using a programme with 1 hour rests at 50°C and 65°C. The extracts obtained from these grists were increased above those obtained from grists of lager malt alone and the viscosities of the worts were reduced when the mashes were supplemented by preparations of bacterial enzymes. Enzyme additions also improved extract recoveries from all-malt mashes and reduced the viscosities of the derived worts.Usinga temperature programmed mashing cycleandsupplementary enzymes beers were prepared from a lager malt grist and grists in which the lager malt was partly replaced, by 30%, with extruded barley or extruded wheat, or extruded maize or wheat flour pellets. In every case wort was recovered relatively easily, the worts fermented normally and the beers were all fully acceptable, although their flavours did differ. However, in contrast to results of preliminary brewing trials, the head retentions of the beers made with adjuncts were unusually low, possibly because of particular enzyme additions.

Radial diffusion tests were used to detect p-glucan-, starch-, protein-, pentosan-and triglyceridedegrading enzyme activities in a variety of commercial enzyme preparations. Some implications of the presence of unexpected enzymes in particular preparations are discussed.A series of laboratory mashes were made with grists of milled barley, extruded barley, and extruded wheat, using various enzyme additions and temperature-time programmes. In addition a limited number of experiments were made with pale ale barley malt, and wheat flour pellets. Extract yields varied from acceptable to outstandingly good. The most intensive mashing system, with enzyme supplementation, increased the extract yield of an all -malt mash by 3-9% relative to the control mashing programme. Some worts were evaluated for Total Soluble Nitrogen (T.S.N.), Free Ami no Nitrogen (F.A.N.) and viscosity. Slow wort-filtration rates occurred in some mashes although the viscosities of the worts from these mashes were low. This problem did not occur in any of the samples intensively mashed over a S h period. The results apparently exclude the possibility that a simple, reliable technique for estimating the extract yields of adjuncts could be devised, using these enzyme preparations.