A carboxypeptidase has been purified from germinated barley. The preparations are homogeneous in ultracentrifugal analysis. I n disc electrophoresis traces of impurities can be detected a t high concentration. The purified enzyme liberates carboxyl-terminal amino acid residues from a wide range of carbobenzoxy (Z)-dipeptides. Peptides containing proline are not attacked. It does not possess any endopeptidase, aminopeptidase, or dipeptidase activity. The enzyme has a pH optimum a t pH 5.2, is inhibited by di-isopropylphosphofluoridate and p-chloromercuribenzoate, and has a molecular weight of about 90,000.I n connection with a search for specific synthetic substrates for barley endopeptidases, we observed that freeze-dried enzyme preparations from germinated barley hydrolysed a substrate for collagenase, Z-Gly-Pro-Gly-Gly-Pro-Ala [I], with rapid liberation of the carboxyl-terminal alanine and slower release of the adjacent dipeptide, glycyl-proline [2]. Appaparently, the enzyme preparation contained a carboxypeptidase-like enzyme and an endopeptidase with collagenase-like or very broad specificity.The presence of carboxypeptidase-like activity was confirmed with a series of carbobenzoxy-dipeptides. Several of these, including Z-Leu-Gly, Z-Glu-Tyr, and Z-Pro-Trp, were hydrolysed by the enzyme preparations, Z-Phe-Ala being the compound most rapidly broken down.As the activities were very high and no information on seed carboxypeptidases is available, an endeavour was made to purify the enzyme.We have now isolated the Z-Phe-Ah-hydrolysing enzyme in apparently pure form. The preparation hydrolyses most of the Z-dipeptides tested, and does not possess any endopeptidase, aminopeptidase, or dipeptidase activity. However, it is totally inactive against the original sub strate, Z -Gly -Pro-Gl y-Gl yPro-Ala, and hydrolyses some of the Z-dipeptides with a lower relative rate than the crude enzyme preparations. Apparently barley contains two or possibly several carboxypeptidase-like enzymes, and we have isolated one of them. EXPERIMENTAL PROCEDURES MaterialsPirkka barley (a Finnish 6-row variety) and Pirkka "high enzyme" malt were obtained from Lahden Polttimo Oy (Lahti, Finland). Freeze-dried green malt was prepared from Pirklca barley, as has been described before [3].Reagents and column materials were purchased from the following sources : peptides, Yeda Research and Development Company Ltd., Sigma Chemical Company, Fluka AG, and Hoffman-LaRoche & Co. ; di-isopropylphosphofluoridate, Sigma; DEAE-cellulose (standard, 0.93 mequiv./g), Carl Schleicher & Schull Co. ; CM-cellulose (0.7 mequiv./g), Macherey, Nagel & Go.; Sephadex G-100 and G-200 (for gel filtration), Pharmacia. Other reagents were of reagent grade, except for ammonium sulphate and acetone which were purum grade. Carboxypeptidase AssayZ-Phe-Ala was used as substrate and the alanine liberated was measured by the ninhydrin reaction as follows.1 ml of substrate solution, 2 mM Z-Phe-Ala in 50 mM sodium acetate buffer, pH 5.2, containing 0.5mM EDTA, was added to 0.1 ...

A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.

The metabolism of the germinating barley seed is a complicated physiological process. From the point of view of industrial malting, the syntheses of amylolytic and proteolytic enzymes as well as the degra dation of endospermal structures are extremely important. In order to control his process the maltster needs to have a good understanding of these processes as well as of the regulatory mechanisms which control them. When a good malting barley is allowed to germinate under traditional conditions for the time needed, a good malt can be produced without this scientific background. The present day brewing industry, however, has to work with barleys which are not of the traditional malting type and further more accelerated processes are an economic necessity. Under these circumstances, the maltster needs all scientific help he can get. Biochemical and physiological research on germination provides him with the tools he needs for process control and optimisation.

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Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T0), and four of the 90 T0 spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T1). The four transgenic shoots of Toivo (the T0 plant) produced 25 plantlets as T1 progeny. Altogether fifteen of these T1 plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T2 progeny.