Renaturation and activation of calf prochymosin produced in an insoluble form in Escherichia coli

Calf prochymosin produced in Escherichia coli cells harboring the expression plasmids was insoluble and formed large inclusion bodies, which were solubilized by 8 M urea. The conditions allowing correct refolding of denatured prochymosin were investigated. Dialysis at pH 10 in the presence of 500 mM NaCl was found to give the maximum renaturation, and subsequent acidic treatment for autocatalytic processing of refolded prochymosin allowed almost 100% recovery of chymosin.     

Predator-avoidance behavior in anuran tadpoles: a new bioassay for characterization of water-soluble cues

Most surveys of large wood in streams are conducted by counting and measuring every piece of large wood within a reach, a technique that is effective but time-consuming. In this study we evaluated an alternative method that takes less time and can be employed in studies in which an estimate of total large wood volume along a stream reach is the primary metric of interest. In first- through third-order streams we estimated in-stream large wood volume and large wood frequency, comparing large wood census estimates to those from a modified a line-intercept technique that has been commonly used in terrestrial forest surveys. Estimates of large wood volume from line transects located in the geographic center of the stream (parallel to stream axis and equidistant from bankfull margins) were highly correlated with those from the wood census (P < 0.001, r ² = 0.88, Pearson’s r = 0.935), but produced slightly greater estimates of large wood volume (regression slope = 1.28, SE = 0.16). Line-intercept estimates of large wood frequency (number per 100 m of stream) were significantly correlated to the wood census counts, but the line-intercept method underestimated frequency by about 50% (P = 0.016). Differences in the estimated large wood volume between line-intercept and wood census surveys were associated with variability in the diameter of the large wood, but unrelated to stream bankfull width, for the range of stream sizes evaluated in this study (≈ 2 to 11 m). Our results suggest that in small constrained streams, line-intercept surveys are an effective method for estimating in-stream large wood volume and that these estimates better approximate results from whole-stream census techniques where the diameter of in-stream wood is relatively consistent. Handling editor: K. Martens     

Cloning and characterization of the carbapenem biosynthetic genes from Streptomyces fulvoviridis

Carbapenem non-producing mutants were isolated from Streptomyces fulvoviridis and divided into six cosynthesis groups. By using one of the mutants as the host and plasmid pIJ385 as the vector, we cloned carbapenem biosynthetic genes from the parental S. fulvoviridis strain. A cloned 6-kb DNA fragment complemented the defects of three mutants each of which had a mutation in different genes. Southern blot hybridization using the cloned 6-kb fragment as probe showed the presence of the nucleotide sequences homologous to the probe in other carbapenem-producing Streptomyces spp. In addition, Streptomyces griseus, a carbapenem non-producer, possessed the sequence homologous to the probe and showed co-synthesis phenomena with some of the carbapenem non-producing mutants of S. fulvoviridis.     

Incorporation of glycosylated amino acid into protein by an in vitro translation system

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.     

Characterization of an alkaline protease from Bacillus sp. no. AH-101

Summary The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes. Offprint requests to: H. Takami     

Microbial Blood-coagulating Protease Screening Test and Some Properties

A potent fungus for blood-coagulating enzyme was isolated from soil during screening tests of about 1500 strains of microorganisms. The fungus was identified as a strain belonging to Cephalosporium. This enzyme could be produced effectively by solid culture on wheat bran and partially purified to about 800 fold. This enzyme was one of the alkaline proteases. It was stable over a pH range of 7~10 and was activated by Ca²⁺ The enzyme could not coagulate 1% fibrinogen solution. The enzyme mixed with diluted blood plasma coagulated the fibrinogen solution. The enzyme also coagulated the citrated plasma from humans, bovines, mice, goats and hens.     

On open access and optimal landings tax

This paper develops two types of simple models on the dynamic interaction between the stock of fish and the effort expended by fishers: continuous-time/discrete-time models in which a landings tax is incorporated as a control variable available to the management authority. The continuous-time model can describe several ideal options of the optimal tax program; however, unfortunately, it is incapable of choosing the best option. Hence, using the alternative tractable discrete-time model and a computational method, the remaining task of determining a unique optimal tax program is accomplished. The fishery thus managed exhibits a regulated open access.