Kiyoshi Mizusawa

Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae.

We cloned the Aspergillus oryzae tannase gene using three oligodeoxyribonucleotide (oligo) probes synthesized according to the tannase N-terminal and an internal amino acid (aa) sequence. The nucleotide (nt) sequence of the tannase gene was determined and compared with that of a tannase DNA complementary to RNA (cDNA) by means of reverse transcriptase PCR. The results indicated that there was no intron in the tannase gene and that it coded for 588 aa with a molecular weight of about 64,000. The tannase low-producing strain A. oryzae AO1 was transformed with the plasmid pT1 which contained the tannase gene, and tannase activities of the transformants increased in proportion to the number of copies. Tannase consisted of two kinds of subunits, linked by a disulfide bond(s) with molecular weights of about 30,000 and 33,000, respectively. We purified these subunits and determined their N-terminal aa sequences. The large and small subunits of tannase were encoded by the first and second halves, respectively. Judging from the above results, the tannase gene product is translated as a single polypeptide that is cleaved by post-translational modification into two tannase subunits linked by a disulfide bond(s). We concluded that native tannase consisted of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300,000.

Purification and some properties of cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus

An intracellular cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus E-244 isolated from soil was purified to a homogeneous state by means of Triton X-100 extraction, DEAE-Sepharose column chromatography, hydrophobic and molecular-sieve HPLC. The enzyme was estimated to have an Mr of 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 144,000 by HPLC gel filtration on TSK gel G 3000 SW. It had a pH optimum of 8.0, and the enzyme, stable at 25 degrees C and pH 5.5-9.5 for 24 h, was inactivated at 50 degrees C for 10 min. The enzyme hydrolyzed beta-cyclodextrin more effectively than linear maltooligosaccharides such as maltopentaose, maltohexaose and maltoheptaose or polysaccharides such as starch, amylopectin, amylose and pullulan.

A new amidinohydrolase, methylguanidine amidinohydrolase from Alcaligenes SP. N-42

Me~~~~uan~dine is known to ~~~~~~1~~~ in body fluids of uremic patients [I ,2] and has been proved a uremic toxin [3]. It was found in several foods [4,S] and nitrosated under acidic conditions to give methylnitrosocyanamide with strong mutagenicity and carc~ogenicity f6]_ We attempted to isolate the microorganisms capable of producing methyiguanidine-decomposing enzyme which may have a potential use for clinical assay and/or detoxification. Here, the purification and iden~~ca~ion of a new enzyme, methylgu~id~~e ~idinohydrolase from ~~e~~~e~e~ sp. N42, which catalyzes specifically the hydrolysis of methylguanidine to yield methylamine and urea, is described.

Studies on the Proteolytic Enzymes of Thermophilic Streptomyces Part II:Identification of the Organism and Some Conditions of Protease Formation

Taxonomical characteristics of a thermophilic streptomycete which had been reported to produce a relatively thermostable proteinase were examined, and it was found to belong to a variety of Streptomyces rectus for which the name Streptomyces rectus var. proteolyticus was suggested. Some cultural conditions for proteinase formation by this strain were investigated. The formation of proteinase was greatly stimulated by the addition of Mn·· and affected by the composition of medium. The proteinase formation occurred during the stationary phase of growth at 50°C, while intracellular peptidase was formed in early logarithmic phase. No presence of precursor of the proteinase was suggested.

Hydrolysis of branched cyclodextrins by a cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus E-244

The action of a cyclodextrin‐hydrolyzing enzyme from Bacillus sphaericus E‐244 on branched α‐ and β‐cyclodextrins was investigated. Glucosyl‐α‐cyclodextrin (6‐O‐α‐D‐glucosylcyclomaltohexaose) and maltosyl‐α‐cyclodextrin (6‐O‐α‐D‐maltosylcyclomaltohexaose) were hydrolyzed to 63‐O‐α‐D‐glucosylmaltohexaose and 63‐O‐α‐D‐maltosylmaltohexaose, respectively. Glucosyl‐β‐cyclodextrin (6‐O‐α‐D‐glucosylcyclomaltoheptaose) and maltosyl‐β‐cyclodextrin (6‐O‐α‐D‐Maltosylcyclomaltoheptaose) were also transformed mainly to 64‐O‐α‐D‐glucosylmaltoheptaose and 64‐O‐α‐D‐maltosylmaltoheptaose, respectively. These results suggest that the cyclodextrin‐hydrolyzing enzyme cleaves branched α‐ and β‐cyclodextrins at an α‐1,4 linkage which is located furthest from the branching point on the cyclodextrin ring.

A new enzymatic assay of urinary guanidinoacetic acid.

We describe a new enzymatic determination of urinary guanidinoacetic acid (GAA) with guanidinoacetate kinase (ATP: guanidinoacetate N-phosphotransferase, EC 2.7.3.1), which does not require a blank to correct for endogenous constituents (ADP and pyruvate). In the first step, pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) were used to eliminate endogenous constituents (ADP and pyruvate) in the presence of phosphoenolpyruvate and NADH. In the second step, urinary GAA was phosphorylated in the presence of ATP by guanidinoacetate kinase to form phosphoguanidinoacetate and ADP. The resultant ADP was sequentially measured at 340 nm in a coupled reaction catalyzed by pyruvate kinase and lactate dehydrogenase. The standard curve was linear up to 20 mg/dl for standard solutions of GAA. Analytical recovery of GAA added to normal urines ranged from 97.0 to 103.2% (mean 100.7%). The within-run and between-run studies gave CV values of less than or equal to 3.6% and less than or equal to 4.8%, respectively. No significant interference by endogenous urinary compounds were observed with the proposed method under this study. The results obtained by the present method correlated well with those obtained by a high-performance liquid chromatographic method. This method is accurate and simple, and less time-consuming than those previously reported. We determined the concentrations of GAA in 24-h urine samples by the proposed method, and observed that the urinary excretion of GAA decreased markedly in patients with renal failure.