Shunichi Akiba

Purification and characterization of a protease-resistant cellulase from Aspergillus niger

Abstract An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β- d -glucopyranoside and p-nitrophenyl-β- d -cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg2+ and Cu2+ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.

Cloning of a gene encoding a highly stable endo-β-1,4-glucanase from Aspergillus niger and its expression in yeast

A gene encoding an endo-beta-1,4-glucanase, which is highly resistant to high temperature, protease and surfactant treatment, was isolated from Aspergillus niger IFO31125 and designated as eng1. The deduced amino acid sequence encoded by eng1 showed high homology with the sequence of a not-well-characterized cellulase encoded by eglB which has not yet been shown to be a stable enzyme. To confirm the sequence of the gene encoding the highly stable endo-beta-1,4-glucanase, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The enzyme retained 56% of the initial activity after 1 h of incubation at 80 degrees C and was stable in the range of pH 3.0-10.0. The optimal temperature for enzyme activity was 70 degrees C and the optimal pH was 6.0. The enzyme was highly protease-resistant and retained more than 80% of the initial activity after protease treatment for 3 d at 40 degrees C. The enzyme was also resistant to various surfactants. From these results, eng1 was confirmed to encode a very stable endo-beta-1,4-glucanase.

The N-terminal amino acid of apolipoprotein D is putatively covalently bound to 3-hydroxy-3-methyl hexanoic acid, a key odour compound in axillary sweat

Axillary sweat is odourless when freshly collected at the surface of human skin, but it contains non‐odoriferous precursors, which can be transformed into odorous substances by bacteria. E‐3‐methyl‐2‐hexanoic acid (3M2H) is one of the key odorous substances, but there are two contradictory reports about its precursor form. One report states that 3M2H linked non‐covalently to apolipoprotein D (apoD) is the precursor, while a second report states that 3M2H–Gln identified in human axillary sweat is the precursor. Recently, 3‐hydroxy‐3‐methyl hexanoic acid (HMHA) and 3‐methyl‐3‐sulfanylhexane‐1‐ol (3M3T) have also been identified and reported as characteristic components found in apocrine sweat. To better understand the formation of axillary odours and the structural relationships between these compounds and apoD, we characterized the linkage between odorous substances and apoD in human axillary secretions. ApoD was purified from human axillary secretions collected from 50 healthy female volunteers and was then digested by trypsin and analysed by MALDI‐TOF mass spectrometry. A Mascot search showed that 8 peaks identified in the trypsin‐digested samples correspond to the masses calculated for theoretically digested apoD sequences and the purified protein was assigned as a precursor of apoD [Homo sapiens]. One spectrum corresponded to the theoretical peak of HMHA linked covalently to the N‐terminal fragment of apoD. In contrast, no spectrum corresponded to the theoretical peak of a 3M2H adduct or to an unmodified N‐terminal fragment of apoD. These results indicate a possibility that HMHA binds covalently to the N‐terminal amino acid of apoD in human axillary secretions.

Transglycosylation activity of the endo-β-1,4-glucanase from Aspergillus niger IFO31125 and its application

Endo-beta-1,4-glucanase from Aspergillus niger was found to have an endo-type transglycosylation activity. The enzyme effectively transferred cellooligosaccharide residues to various 1-alkanols in the presence of cellopentaose as the oligosaccharide donor. By incubating the enzyme with 1-octanol and cellopentaose in the presence of acetonitrile, 1-octyl-cellotrioside was synthesized. The product was isolated by silica gel column chromatography and analyzed by mass spectrometry. p-NP-beta-Glc, p-NP-beta-Gal, p-NP-beta-GlcNAc, Ser, and Thr were also possible acceptors for the transglycosylation activity of the enzyme.

The influence of incontinence on the characteristic properties of the skin in bedridden elderly subjects

The mechanisms of skin breakdown induced by incontinence have been proposed from a variety of experimental studies. However, studies on the influence of skin properties caused by incontinence of bedridden subjects are very limited.