Studies on the Chitinolytic Enzymes of Black-koji Mold

It has been found that glycol chitin is a suitable substrate for the viscometric determination of chitinase activity, because the viscosity of its aqueous solution is not affected by the presence of added salt and the changes of pH, differing from chitosan acetate and carboxy-methyl chitin used by earlier workers. Using this substrate the viscometric activity is determined, basing on the observation that the time required to halve the viscosity of reaction mixture is inversely proportional to the amount of enzyme used.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

It was found that the purified chitinase preparation acts upon glycol chitin resulting in the decomposition to constituent aminosugar, the saccharifying activity being determined by application of the Morgan-Elson reaction. The enzymatic properties of the mold chitinase were investigated by measuring liquefying activity and saccharifying activity. Distinct differences were observed between the two activities, and especially liquefying activity was more stable than saccharifying activity against heat treatment. The chitinase preparation whose saccharifying activity was inactivated by heating was able to decrease the viscosity of glycol chitin solution, with an insignificant production of aminosugar.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

The measurement of N-acetyl-β-glucosaminidase activity upon β-MAGA was carried out in order to survey oligosaccharidase fraction in the chitinolytic enzymes of Aspergillus niger. N-Acetyl-β-glucosaminidase was purified in parallel with chitobiase activity, being separated from chitinase activity, and some properties of the enzyme in the hydrolysis of β-MAGA and DACE were investigated. The enzyme hydrolysed more rapidly S-glueosaminidie bonds in DACE than that in β-MAGA, but did not decompose α-MAGA.     

Studies on the Chitinolytie Enzymes of Black-koji Mold

In an attempt to separate the enzyme system participating in the decomposition of glycol chitin to constituent aminosugar, the purification of chitinase of Aspergillus niger was carried out by detemining both liquefying and saccharifying activities. Using fractionation with ammonium sulfate and column chromatography by hydroxylapatite, the chitinase system of the mold was separated into different enzyme fractions, which were required for the complete hydrolysis of glycol chitin. It was found that one of these enzymes caused a rapid decrease in viscosity of glycol chitin solution, another enzyme possessed N-acetyl-β-glucosaminidase activity upon N, N′-diacetylchitobiose and β-methyl-N-acetylglucosaminide, and that glycol chitin was decomposed to constituent aminosugar by a successive action of the two different enzymes.     

Studies on the Amylolytic System of the Black-koji Molds

The amylase system of black-koji molds was fractionated, in respect to its activity, into two fractions, the dextrinogenic and saccharogenic. Their separate and combined activities to digest raw starch were investigated. Contrary to the knowledge established so far, the dextrinogenic amylase fraction was found to be capable of digesting the raw starch though only weakly, while the saccharogenic amylase fraction much more strongly. When the two fractions were combined, digestion proceeded with twice or thrice the velocity of the, sum of each component. This accelerating interaction, which never occurs in cooked-starch digestion, is worthy to be stressed as a characteristic of raw starch digestion by this amylase system. The raw starches of corn, wheat and glutinous rice are in this way, digested completely to glucose. Among them, glutinous rice starch displays peculiar facility in the digestion.     

Studies on the Amylolytic System of Black-koji Molds

The saccharogenic amylase fraction was prepared from a black-koji amylase system, and its debranching activity was investigated. From its different attitude towards various chemical procedures, such as (NH₄)₂SO₄ fractionation, corn starch adsorption, and paper electrophoresis, it is suggested that two saccharogenic amylases one with and the other without debranching activity, may exist in the saccharogenic amylase fraction.     

Studies on the Amylolytic System of the Black-koji Molds

Saccharogenic and dextrinogenic amylase fractions were prepared from Black-koji amylase system and their actions investigated with a number of different substrates.

It was found that saccharogenic amylase fraction completely hydrolyzes glutinous rice starch and glycogen to glucose, without leaving any limit dextrin. On the other hand, this enzyme fraction converts potato starch to an extent of about 90% theoretical glucose, the remainder being left as limit dextrin, which is colored purple by iodine. The complete hydrolysis of the branched substrates except potato starch shows that the saccharogenic amylase fraction is capable of hydrolyzing the l,6-α-d-glucosidic linkage besides the 1,4-linkage, while the branched fraction of potato starch may contain some sort of anomaly to the enzyme. Dextrinogenic amylase fraction hydrolyzes starch and glycogen just as malt α-amylase.     

Studies on Mold Protease

An acid protease of Rhizopus chinensis was purified by sequential chromatographies on columns of Duolite A-2, Sephadex G-100 and CM-cellulose, and crystallized from aqueous acetone solution. The preparation was shown to be monodisperse on column chromatography of ion-exchange sephadex and on ultracentrifugal analysis. The enzyme was most active at pH values between 2.9 and 3.3 and was stable over the range of pH 2.8 to 6.5. The protease was markedly inactivated by ferric ions and sodium lauryl sulfate, whereas it was affected by neither sulfhydryl reagents nor metal-chelating agents. In milk-clotting activity, the acid protease was shown to be one of the most potent enzymes among those of fungal origin. Substrate specificity experiments on several synthetic peptides indicated that the peptide bonds susceptible to the action of the enzyme were mainly those involving amino group of aromatic amino acids.     

Studies on Mold Proteases

The substrate specificity of the crystalline acid protease obtained from Rhizopus chinensis was determined using B-chain of oxidized beef insulin and numerous synthetic peptides, comparing with that of several acid proteases from various sources. The peptide bonds susceptible to the action of Rhiz. acid protease were found to be mainly those involving the amino group of bulky amino acids. The enzyme split the B-chain of oxidized insulin at twelve sites of the peptide linkages and a certain similarity in the specificity was observed among the three acid proteases, Rhiz. protease, rennin and pepsin, all of which were known to show potent milk clotting activities.     

Studies on Mold Dextranases

Nine strains capable of producing dextranase were isolated from soil. Among them, a strain belonging to the genus Aspergillus was chosen as the best producer of the enzyme. The mold produced greater amounts of dextranase than those found in some strains in the genus Penicillium, when grown aerobically at 28°C for 5 to 6 days in medium containing 1% dextran, 1% NaNO₃ or polypeptone, 0.2% yeast extracts, 0.4% K₂HPO₄ and small amounts of inorganic salts, pH 8.5. From the comparative taxonomic experiments, the mold used here was identified to be a strain belonging to Aspergillus carneus.     

Studies on Sugar-isomerizing Enzymes

The effect of a borate on the isomerization reaction between glucose and fructose which is catalyzed by a glucose isomerase was investigated. The yield of fructose was dependent on both the ratio of sugar to the borate and pH. A maximum of 88 to 90% of glucose was converted into fructose when the isomerization reaction was carried out at around pH 7.5 and in the presence of an appropriate amount of the borate which forms a complex between one molecule of sugar and one molecle of boric acid.     

Studies on Bacteriolytic Enzymes

About 100 soil samples were subjected to screening for microorganisms which were capable of producing lytic enzyme toward Staphylococcus aureus. A strain belonging to Streptomyces was isolated and found to produce lytic enzyme(s) noninduciblly, when grown aerobically at 37°C for 25 hr in a medium containing 7.5% soybean cake extract, 2% dextrin, 0.6% K₂HPO₄, 0.02% each of MgSO₄·7H₂O and KCl, pH 7.0. The crude enzyme preparation was active at pH values of 8.5 and 5.8 toward S. aureus, B. subtilis, L. bulgaricus and Str. faecalis but was completely inert against M. lysodeikticus, indicating the enzyme(s) to be distinguished from other bacteriolytic enzymes of Streptomyces so far reported.     

Studies on the Enzymes Acting on Araban

Arabanase was purified by various procedures of gel filtrations and column chromatographies from the submerged culture filtrate of Aspergillus niger grown on the medium containing beet-araban as a carbon source, and the properties of the enzyme was examined. The purified enzyme was homogeneous protein in ultracentrifugal analysis, the optimum pH for enzymatic action was 4.0. The enzyme was thermostable at pH 6.0, namely, even after heating at 98°C for 10 minutes, 20.8 per cent of the initial activity still remained. The enzyme hydrolyzed beet-araban with producing l-arabinose.     

Studies on the Mechanism of Mold Protease Production

Inactive protease in the cell free extracts obtained from growing cells of Aspergillus sojae KS was collected in a supernatant of ultracentrifugation at 14×l0⁴ g, and in fractions obtained by acetone of 35~50 per cent and by ammonium sulfate of 0.5~0.6 saturation.

The inactive protease has the same resistance against pH or heat treatments as active protease has. The activation of the inactive protease was maximal between pH 5~6, and was accelerated by several kinds of protease, and was not affected by thioglycollate and KCN.     

Studies on the Mechanism of Mold Protease Production

The activating factors of the inactive protease in cell-free extracts obtained from growing mycelia of Aspergillus sojae KS were studied. It was found that the several kinds of metals were involved in activation, and the role of these metals on the activation was investigated. The velocity of the activation was maximal around pH 10 as well as around pH 5. It was proved that a kinase (enzyme) capable of activating the inactive protease in alkaline solution does exist in the cell-free extract.     

Studies on the Proteolytic Enzymes of Black Aspergilli

In order to investigate the production of acid protease, the cultivation conditons were studied with black Aspergilli belonging to Kuro-Koji mold group by solid cultivation (Koji-culture).

It was observed that the production of acid-protease by A. saitoi R-3813 mut. UV-13 was markedly increased by adding an adequate amount of an inorganic nitrogen compound to the cultivation medium. Concerning the production of acid protease by adding an inorganic nitrogen compound, one hundred and eighty-four strains of Kuro-Koji mold group and eleven strains of other Aspergilli were tested.     

Studies on Pectic Enzymes of Microorganisms

Conditions for the production of endo-polygalacturonase (endo-PG) with Aspergillus saitoi IAM 2217 in the submerged culture was examined. This strain was selected as the most potent producer of endo-PG. Endo-PG of this strain was produced in the absence of pectin, but the addition of pectin increased endo-PG activity when inoculated with proliferated mycelia.

As far as examined with a modified Czapek medium (ordinary constituents + pectin and ammonium tartrate), the addition of organic nitrogen sources, such as corn steep liquor, markedly reduced the enzyme producibility. As for the carbon and nitrogen amount in the medium, sucrose: 4%, pectin: 2%, NaNO₃: 1.15%, C/N = 10, gave the best result among tested.     

Studies on the Proteolytic Enzymes of Black Aspergilli

The production of acid protease, as well as that of other enzymes, involves many variable factors, such as rate of aeration and agitation, temperature control during the operation, components of the media and type of antiform agents. The proper control of such factors can be achieved through effective utilization of a well-equipped pilot plant, designed specifically for the acid protease research and development. The fermentation unit described ranges in capacity from 500 ml shake-flask to 20-liter jar fermentor. Detailed studies on the various conditions for the production of acid protease by Aspergillus usamii, Aspergillus saitoi and other related species are described. It was found that high rates of agitation and aeration were required to obtain acid protease in maximum yield.     

Studies on Pectic Enzymes of Microorganisms

To pick out potent strains which specifically produce one of several pectic enzymes, endo- and exo-polygalacturonase, pectin esterase, macerating, and apple juice clarifying activities were examined with regard to 344 strains of mold (containing 71 strains of phytopathogenic mold) grown on a bran culture medium and 56 strains of shakingly cultured yeast. As the result of screening, Asper gillus saitoi and Penicillium islandicum were isolated as potent specific producers of endo-polygalacturonase. And the composition of pectic enzymes of mold was found to be rather genus or species specific. So far as examined in crude enzyme systems, there was no parallelism between anyone of pectic enzyme activities and apple juice clarifying or macerating activities.