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.     

Calorimetric versus Growth Microbial Analysis of Cellulase Enzymes Acting on Cellulose

Assay of cellulase enzymology on cellulose was investigated by two methods: (i) plate colony counting to determine microbial growth and (ii) microbial calorimetry. These methods were chosen because they accept raw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays. Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 μM concentrations of cellulolytic lower sugars (glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5 mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requires completion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetric method to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated with Escherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiella sp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heat proportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- and endocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetric means for demonstrating enzyme synergism.     

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 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 Proteolytic Enzymes of Thermophilic Streptomyces

Proteolytic enzymes derived from thermophilic streptomyces sp. strain 1689 were purified and some properties were studied. A 8-fold purification was obtained from the culture supernatant by ammonium sulfate fractionation, acetone precipitation, and chromatography on CM-Sephadex. Two proteinases of almost identical properties were fractionated on CM-Sephadex chromatography. The purified preparations appeared to be homogeneous on ultracentrifugation. The optimum pH for proteolytic activity on casein was found to be pH 10.6~10.8. The stability was considerably increased by the addition of Ca⁺⁺, and the proteinases exhibited d relatively high thermal stability. Enzyme activity was inhibited by oxidizing agents, PCMB, potato inhibitor, DFP, and heavy metal ions. Na⁺, K⁺, Mg⁺⁺, and Fe⁺⁺ showed an activating effect.     

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 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 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 Proteolytic Enzymes of Black Aspergilli

In order to determine the specificity of Aspergillus Saitoi protease, the hydrolyzate of B-chain of insulin oxidized by this enzyme was investigated on paperchromatography according to the 2,4-dinitrofluorobenzene technique. Specificity was compared with pepsin and other proteolytic enzymes.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

The mode of degradation of glycol chitin and chitin by two enzyme fractions separated from Aspergillus niger was investigated. One of the enzyme rapidly cleaved the endo-β-glucosaminidic bonds in the polysaccharide chain, forming chitodextrin and oligosaccharides, while the other produced monosaccharide as a main product in the degradation. The successive action of the two enzymes was also examined. Intermediate products in the enzymatic degradation were surveyed using paper and column chromatography. Also, the over-all pattern of degradation of glycol chitin and chitin by the chitinase system of Aspergillus niger was discussed.     

Studies on the Proteolytic Enzymes of Black Aspergilli

The specificity of crystalline Asp. Saitoi proteinase on oxidized lysozyme has been investigated by application of the Sanger DNP-method.

It was found that this proteinase has a much broader specificity as compared with pepsin and Bac. subtilis proteinase.     

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 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 Pectolytic Enzymes of Molds

Exopolygalacturonase from Coniothyrium diplodiella has been purified by ammonium sulfate fractionation, chromatography on DEAE-cellulose and column zone electrophoresis. The enzyme was concentrated about 5-fold with a yield of 0.24% on the basis of polygalacturonase activity per weight of total nitrogen. The purified enzyme was homogenous On free-boundary electrophoresis. The enzyme was most active in the pH range 4.0~4.5. The enzyme was stable at 50°C and pH range of 3.5~6.0, but inactivated at higher than 55°C. Hydrolysis of pectic acid by the enzyme went to completion via galacturonic acid liberation from the end of the chain, but pectin was little affected by the enzyme.     

Studies on Pectolytic Enzymes of Molds

The clarification of apple juice has been studied using six pectolytic enzymes produced by Coniothyrium diplodiella, endo-PG (polygalacturonase) I, II and III, exo-PG and PE (pectinesterase) I and II. Each of these six enzymes had no effect on the clarification of apple juice when acted alone, whereas mixtures of any one of endo-PGs and PEs were all able to clarify the juice. Mixtures of exo-PG and either of PEs has no effect on the clarification. Clarifying activities of PG-PE mixtures were varied with the kind of endo-PG used in each mixture and not with the kind of PE. Clarifying activity of PG-PE mixture depended on either endo-PG or PE activities when the other was kept constant.

Crude enzyme from the mold and a mixture of the four PGs and PE in the ratio of the crude enzyme had essentially identical effect on apple juice as well as on artificial pectin and pectic acid.     

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.     

Studies on Pectic Enzymes of Microorganisms

Endo-polygalacturonase (endo-PG) of Aspergillus saitoi was purified through ammonium sulfate fractionation, Amberlite IRC-50 column chromatography, and several combinations of Sephadex column chromatography.

The purified endo-PG, which was almost homogeneous ultracentrifugally and electrophoretically, had the sedimentation constant of 2.2 S and the absorption maximum at 277 mμ. Its optimum pH and temperature were 4.8~5.0 and 45°C, respectively, and it was most stable between pH 4.0 and 6.0, but over 90% of the activity was lost at 50°G for 10 min.

The purified enzyme was a typical endo-PG, and hydrolyzed about 60% and 17% of glycosidic linkage of polygalacturonic acid and pectin, respectively. This enzyme preparation had no pectinesterase, trans-eliminase, and apple juice-clarifying activities, but macerated potato tuber slices singly.     

Studies on Pectolytic Enzymes of Molds

Experiments have been made on fractionation of the pectolytic enzymes produced by Coniothyrium diplodiella. It has been observed that 30 to 35% of the polygalacturonase (PG) activity of the pectolytic enzymes of the said microorganism is salted out with ammonium sulfate, and this portion contains cndo-PG I, endo-PG II and pectin esterase (PE) (with a trace of exo-PG). The endo-PG I accounts for 60 to 65% of the total PG activity, and the endo-PG II, 25 to 30%. Both types of endo-PG scarcely act on pectin, and hydrolyze pectic acid to the extent of 65 to 70%.