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.     

Pectic enzymes associated with Penicillium digitatum decay of citrus fruit

Pectin methylesterase was the only pectic enzyme detectablein extracts from rind of sound or Penicillium digitatum-infectedoranges. No pectic enzymes were detected in juice from soundor infected fruit. Extracts from infected rind, and juice frominfected fruit, had macerating activity. Chromatographic analysesof rind extracts, and juice from infected fruit, showed galacturonicacid as a possible product of the degradation of pectic substances.Orange juice contained a thermo labile inhibitor of pectic ‘chain-splitting’,and macerating enzymes.     

Studies on Pectolytic Enzymes of Molds

Two hundred and fifty strains of molds including plant pathogenic microorganisms were cultured on solid media, and the production of pectolytic enzymes was followed by the clarification of fruit juice. Forty-four of them were found to have the action of clarifying fruit juice.

Out of the said 44 strains, the following 7 strains, Coniothyrium diplodiella, Agaricus capentris, Botrytis cinerea, Penicillium citrinum, Sclerotinia libertiana, Carpenteles javanicus and Aspergillus niger, were choosen as producers of effective pectolytic enzymes, and C. diplodiella proved the most active of all in clarifying fruit juice and hydrolyzing pectin or pectic acid.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

Pectic Enzymes and Phenolic Substances in Apples Rotted by Fungi

The activities of pectic enzymes in extracts from sound applesand from apples rotted by different fungi are described. Sclerotiniafructigenaand Botrytis cinerea rots had little or no polygalacturonaseor macerating enzyme activity, but Penicillium expansum rotswere very active in these respects. Extracts from each of therots had very high pectinesterase activity, and contained galacturonicacid. None of the rots had any cellulase activity. Each of thefungi produced polygalacturonase, macerating enzymes, and pectinesterasein liquid media. The effects of adding extracts of apples tothese media are described. Filtrates from cultures of S. fructigenaand P. expansum liberated galacturonic acid from apple fruitfibre which had been thoroughly extracted with cold water. The phenolic jsubstances present in healthy and rotted tisueswere estimated. B. cinerea and S. fructigena rots containedvery little, but P. expansum rots contained as much as healthytissue which had been allowed to brown. An extract of healthyapple tissue reduced the activity of the polygalacturonase ina culture filtrate of S. fructigena. The substances responsiblefor this were tentatively identified as leuco-anthocyanins whichhad been changed to other compunds following the action of polyphenoloxidase.Thej significance of these results is discussed.     

Macerating Activity of Streptomyces fradiae IFO 3439

Streptomyces fradiae IFO 3439 elaborated enzymes with macerating activity toward various plant tissues. The optimum pH of the macerating activity was about 8.0 when the crude enzyme preparation acted on disks of potato tuber or pieces of Ganpi (Wikstroemia sikokiana Fr. et Sav.) bark. Pectolytic activities in this preparation toward free pectin or poly-galacturonic acid were considerably lower than those of fungi or bacteria. However, when the crude enzyme preparation acted on native pectin in Ganpi bark, about 90 per cent of the galacturonic acid residues were recovered as the polygalacturonides having a still high degree of polymerization. These results suggested that the crude enzyme of S. fradiae solubilized Ganpi pectin, degrading it to only a very small extent.     

Effect of oxygen transfer rate on the composition of the pectolytic enzyme complex of Aspergillus niger

Optimal agitation and aeration conditions [assuring O₂ transfer rates (OTR) from 12 to 179 mmol L^?1 h^?1] were determined for pectin lyase (PL) synthesis of an Aspergillus niger strain. Components of the pectolytic enzyme complex were also investigated in order to determine whether their O₂ demand is identical with or different from that of pectin lyase. Should the latter be the case, a possibility would be given to produce enzyme complexes of different agitation and aeration conditions. According to our results, mycelium yield of Aspergillus niger attained a maximum at an OTR of 100 mmol L^?1 h^?1. The yields of the various pectolytic enzymes reached maxima at different OTRs. Pectin lyase production was the highest (0.555 µmol min^?1 mL^?1) at an OTR of 60 mmol L^?1 h^?1. Endopolygalacturonase (PG) production showed a maximum at the OTR of 49 mmol L^?1 h^?1 with a second peak at 100?135 mmol O₂ L^?1 h^?1. Pectin esterase (PE) synthesis showed a maximum at on OTR of 12?14 mmol L^?1 h^?1, while both apple juice clarifying and macerating activities gave two maxima at 14 and 60 mmol L^?1 h^?1 due to the optima of PE and endo-PG. Macerating activity showed a high value at OTR optimal for PL production as well.     

Pectic Enzymes in the Clarification of Apple Juice

To know the role of pectic enzymes in the clarification reaction of apple juice, a simplified model for apple juice, that is, aqueous re-suspension of ultracentrifugal precipitates of apple juice, was employed. It was found that the precipitates (i.e., suspended materials) contained 36% of protein and that the surface of the suspended materials was negatively charged at pH 3.5. Positively charged colloids at pH 3.5 such as gelatin enhanced the clarification reaction or mutually coagulated with the suspended materials. While negatively charged colloids at pH 3.5, such as sodium alginate completely inhibited the clarification reaction. The direct participation of pectic enzymes in the clarification of apple juice was shown, and a supposed mechanism of the enzymic clarification was presented.     

Pectolytic enzymes in inoculated and uninoculated red clover seedlings

Summary Pectolytic enzymes were studied in inoculated and uninoculated red clover (Trifolium pratense) seedlings grown aseptically in flasks containing distilled water or nitrogen-free salts media. Enzyme activity in root exudates and root extracts depended on the conditions of seedling growth. Tests with sodium polypectate and citrus pectin indicated the presence of two enzymes, specific for pectic acid and pectin respectively. Both enzymes were produced by uninoculated seedlings, and in seedlings inoculated with Rhizobium trifolii, R. leguminosarum or R. lupini, enzyme activity was not correlated with infectivity of the strains. re]19720814     

Studies on Pectolytic Enzymes of Molds

The process of apple juice clarification by pectolytic enzymes has been successfully observed turbidimetrically and macroscopically by heating of reaction mixtures. It has been shown that the process of apple juice clarification varies with the varieties and conditions of apple juices as well as with the sources of enzyme preparations. From a study of the turbidimetry of apple juice clarification, α method for determination of clarification values been described.     

Pectic enzyme production and morphogenesis inHelminthosporium atypicum

The production of pectic enzymes byHelminthosporium atypicum and its morphogenesis on different media were studied. It was observed that the fungus produces pectic enzyme (macerating enzyme) adaptively. Increasing concentrations of glucose had an inhibitory effect on enzyme production. Glucose promotes profuse growth and early sporulation whereas presence of pectin slows down the growth and delays sporulation. Delay in sporulation is the effect of presence of pectin and not of the low pH of the medium. It is also suggested that in the case ofH. atypicum low pH of the medium does not allow the fungus to utilize a carbon source as efficiently as at higher pH.     

Studies in the Physiology of Parasitism: XX. The Pathogenicity of Botrytis cinerea, Sclerotinia fructigena, and Sclerotinia laxa, with special reference to the part played by pectolytic enzymes

1. Though Sclerotinia fructigena, S. laxa, and Botrytis cinereacause rotting of apple tissue and death of the protoplasts,little or no pectolytic activity was detectable in extractsof the rotted tissue. 2. Pectic materials were extracted from normal and parasitizedapple tissue in three fractions and precipitated as calciumpectate. There was a loss of total, total insoluble, and solublepectic substances in the invaded tissues. This was most markedwith B. cinerea and S. laxa and least with S. fructigena. 3. Pectolytic activity was measured by methods involving (a)maceration of plant tissues, (b) viscosity and reducing groupdeterminations in pectic substrates, (c) increase in acidityof pectin. By these methods it was shown that pectolytic enzymeswere produced by all three fungi in synthetic media. With S.fructigena, which was the only fungus studied in detail, replacementof glucose by pectin increased the formation of pectolytic enzymes. 4. When various apple extracts were used as culture media, littleor no pectolytic activity was detectable. With all three fungithe presence of apple juice in a culture medium, which by itselfwas suitable for enzyme formation, resulted in the suppressionof pectolytic activity. 5. Oxidized apple juice had a pronounced effect in deactivatingcertain pectolytic enzymes, an effect which was especially markedwith B. cinerea. This points to an interaction between the pectolyticand oxidizing systems and introduces a new line of approachto the study of the biochemical interaction between host andparasite.     

Paenibacillus amylolyticus 27C64 has a diverse set of carbohydrate-active enzymes and complete pectin deconstruction system

A draft genome of Paenibacillus amylolyticus 27C64 was assembled and a total of 314 putative CAZymes in 108 different families were identified. Comparison to well-studied polysaccharide-degrading organisms revealed that P. amylolyticus 27C64 has as many or more putative CAZymes than most of these organisms. Four different pectic substrates and xylan supported growth but cellulose was not utilized. Measurement of enzyme activities in culture supernatants revealed low levels of cellulase activity, high levels of xylanase activity, and pectinase activities that adapted to the specific polysaccharides provided. Relative expression levels of each putative pectinase in cells grown with and without three different pectic substrates were evaluated with RT-qPCR and distinct sets of genes upregulated in response to homogalacturonan, methylated homogalacturonan, and rhamnogalacturonan I were identified. It is also noted that this organism’s pectinolytic system differs from other well-studied systems and contains enzymes which are of value for further study.

     

Production of macerating enzymes of mandarin orange peel by fungal cultures

Summary Thirty-nine fungal cultures belonging to the genera of Aspergillus, Podospora, Sordaria, Cbaetomium, Iodophanus, Scleotinia, Coniella, Pellicularia and others, were examined for the production of enzymes which macerate the mandarin orange peel using a wheat bran as substrate. An isolated strain of Aspergillus niger was an excellent producer of macerating enzymes compared to other organisms tested. The peel of the mandarin orange could be macerated by the crude enzymes produced by isolated A. niger. The maceration of 1 g of peel/24 h yielded 0.57 g of reducing sugars. Expressed differently, 83% of solid peel materials were released from the peel into the water/24 h under the following conditions: a peel concentration of 8 g peel/l, a crude enzyme concentration of 1 g protein/l, a temperature of 40°C, a pH of 5, a 24 h incubation time and 120 rpm reciprocal shaking. The test of the macerating activity of commercially available hydrolases on the orange peel showed that the two samples of pectinase originating from A. niger had about the same activity as isolated A. niger whereas the two samples of cellulase originating from Trichoderma viride had remarkably lower activities than A. niger.     

Dextransucrase production using cashew apple juice as substrate: effect of phosphate and yeast extract addition

Cashew apples are considered agriculture excess in the Brazilian Northeast because cashew trees are cultivated primarily with the aim of cashew nut production. In this work, the use of cashew apple juice as a substrate for Leuconostoc mesenteroides cultivation was investigated. The effect of yeast extract and phosphate addition was evaluated using factorial planning tools. Both phosphate and yeast extract addition were significant factors for biomass growth, but had no significant effect on maximum enzyme activity. The enzyme activities found in cashew apple juice assays were at least 3.5 times higher than the activity found in the synthetic medium. Assays with pH control (pH = 6.5) were also carried out. The pH-controlled fermentation enhanced biomass growth, but decreased the enzyme activity. Crude enzyme free of cells produced using cashew apple juice was stable for 16 h at 30°C at a pH of 5.0.     

Stability Study of Crude Dextransucrase from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> NRRL B-742

In the present work, the stability of crude dextransucrase from Leuconostoc citreum B-742 was evaluated in synthetic and in cashew apple juice culture broth. Optimum stability conditions for dextransucrase from L. citreum B-742 were different from the reported for its parental industrial strain enzyme (L. mesenteroides B-512F). Crude dextransucrase, from L. citreum B-742, produced using cashew apple juice as substrate, presented higher stability than the crude enzyme produced using synthetic culture medium, showing the same behavior previously reported for dextransucrase from L. mesenteroides B-512F. The crude enzyme presented good stability in cashew apple juice for 48 h at 25°C and pH 6.5.     

Application of Pectic Enzymes to Maceration of Plant Tissues for Microscopic Study

A new method of maceration of relatively soft plant parts, such as petioles, fruits, and fruit skins, which depends upon the treatment of the material with pectic enzyme solutions is described. The commerical preparation Pectinol W, (manufactured by Rohm and Haas Co., Washington Square, Philadelphia S, Pa.) as well as pectic enzymes secreted by growing Aspergillus species upon pectin-containing media were effective in macerating a variety of plant materials.     

Immobilized pectolytic enzymes on acrylic supports

The paper deals with the pectolytic enzymes immobilization on different acrylic supports, and the application of immobilized preparations in the apple juice pectinization process. The correlation between the protein content (C_p) and specific catalytic activities of immobilized enyzme preparations suggest a specific immobilization process only in the case of PONILEX ASH type acrylic supports. The active immobilization degree on PONILEX ASH type supports of Ultrazym 100 G and technical pectinase extract ranged from 99.80 to 296.40% for the Pectinesterase (PE) activity, and from 101.85 to 252.94% for the chain splitting (CS) activity, proving that the ionic immobilization process is a selective one. The simulated operational stability of the immobilized pectolytic enzymes tested by the PE and CS activity values proves the preservation of enzyme catalytic activity.     

福寿螺和田螺消化酶活性比较

为比较福寿螺(Pomacea canaliculata(Lamarck,1828))和当地中国圆田螺(Cipangopaludina chinensis(Gray,1832))消化能力的差异,探索福寿螺成功入侵的机制,以田螺为对照,测定了1—4龄的福寿螺和田螺的胃和肝脏的消化酶——纤维素酶(羧甲基纤维素法)、淀粉酶(3,5-二硝基水杨酸法)和脂肪酶(滴定法)的活性。结果表明:1)相同年龄的福寿螺胃和肝脏中的消化酶活性明显高于田螺。其中,纤维素酶活性分别高出1.00—2.11倍、1.66—2.84倍;淀粉酶活性分别高出1.53—3.47倍、1.47—1.80倍;脂肪酶活性分别高出2.07—4.73倍、6.13—9.93倍。2)在生长发育过程中,福寿螺胃和肝脏中的消化酶活性变化幅度(51.2%—131.2%)明显高于田螺(23.3%—47.1%)。3)福寿螺的各种消化酶之间存在协同作用。如福寿螺的淀粉酶活性与脂肪酶活性呈极显著正相关(胃中r=0.736**、肝脏中r=0.867**)。此外,胃中的淀粉酶活性还与纤维素酶活性呈显著正相关关系(r=0.696*)。相应地,田螺胃中的淀粉酶和脂肪酶之间也存在显著的正相关关系(r=0.706*),而肝脏中的纤维素酶与脂肪酶活性呈显著负相关(r=-0.593*)。4)福寿螺对纤维素类和淀粉类物质都有较强的消化能力,且能较好地消化脂肪类物质,而田螺能消化纤维素类和淀粉类物质,对脂肪的消化能力却很弱。福寿螺的纤维素酶和淀粉酶活性分别是田螺的2.42和1.88倍,脂肪酶活性达到了5.66倍。可见,福寿螺具有较高的消化酶活性,且各消化酶之间存在正协同性。这可能是导致福寿螺食量大、食性杂,使其能快速生长和成功入侵的重要原因之一。     

Exopectic Acid Transeliminase of an Erwinia

A species of Erwinia was found to produce no other pectolytic enzyme than the two transeliminases of exo-types, namely, an oligogalacturonide transeliminase and an exopectic acid transeliminase. Of the two enzymes, the exopectic acid transeliminase was isolated and its properties were investigated. The results are as follows: (1) Pectic acids having an unsaturated galacturonic acid residue at the non-reducing end of the molecule are susceptible but oxidized or reduced pectic acids resistant to the enzyme action. (2) The enzyme has no activity toward pectinic acid and polymethylpolygalacturonate methyl glycoside. The limit of the enzymatic degradation for citrus pectic acid is 43.8%. (3) The rate of the enzyme activity was maximal with tetragalacturonic acid and followed by acid-soluble pectic acid, acid-insoluble pectic acid, pectic acid and trigalacturonic acid. Unlike the oligogalacturonide transeliminases of Pseudomonas sp. (strain S2) and Erwinia aroideae, the present enzyme shows a considerably high activity toward pectic acids of high molecular weight. (4) The pH-activity curves vary with the buffer employed. (5) The enzyme is activated by Co²⁺ and Mn²⁺ but powerfully inhibited by Cu²⁺ and Hg²⁺. Ca²⁺ has no significant effect on the enzyme activity.