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.     

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.     

Pectic Enzymes in the Clarification of Apple Juice

Pectinesterase of Sclerotinia arachnidis was purified by 44 times. The clarification of apple juice with the purified pectinesterase and the formerly purified endo-polygalacturonase of Aspergillus saitoi was examined. The coexistence of pectinesterase and endo polygalacturonase was necessary for the complete clarification of apple juice. During the clarification reaction rapid decrease of viscosity and resultant coagulation of the suspended materials in apple juice were observed. The coagulated materials slowly precipitated leaving clear juice.

It was suggested from the result of electrophoresis that the positive charge existed inside of the suspended materials. The decrease in viscosity was supposed to correlate to pectin’s depolymerization which would result in revealing of positive charge inside. The probable mechanism of coagulation was attributed to the electrostatic neutralization between positive charge thus appeared and negative charge of still undegraded pectin.     

Studies in the Physiology of Parasitism: XVIII. Pectic Enzymes secreted by Bacterium aroideae

Cell-free filtrates from cultures of Bacterium aroideae on asimple synthetic medium contained an enzyme, provisionally termeddepolymerase, which rapidly reduced the viscosity of pectinsolutions, and protopectinase which macerated slices of potatotuber tissue. These filtrates had little pectin-esterase activity. The activity of depolymerase was directly proportional to enzymeconcentration; the activity of protopectinase was approximatelyproportional to the square root of the enzyme concentration.Crude solutions were partially purified by acetone or ethanolprecipitation; ammonium sulphate was less satisfactory as aprecipitating agent. Enzyme preparations which rapidly reduced the viscosity of pectinand pectate solutions were relatively inactive when assayedfor polygalacturonase. activity by measuring reducing groupsliberated. After prolonged incubation some 20 and 40 per cent.hydrolysis of solutions of pectin and pectate respectively wasobtained. The pH optimum for depolymerase activity was near 9.0, the enzymewas activated by Ca⁺⁺ but not by a number of other cations;the loss of activity following dialysis was largely restoredby adding Ca⁺⁺. The enzyme was rapidly inactivated at temperaturesabove 60° C. and at pH 2.7. The properties of protopectinase generally resembled those ofdepolymerase. Analysis of the breakdown products following enzyme degradationof pectin and pectate solutions by paper chromatography showedthat galacturonic acid was not produced but that a number ofother products were formed, including one of fairly low molecularweight. The differences between the pectic enzymes of B. aroideae andthose from other sources, and the possible identity of depolymerase,polygalacturonase, and protopectinase are discussed.     

海洋微生物酶研究进展

海洋中蕴含着丰富的微生物资源,海洋微生物逐渐成为新型酶制剂的重要来源。多种海洋微生物如细菌、放线菌和真菌能够产生活性酶。对海洋微生物来源的多糖酶、脂类水解酶、蛋白酶和漆酶等的研究进展进行了综述,以期为海洋微生物资源利用研究提供参考。     

Milk-Clotting Enzymes From Microorganisms

A total of 230 cultures of fungi and 43 cultures of bacteria, isolated from such sources as soil, butter, and milk, were screened for their milk-clotting activity. The fungi were cultivated on semisolid media, and the bacteria were grown in milk media in shake culture. Phytic acid, added as calcium phytate, was found to stimulate production of the enzyme in most of the bacterial isolates. Proteolytic activity was invariably found to be associated with the milk-clotting enzyme in bacterial isolates. There was considerable variation in the ratio of the two enzymes from strain to strain.     

The Effects of Pectic Enzymes and Phosphatidases on Potato Tuber Tissue

Extracts from rots of potato tubers caused by Erwinia atrosepticaand Corticium praticola were fractionated by precipitation withammonium sulphate and by gel filtration. For the various fractionsof the E. atroseptica extracts there was a close relation betweenthe activity of pectate franj-eliminase and capacity to increasethe permeability of protoplasts as assessed by loss of electrolytes.There was no such relation with phosphatidase acting on lecithin. For certain fractions of C. praticola extracts there was a similarclose relation between increase in permeability and activityof a polygalacturonase but for other fractions with low polygalacturonaseactivity there was a better relation with phosphatidase thoughall fractions that caused increase in permeability did havesome polygalacturonase activity. Phosphatidases which probablyplay no part in the killing of cells in E. atroseptica rotsmay, therefore, have some role in the killing of cells in C.praticola rots though they are likely to be less important thanpectic enzymes. Extracts from E. atroseptica rots caused marked increases inuptake of oxygen by tuber discs. Dialysis decreased and heatingeliminated this increase and had corresponding effects on permeability.However, after fractionation with ammonium sulphate, fractionswith high trans-eliminase activity had little effect on oxygenuptake whereas fractions with low trans-eliminase had littleeffect on permeability and greatly increased oxygen uptake. Similar results were obtained with C. praticola rot extracts.In contrast, nigericin and Triton X-100 both increased permeabilityand caused large increases in oxygen uptake The significance of these results is discussed especially inrelation to the killing of protoplasts by extracts from bothtypes of rot.     

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.     

低温微生物及其酶类的研究概况

广泛分布在地球寒冷生境 ,如南北两极、高山、深海以及冰川中的低温微生物 ,不但为研究低温生态系统、生命起源与进化以及生物适冷机制提供了丰富的材料 ,同时在生物工程方面也具有潜在的巨大开发价值。国内外越来越多的科研人员对低温微生物及其产物的研究表现出了浓厚的兴趣。关于细胞膜和低温酶的研究 ,是目前微生物适冷机制研究中的 2个热点。就低温微生物的研究现状和适冷机制以及低温酶类的研究进行了综述。     

Studies on Intercellular Pectic Strands of Leaf Palisade Parenchyma

Regular rows (‘scala’) of pectic strands of uniformthickness interconnect palisade cells of leaves of a wide rangeof plant species, the leaf lamina of which possesses a regularpalisade layer. As the strands can be viewed by scanning electronmicroscopy of fractured, uncoated, fresh frozen leaves, theyare not artefacts. The techniques of electron probe analysis have been used toexamine fresh frozen leaves of holly in which there are regularpectic scala. Evidence is presented to support the view alreadyput forward, to explain the origin of the strands, that theyare deficient in calcium. The strands are shown to be rich inpotassium which, like the potassium in leaf cells, can be readilyleached with water. The advantages of fresh frozen plant material for elementalelectron probe analyses as well as problems arising from surfaceirregularities and surface charging, are discussed. pectic strands, leaf palisade parenchyma, electron-probe micro analysis, calcium, potassium     

Cold-Adapted Enzymes from Marine Antarctic Microorganisms

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the viscosity of the medium, which is also significantly enhanced by low temperatures, contributes to slow down reaction rates. This review analyses the different challenges and focuses on a key element of life at low temperatures: cold-adapted enzymes. The molecular characteristics of these enzymes are discussed as well as the adaptation strategies which can be inferred from the comparison of their properties and three-dimensional structures with those of their mesophilic counterparts. As these enzymes display a high specific activity at low and moderate temperatures associated with a relatively high thermosensitivity, the interest in these properties is discussed with regard to their current and possible applications in biotechnology.     

Studies in the Physiology of Parasitism: XXV. Some Propertles of the Pectic Enzymes secreted by Pythium debaryanum

The pectic enzymes of Pythium debaryanum have been comparedwith those from two other soft-rot causing organisms, Erwiniaaroideae and Botrytis cinerea, by their effects (viscosity reduction,acid production, and reducing power) on 1 per cent, solutionsof (a) high methoxyl pectin, (b) sodium polypectate, and (c)sodium pectate (2 types). The Pythium debaryanum preparationdiffered particularly in giving no increase in reducing poweror evidence of galacturonic-acid-like derivatives. It maceratedthe walls of potato-tissue freely but had nopolygalacturonaseor pectinesterase activity.     

Production of Alkaline Enzymes by Alkalophilic Microorganisms

Bacillus No. A–40–2 isolated from soil produced an alkaline amylase in alkaline media. The characteristic point of this microorganism was especially good growth in alkaline media, and no growth was detected in neutral media such as nutrient broth. The alkaline amylase of Bacillus No. A–40–2 was purified by DEAE-cellulose and hydroxyl apatite columns. The amylase was most active at pH 10.5 and stable pH was about 8.5. Calcium ion was effective to stabilize the enzyme especially at high temperatures. The sedimentation constant was about 3.8 S and molecular weight estimated by the Sephadex gel-filtration method was about 70,000. The enzyme was inactivated by urea, sodium laurylsulfate and sodium dodecylbenzene sulfonate. EDTA, PCMB and DFP did not show inhibitory effect. The enzyme hydrolyzed about 70% of starch and yielded glucose, maltose and maltotriose. If the enzyme is a single entity, this alkaline amylase is a type of saccharifying α-amylase.     

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.     

Studies on Xylanase from Microorganisms

As xylanase-producing microorganisms, 64 strains belonging to the genus Streptomyces were isolated from the barn-yard manures, silages and litters collected in Hokkaido district. Among these isolates the strain 102–1–4, which was found to be a new species under taxonomical studies and named Streptomyces xylophagus nov. sp., had the most outstanding ability for the enzyme production. In addition to the isolates, 38 strains of Streptomyces and 480 strains of filamentous fungi which have been preserved in our culture collection were also examined on their ability to produce the enzyme. 1) Among the strains of Streptomyces tested, only two strains, St. albogriseolus IAM 0031 and St. olivaceus IAM 0025 were found to have the ability, but their abilities were less than that of St. xylophagus nov. sp. 2) Out of 480 strains of fungi tested, Chaetomium, Schyzophyllum, Trametes, Echinodontium, Alternaria, Cepharosporium, Cercospora, Gibberella, Glomerella and Macrosporium produced the enzyme. Especially, Ch. trilateral 2264 was the most excellent.     

Studies on Xylanase from Microorganisms

X-Ray diffraction analysis of the α-cyclodextrin complexes with a number of organic guest molecules were carried out. Several different kinds of the X-ray diffraction patterns were obtained. It was found that different guest molecules enclosed within the void of the dextrin cause large changes in the diffraction patterns of the complexes. However, most of the diffraction patterns could be reasonably interpreted in terms of the hexagonal unit cells with minor differences in the unit cell dimensions ranging a = b = 27.0 ~ 27.8 Å and c = 14.7 ~ 16.7 Å. The crystal structure of the complexes could be accounted for by a closest packing of channel cylinders that are made by coaxial alignments of the dextrin molecules and the cage structure in the crystal, in which the dextrin molecules align non-coaxially, may not be plausible.