Mannanase Production Research Articles

Optimization of xylanase and mannanase production by Bacillus circulans strain BL53 on solid-state cultivation

A 2 3 central composite design (CCD) was applied to determine the optimal conditions of cultivation time, aeration and temperature to xylanase and mannanase production by Bacillus circulans BL53, a strain isolated from the Amazonian environment. The results suggest that xylanase production by B. coagulans BL53 is higher at a moderate cultivation aeration and at low temperatures and following CCD modeled conditions, we were able to increase 2.5-fold enzyme activities previously obtained and published by our group. For mannanase, the results showed that production was increased at higher aeration rate, and lower temperature. The highest enzymatic activity experimentally obtained according to the CCD was 3.7-fold higher than previous conditions employed. The predicted optimal parameters were tested in the laboratory and the final xylanase and mannanase activities obtained were very close to the predicted value (0.928 U mg −1, predicted; 0.953 U mg −1, tested; and 0.544 U mg −1, predicted; 0.540 U mg −1, tested, respectively). Our results show that optimization of the enzymes production is an useful way of obtaining concentrated enzyme extracts from solid-state cultivation of industrially important microorganisms.

Enhanced mannanase production by submerged culture of Aspergillus niger NCH-189 using defatted copra based media

Copra and other mannans including locust bean gum, guar gum, konjac flour, copra and defatted copra were used to produce extracellular mannanase by shaken flask cultures of Aspergillus niger NCH-189 in this study. The best carbon source for mannanase production was defatted copra, which provided more nitrogen source and mannan content. The peak mannanase activity at 28 U ml −1 was obtained on the day 3 at 30 °C, which was four times of those obtained from other carbon sources. Presence of oil in copra depressed the mannanase production of the fungus and the amount should be less than 3% (w/w). The copra suspension could be sequentially treated by boiling and refrigeration, followed by using n-hexane to remove copra oil.

Production of polysaccharide hydrolases in the genus Rhizopus.

Polysaccharide hydrolase activity was assayed in a group of 28 selected Rhizopus strains. The production of lichenases, mannanases, cellulases, xylanases, amylases and pullulanases was demonstrated using the gel-testing method during growth of the strains on suitably meshed polysaccharide gels.

Production of xylanases, mannanases, and pectinases by the thermophilic fungus Thermomyces lanuginosus

A group of 17 strains of the thermophilic fungus Thermomyces lanuginosus was examined for the production of xylanases, β-mannanases, arabinanases, and pectinases. All strains were found to be xylanolytic, and several were proven to be outstanding producers of microbial xylanase on glucuronoxylan and corn cobs. The strains hyperproducing xylanase secreted low amounts of xylan-debranching enzymes and did not produce β-mannan and arabinan-degrading enzyme systems. Only the strains showing lower xylanase production exhibited a higher degree of xylan utilization and also the ability to produce a mannanolytic enzyme system. One of the mannanolytic strains was found to be capable of producing arabinan-degrading enzymes. This strain also showed the best production of pectinolytic enzymes during growth on citrus pectin or sugar beet pulp. Some of the strains have good potential for use as sources of important industrial enzymes of high thermal stability.

Endo- β-1,4- d-mannanase is efficiently produced by Sclerotium ( Athelia) rolfsii under derepressed conditions

A number of wild-type isolates of Sclerotium ( Athelia) rolfsii and S. coffeicola were studied for their ability to produce endo- β-1,4-mannanase, endo- β-1,4-xylanase, and endo- β-1,4-glucanase activity when grown on cellulose- or glucose-based media. Whereas the presence of the inducer cellulose was strictly necessary for increased xylanase and endoglucanase production by both S. rolfsii (208 and 599 U ml −1, respectively) and S. coffeicola (102 and 330 U ml −1, respectively), elevated activities of mannanase (up to 96.6 U ml −1) were formed even when employing glucose as the only carbohydrate substrate. Significant production of mannanases as well as of auxiliary mannan-degrading enzymes ( β-mannosidase, β-glucosidase, α-galactosidase, acetyl esterase) was only observed, however, under derepressed conditions, i.e. after glucose had been consumed from the medium. By applying a fed-batch strategy, in which a glucose solution was continuously fed to a cultivation of S. rolfsii CBS 191.62 so that the glucose concentration in the medium never exceeded a certain low, critical value, production of mannanase could be almost doubled as compared to a batch cultivation on glucose (462 versus 240 U ml −1). Mannanase preparations produced by several S. rolfsii and S. coffeicola strains under inductive and noninductive conditions (i.e. using cellulose or glucose as the substrates, respectively) were further analyzed with respect to the patterns of isoformic mannanases formed under these different growth conditions. Multiple mannanases were secreted by all isolates investigated. Certain mannanase isoenzymes were only formed by S. rolfsii in the presence of the inducer cellulose, indicating a complex and separated regulation of the synthesis of mannanase isoenzymes in this strain.

Efficient production of mannan-degrading enzymes by the basidiomycete Sclerotium rolfsii.

Sclerotium rolfsii CBS 191.62 was cultivated on a number of carbon (C) sources, including mono- and disaccharides, as well as on polysaccharides, to study the formation of different mannan-degrading enzyme activities. Highest levels of mannanase activity were obtained when alpha-cellulose-based media were used for growth, but formation of mannanase could not be enhanced by employing galactomannan as the only carbon source. Although both xylanase and cellulase formation was almost completely repressed when S. rolfsii was grown on more readily metabolizable carbohydrates, including glucose or mannose, considerable amounts of mannanase activity were secreted under these growth conditions. Enhanced mannanase production only commenced when glucose was depleted in the medium. The maximal mannanase activity of 240 IU/mL obtained in a laboratory fermentation is remarkable. Mannanase activity formed under these derepressed conditions could be mainly attributed to one major, acidic mannanase isoenzyme with a pI value of 2.75.

Production of Hemicellulose- and Cellulose-Degrading enzymes by various strains ofSclerotium Rolfsii

A number of wild-type isolates of Sclerotium rolfsii were screened for their capacity to produce lignocellulolytic enzymes when grown on a cellulose-based medium.S. rolfsii proved to be an efficient producer of hemicellulolytic enzymes under the conditions selected for this screening, although there was a great variability in enzyme activities formed by the different isolates. In addition to xylanase and mannanase, which were produced in remarkably high levels, a number of accessory enzymes, which are important for the complete degradation of substituted hemicelluloses and include a-arabinosidase, acetyl esterase, and a-galactosidase, are formed by S. rolfsii. Efficient production of xylanase and mannanase was achieved when cellulose-based media were used for growth. Under these conditions, enhanced levels of endoglucanase were formed as well. Formation of xylanase and mannanase could be more specifically induced when using xylan or mannan as growth substrates, although the enzyme activities thus obtained were significantly lower compared to cultivations on cellulose as main inducing substrate.

Improved Production of Mannanase by Streptomyces lividans

Replacement of the natural promoter of the (beta)-mannanase gene of Streptomyces lividans by lacp resulted in a 15-fold increase in enzyme production over that of the previously reported clone S. lividans IAF36, a clone carrying multiple copies of manA, and a 350-fold increase over that of the wild-type strain S. lividans 1326. In addition, the use of lacp in the shuttle vector pIAF199 allowed synthesis of the enzymes on carbon sources that did not contain mannan, such as xylan and whey, which offers interesting possibilities for industrial production of the enzyme.

Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production

A locally isolated strain, Bacillus subtilis NM-39, was selected as an active mannan-utilizing bacterium based on high saccharifying activities on coconut residue and locust bean gum galactomannan. The optimal pH and temperature ranges for activity of the crude enzyme were 5.0 to 6.0 and 50 to 60°C, respectively. The organism gave maximum mannanase activity when grown in liquid mineral salts medium containing 1% (w/v) each of coconut residue and soybean flour, as carbon and nitrogen sources, respectively, at pH 7.0 and in aerobic growth for 28 h at 37°C. High saccharifying activity on coconut mannan was also observed.

Production of \-mannanases by Trichoderma harzianum E58

The extracellular mannanase and endoglucanase activities of Trichoderma harzianum E58 were followed during growth of the fungus on 1% (w/v) mannose, Avicel, locust bean gum, konjac powder or the water-soluble fraction from stream-treated white spruce (SWS). Peak galactomannanase activities of 0.60 IU/ml and 0.66 IU/ml were detected in culture filtrates after 6–8 days growth on locust bean gum and Avicel respectively. When SWS or konjac powder were used as substrates, lower but relatively constant levels of activity were detected between 2 and 11 days of growth. Growth of the fungus on mannan-rich locust bean gum resulted in the highest specific glucomannanase and galactomannanase values. Although growth on 1% mannose failed to induce any mannanase activity, when 0.5% galactomannan was added with mannose, mannanase activity was detected in the culture filtrate. This indicated that mannanase production was not repressed in the presence of mannose. Samples were taken from each culture at the time of maximum galactomannanase activity. A protein profile obtained by isoelectric focusing was followed by a zymogram overlay to detect bands exhibiting galactomannanase, glucomannanase and endoglucanase activities. Several bands showed mannanase and endoglucananase activity. One band at pI 6.55 revealed both gluco- and galactomannanase activity and was free of detectable cellulase activity.

Extracellular mannanases and galactanases from selected fungi

Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of β-mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, beta-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. beta-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. beta-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65 degrees C, 65 degrees C, 60 degrees C and 55 degrees C respectively. Galactanases retained more than 95% activity at 55 degrees C after 3 h. The pH optima of mannanases ranged from 6.5-6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.

Mannanase from Lactuca sativa: Metabolic requirements for production and partial purification

Endo- (1→4)-β-mannanase is produced by endosperms isolated from ungerminated Grand Rapids lettuce achenes when they are incubated in a sufficiently large volume of buffer (LV); this production is inhibited if incubation takes place either in a small volume (SV) or with ABA included in the large volume (LV + ABA). This report describes changes in protein and RNA synthesis concominant with mannananase production, the effects of inhibitors specific to these metabolic events on enzyme production, and partial purification of mannase from lettuce endosperms. After 18 hr or more of incubation, protein synthesis is greater in endosperms incubated in a LV than in endosperms incubated in either a SV or a LV + ABA. The peak of protein synthesis in endosperms in a LV precedes that of mannanase activity by ca 4 hr. Mannanase production is cycloheximide-sensitive, indicating that increased mannanase activity is dependent of protein synthesis. RNA synthesis is invariant with incubation conditions. Nevertheless, mannanase production can be reduced, although not prevented, by either actinomycin D or α-amanitin; these results suggest a transcriptional requirement for mannanase production. Partial purification of mannanase was achieved by DEAE-chromatography and affinity chromatography. Mannanase activity in the incubation medium of isolated endosperms can be attributed to two polypeptides of apparent M r of 46 000 which may represent isozymes of the enzyme. These M r values are similar to those reported for mannanases from diverse sources. Mannanase is only a minor product protein synthesis in isolated lettuce endosperms.

De no vo synthesis of mannanase by the endosperm of Lactuca sativa

The increase in endo-(1→4)-β-mannanase activity in isolated lettuce endosperms was inhibited by 1 μg/ml cycloheximide added before or during enzyme production. In vivo density labelling using 50% D 2O increased mannanase buoyant density from 1.308 to 1.319. The production of the enzyme is therefore based upon de novo synthesis. Mannanase synthesis in isolated endosperms was prevented by addition of 10 μM cordycepin or 20 μg/ml actinomyein D during the ca 4 hr period before enzyme production began. These inhibitors had little efrect at later times. Enzyme production was similarly inhibited when endosperms were isolated from non-germinated seeds at 24 hr, instead of 4 hr, after sowing. It is suggested that (i) mannanase production is dependent on the completion of transcriptional events and (ii) in germinating seeds these events are part of the control mechanism through which the germinating axis initiates mannanase production. Mannanase secreted by isolated lettuce endosperms has been characterized by native gel filtration chromatography and two-dimensional gel electrophoresis. The secreted mannase was present as three charge-isomers, with pIs between 4.75 and 4.9. Estimates of M r by chromatography and electrophoresis were 19 and 46 000, respectively. Reasons for favouring the 46 000 estimate are presented.

Production of mannan-degrading enzymes

Production of mannanase by four hemicellulolytic microorganisms was studied. The highest mannanase activity was produced byBacillus subtilis. β-Mannosidase and α-galactosidase were not detected inB. subtilis culture filtrate. The hydrolysis of galactomannans was limited by the increasing degree of substitution of the substrate. No monomeric sugars were produced in the hydrolysis withB. subtilis culture filtrate.

Abscisic Acid Is an Endogenous Inhibitor in the Regulation of Mannanase Production by Isolated Lettuce (Lactuca sativa cv Grand Rapids) Endosperms

The production of mannanase, a cell-wall-degrading carbohydrase, can be manipulated in isolated lettuce (Lactuca sativa cv Grand Rapids) endosperms by changes in the volume of buffer in which they are incubated. The enzyme is produced when endosperms are incubated in a large volume, but not when incubated in a small volume, which is suggestive that an endogenous, diffusible inhibitor of mannanase production is being lost from the endosperms in a large volume (JD Bewley, P Halmer 1980/1981 Israel J Bot 29: 118-132). We have investigated the possibility that the phytohormone abscisic acid (ABA) is involved in this regulation of mannanase production in isolated lettuce endosperms. We find several correlations between the presence of the endogenous inhibitor and of ABA, i.e. (a) a ;leachate' prepared from isolated lettuce endosperms induces synthesis of ABA-specific proteins in barley aleurone layers, indicating that incubation of endosperms in a large volume results in the diffusion of ABA therefrom into the surrounding medium; (b) fractionation of the components of a leachate by either polyvinylpyrrolidone-chromatography of C(18) reversed-phase high performance liquid chromatography fails to separate the endogenous inhibitor from authentic ABA; and (c) changes in the incubation volume of endosperms result in changes in the amount of extractable ABA in the endosperms, as detected by ELISA. These results are consistent with a role for endogenous ABA in the regulation of mannanase production in isolated lettuce endosperms.

Embryo-endosperm interactions in the hydrolysis of lettuce seed reserves.

Red light- and gibberellin-induced seeds of lettuce ( Lactuca sativa L., cv. Grand Rapids) produce copious amounts of endo-β-mannanase after germination is completed. This enzyme is produced and secreted by the endosperm itself and participates in the hydrolysis of its cell walls, which are largely mannans. The resulting oligomannans are further hydrolysed by β-mannosidase, an enzyme produced within the cotyledons. Degradation of the endosperm is completed prior to mobilization of the main storage reserves within the cotyledons. Isolated endosperms produce and release mannanase. This can be prevented by placing the endosperms in a small incubation volume, probably due to insufficient dilution of endogenous leachable inhibitor(s). The inhibition is lifted during germination: it also can be relieved by benzyladenine and gibberellic acid when the cotyledons are present. Abscisic acid can prevent mannanase production by isolated endosperms and it may contribute to the endogenous inhibitory regulation of this enzyme. By using dissection experiments it has been demonstrated that mannanase production by the endosperm is under the control of the embryo, and that both the axis and cotyledons play a role. The cotyledons are capable of lifting the inhibition, but depend upon an axial event early in germination, apparently involving the transmission of diffusible substances.

Mannanase production by the lettuce endosperm

Endo-β-mannanase (EC 3.2.1.78) is produced and secreted by the cells of the endosperm of lettuce (lactuca sativa L.) "seeds" (achenes). In imbibed intact seeds, production is prevented by inhibitors. If the endosperm is incubated alone, these inhibitors can be removed by leaching, allowing mannanase production. Abscisic acid, a component of lettuce seeds, inhibits the production of mannanase in the isolated endosperm, and may be involved in regulation of mannanase production in intact seeds. During germination the inhibition is removed, beginning 4-8 h after red-light irradiation, which was given 4 h from sowing. The cotyledons participate in this process, and are controlled by events occuring in the axis within 4 h from red-light irradiation. This control by the axis apparently depends on the exchange of diffusible substances. Both benzyladenine and gibberellic acid can replace the influence of the axis if the latter is removed, and may therefore be involved in the control by the axis of the rest of the seed.

Alpha-Mannosidase and mannanase of some wood-rotting fungi.

Cultivation media from 11 wood-rotting fungi contained alpha-mannosidase and mannanase activity, alpha-Mannosidase was studied in more detail in Phellinus abietis and mannanase was studied more intimately in basidiomycetes Phellinus abietis, Trametes sanguinea and Pholiota aurivella. Suitable cultivation conditions and optimum conditions for the production of alpha-mannosidase and mannanase were determined. Both enzymes are constitutive; mannanase is extracellular, alpha-mannosidase was found in both mycelium and cultivation medium.

Extracellular enzyme system utilized by the fungus Chrysosporium lignorum for the breakdown of cellulose : I. Studies on the enzyme production

The production of cellulolytic and related enzymes by the fungus Chrysosporium lignorum has been studied with cellulose, xylan, and mannan as carbon sources. The enzymes studied, namely cellulase, xylanase, mannanase, β-glucosidase and aryl-β-glucosidase, were all found in good yield in the culture solution only when cellulose was used as carbon source. The production of cellulase and mannanase was not observed on xylan and only traces of all the enzymes studied were found on mannan as carbon source. The results of concentration, dialysis, and separation of the studied enzymes by ultrafiltration membranes are reported as well as the change in isoelectric points of the enzymes with the time of cultivation.