Growth and enzyme production of Cellulomonas sp. ATCC 21399 on microcrystalline cellulose. Effects of increasing concentration of a mineral medium

Summary The kinetics and production of different extracellular enzyme activities were studied during growth of Cellulomonas sp. ATCC 21399 on 2% Avicel with different concentrations of M9 mineral medium. The lag phase and the doubling time increased with increasing ionic strength of the medium. The highest cell density was obtained during growth at 5 x M9 mineral medium and Cellulomonas grew well at this high salinity. The enzyme activities against carboxymethylcellulose and xylan increased with increasing concentration of M9 medium up to 5 x M9. By contrast, activities against microcrystalline cellulose (Avicel), galactomannan and amylose decreased with increasing concentration of M9 medium. The extracellular proteinase activity increased with increasing concentration of M9 medium, and it is possible that the lability of the cellulolytic and amylolytic enzymes may be due to their susceptibility to proteolytic inactivation by the extracellular proteinases.     

Effect of agitation on growth and enzyme production of Trichoderma reesei in batch fermentation

Growth, enzyme-producing activity and respiratory properties of Trichoderma reesei QM 9414 were examined under various agitation intensities. Two substrates were compared: lactose and Avicel. Pellet formation occurred at all agitation intensities for both substrates. Oxygen dependence at the lower agitation rate varied with the substrate type. With lactose as the carbon source, linear growth was observed, despite a regulation of the dissolved oxygen concentration at 30% saturation. The enzyme production was strongly affected by the agitation. At the higher agitation rates the enzyme production dropped. With Avicel as the carbon source, the production of enzymes surged as soon as the growth was limited by the hydrolysis of Avicel.Growth on Avicel, in the conditions we used, was limited by Avicel hydrolysis. Cubic growth was observed when lactose was the carbon source. A new derivation for a model of the observed cubic growth is proposed and is used to correlate growth, CO₂ production and oxygen consumption in a consistent way, impossible with exponential growth models.     

Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from Cellulomonas sp. ATCC 21399

Two endoglucanases (endoglucanase B and endoglucanase C) without affinity for cellulose were purified from the culture broth of Cellulomonas sp. ATCC 21399 using gelfiltration and ion exchange chromatography. Fused rocket immunoelectrophoresis was used to select the fractions with the highest content of endoglucanase and lowest content of contaminating proteins. The endoglucanases were purified to immunological homogeneity. In addition both endoglucanases were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights of endoglucanase B and endoglucanase C were 67000 and 25000, respectively). Endoglucanase B was homogeneous when studied by isoelectric focusing showing one protein band at pl 4.3. Both endoglucanases lacked activity against microcrystalline cellulose (Avicel) and showed similar endo action on carboxymethylcellulose (CMC). Endoglucanase B had a high specific activity against CMC, H(3)PO(4)-swollen Avicel and xylan, but showed no activity against galactomannan. In contrast, endoglucanase C showed activity against both CMC, xylan, and galactomannan all being polysaccharide substrates linked with beta-1-4-D-glucoside bonds. The specific activity of endoglucanase C against H(3)PO(4)-swollen Avicel was low.     

Electrophoretic and enzymatic studies on the crude extracellular enzyme system of the cellulolytic bacterium Cellulomonas sp. ATCC 21399

Cellulomonas sp. ATCC 21399 produced extracellular enzyme activities against Avicel, H(3)PO(4)-swollen Avicel, carboxymethylcellulose, (1-3, 1-4)-beta-D-heteroglucan, xylan, galactomannan, and amylose drying growth on microcrystalline cellulose. No extracellular cellobiase activity was produced. Crossed immunoelectrophoresis of the crude extracellular enzyme system revealed 15 immunologically distinct immunoprecipitates. The immunoprecipitates of endoglucanase A, endoglucanase B and the xylanase appeared heterogeneous with several optima, whereas the immunoprecipitates of endoglucanase C and the amylase appeared homogeneous. The heterogeneity of endoglucanase A, endoglucanase B and xylanase was also visualized using electrofocusing-immunoelectrophoresis. Electro-focusing could resolve the activity against carboxymethylcellulose into six peaks, whereas only one peak of activity against Avicel was observed. The later peak coincided with the major peak of activity against carboxymethylcellulose with isoelectric point between pH 4.0-5.0.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates.

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

A Thermostable Xylanase from a Thermophilic Acidophilic Bacillus sp.

Bacillus sp. 11-IS, a strain of thermophilic acidophilic bacteria, produced an extracellular xylanase during growth on xylan. The enzyme purified from the culture supernatant solution was homogeneous on disc-gel electrophoresis. The molecular weight was calculated to be 56,000 by SDS-gel electrophoresis. The enzyme had a pH optimum for activity at 4.0, and its stability range was pH 2.0 ~ 6.0. The temperature optimum was 80°C (10-min assay); however, the enzyme retained full activity after incubation at 70°C for 15 min. The enzyme acted on carboxymethyl cellulose (CMC) and cellulose, as well as on xylan. The Michaelis constants for larchwood xylan and CMC were calculated to be 1.68 mg xylose eq/ml and 0.465 mg glucose eq/ml, respectively. The predominant hydrolysis products from larchwood xylan were xylobiose, xylotriose, and xylose; the release of arabinose from rice-straw arabinoxylan was not detected. CMC was cleaved to cellobiose and larger oligosaccharides. Thus, the enzyme is considered to be an endoenzyme which degrades the β-1,4-glycosyl linkages in xylan and cellulose.     

Partial Purification and Some Properties of Mitomycin C Inactivating Factors of Pseudomonas convexa 4–87

Strains producing higher levels of cellulolytic enzymes were selected from among 520 strains of plant pathogenic fungi, Fusarium species, and F. oxysporum strain SUF850 was found to be the best producer. When strain SUF850 was cultured using one of three polysaccharides, Avicel, carboxy- methyl cellulose (CMC) or xylan, as a carbon source, the culture filtrate contained degrading activi- ties toward all three substrates, i.e., irrespective of the carbon source used. From the culture filtrate of Avicel-grown cells, four distinct enzymes were purified to homogeneity, as judged on SDS-PAGE. They were designated as CMCase I, CMCase II, /Miitrophenyl-β-d-cellobiosidase and xylanase, and the characteristics of the individual enzymes were examined and compared.     

Production of \-mannanases by Trichoderma harzianum E58

Summary 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. Offprint requests to: J. N. Saddler     

Comparison of the Influence of Carbon Substrates on the Fibrolytic Activities of Anaerobic Rumen Fungi

Cellulolytic and xylanolytic activities among genera of anaerobic fungi when grown on glucose, xylan and the cellulosic substrates, filter paper and Avicel were compared. All the fungi had basal extracellular fibrolytic activities that could be enhanced by growth on xylan or the cellulosic substrates. However,Piromyces communisstrain 22 andNeocallimastix patriciarumstrain 27 had substantially greater levels of fibrolytic activity thanOrpinomyces joyoniistrain 19-2 orNeocallimastix frontalisstrain RE1. Zymogram analysis suggested both structural and regulatory differences amongst the enzyme systems of the fungi. Numerous and varied enzyme bands were evidenced for all the fungi, with substantial substrate influences seen in the xylanase activities. Most commonly the smaller molecular weight bands, found exclusively extracellularly, appeared under the greatest regulatory control. Endoglucanase activities ofP. communisandO. joyoniidemonstrated similar regulatory control, while those of the twoNeocallimastixstrains did not appear to exert such control. These results suggest that while the enzymatic activities are functionally similar, there are likely significant variations in the enzyme systems of the anaerobic fungi.     

The Anaerobic Fungus Neocallimastix sp. Strain L2: Growth and Production of (Hemi)Cellulolytic Enzymes on a Range of Carbohydrate Substrates

The anaerobic fungus Neocallimastix sp. strain L2, isolated from the feces of a llama, was tested for growth on a range of soluble and insoluble carbohydrate substrates. The fungus was able to ferment glucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch, inulin, filter paper cellulose, and Avicel. No growth was observed on arabinose, galactose, mannose, ribose, xylose, sorbitol, pectin, xylan, glycerol, citrate, soya, and wheat bran. The fermentation products after growth were hydrogen, formate, acetate, ethanol, and lactate. The fermentation pattern was dependent on the carbon source. In general, higher hydrogen production resulted in decreased formation of lactate and ethanol. Recovery of the fermented carbon in products at the end of growth ranged from 50% to 80%. (Hemi)cellulolytic enzyme activities were affected by the carbon source. Highest activities were found in filtrates from cultures grown on cellulose. Growing the fungus on inulin and lactose yielded the lowest cellulolytic activities. Highest specific activities for avicelase, endoglucanase, β-glucosidase, and xylanase were obtained with Avicel as the substrate for growth (0.29, 5.9, 0.57, and 13 IU · mg⁻¹ protein, respectively). Endoglucanase activity banding patterns after SDS-PAGE were very similar for all substrates. Minor differences indicated that enzyme activities may in part be the result of secretion of different sets of isoenzymes. Received: 10 July 1996 / Accepted: 22 July 1996     

Paenibacillus curdlanolyticus Strain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, β-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, β-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

Characterization and substrate specificity of an endo-beta-1,4-D-glucanase I (Avicelase I) from an extracellular multienzyme complex of Bacillus circulans.

An endo-1,4-beta-D-glucanase I (Avicelase I; EC 3.2.1.4) was purified to homogeneity from an extracellular celluloxylanosome of Bacillus circulans F-2. The purification in the presence of 6 M urea yielded homogeneous enzyme. The enzyme had a monomeric structure, its relative molecular mass being 75 kDa as determined by gel filtration and 82 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.4, and the N-terminal amino acid sequence was ASNIGGWVGGNESGFEFG. The optimal pH was 4.5, and the enzyme was stable at pH 4 to 10. The enzyme has a temperature optimum of 50 degrees C, it was stable at 55 degrees C for 46 h, and it retains approximately 20% of its activity after 30 min at 80 degrees C. It showed high-level activity towards carboxymethyl cellulose (CMC) as well as p-nitrophenyl-beta-D-cellobioside, 4-methylumbelliferyl cellobioside, xylan, Avicel, filter paper, and some cello-oligosaccharides. Km values for birch xylan, CMC, and Avicel were 4.8, 7.2, and 87.0 mg/ml, respectively, while Vmax values were 256, 210, and 8.6 mumol x min-1 x mg-1, respectively. Cellotetraose was preferentially cleaved into cellobiose (G2) plus G2, and cellopentaose was cleaved into G2 plus cellotriose (G3), while cellohexaose was cleaved into cellotetraose plus G2 and to a lesser extent G3 plus G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. Xylotetraose (X4) and xylobiose (X2) were mainly produced by the enzyme hydrolysis of xylan. G2 inhibited the activity of carboxymethyl cellulase and Avicelase, whereas Mg2+ stimulated it. The enzyme was completely inactivated by Hg2+, and it was inhibited by a thiol-blocking reagent. Hydrolysis of CMC took place, with a rapid decrease in viscosity but a slow liberation of reducing sugars. On the basis of these results, it appeared that the cellulase should be regarded as endo-type cellulase, although it hydrolyzed Avicel.     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM‐2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl₂ as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H₂ energy was examined with seven H₂‐producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H₂ production performance with the hydrolysate of SCB and xylan. The cumulative H₂ production and H₂ yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H₂/g reducing sugar (or 1.91 mmol H₂/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H₂ production and H₂ yield of 87.02 mL/L and 5.03 mmol H₂/g reducing sugar (or 4.01 mmol H₂/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM)

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.     

Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHΔCBM and EngYΔCBM devoid of the CBM lost activity toward all substrates including CMC. EngKΔCBM and EngMΔCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.     

Hemicellulolytic and cellulolytic functions of the domains of a β-mannanase cloned from Caldicellosiruptor saccharolyticus

Various combinations of the four domains of the multifunctional mannanase from Caldicellosiruptor saccharolyticus have been cloned and expressed in Escherichia coli. The four domains comprise two catalytic domains (1 and 4), and two putative cellulose binding domains (2 and 3). Each of the six gene products (Man1, Man123, Man1234, Man23, Man234 and Man4) was partially purified by heat treatment.The enzymes Man1234, Man123 and Man1 exhibited activity on mannans, and Man1234, Man234 and Man4 exhibited activity on xylan and carboxymethylcellulose (CMC). For the complete enzyme (Man1234) all activities were of the same order of magnitude. Activities were additive against a mixture of mannan and xylan or mannan and CMC (but not xylan and CMC). The expression product Man23 exhibited activity on none of the substrates tested, nor did its presence influence thermostability or significantly reduce the K_m value for any of the substrates. However, when expressed in combination with domains 1 or 4 it greatly increased their activity.We conclude that domain 1 catalyses mannan hydrolysis and domain 4 catalyses xylan and CMC hydrolysis at the same active site: domains 2 and 3 have no obvious function, since they do not reduce substrate K_m nor affect thermostability. However, their effect on rates of substrate hydrolysis may indicate a role influencing the conformation of the adjacent catalytic domains.     

Enzymatic degradation of granular potato starch by <Emphasis Type="Italic">Microbacterium aurum</Emphasis> strain B8.A

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.