Production of pectolytic enzymes – a review

Pectolytic enzymes play an important role in food processing industries and alcoholic beverage industries. These enzymes degrade pectin and reduce the viscosity of the solution so that it can be handled easily. These enzymes are mainly synthesized by plants and microorganisms. Aspergillus niger is used for industrial production of pectolytic enzymes. This fungus produces polygalacturonase, polymethylgalacturonase and pectinlyase. This review mainly concerns with the production of pectolytic enzymes using different carbon sources. It also deals with the effect of operating parameters such as temperature, aeration rate, agitation and type of fermentation on the production of these enzymes.     

The Production of β-Fructofuranosidase from Bifidobacterium spp.

The productions of β-fructofuranosidase from Bifidohacterium longum A1, B. adolescentis G1, and four other strains of Bifidobacteria were investigated. All strains used in this study were grown in modified BL broth containing a mixture of fructooligosaccharides [1^F (1-β-D-fructofuranosyl)_n-1sucrose, GF_n (n = 2 – 5)] as the only carbon source. Hydrolyses of 1-kestose, sucrose, and inulin were detected in the extract of the cell. The highest activity on 1-kestose was detected in the extract of B. longum A1 followed by B. adolescentis G1. The other extracts weakly attacked 1-kestose. The relative activities of the extract of B. adolescentis G1 for 1-kestose, nystose, 1^F-fructosylnystose, sucrose, and inulin were 100, 82.5, 50.8, 28.3, and 15.0, respectively. The relative activities for various substrates differed from invertases (yeast β-fructofuranosidases) and exo-inulinase from Penicillium trzehinskii.     

Production and localisation of carboxymethylcellulase,xylanase and β-glucosidase fromCellulomonas andMicrococcus spp.

Summary Cellulomonas and Micrococcus spp. grew well at 30°C, pH 7.0, and produced carboxymethylcellulase (CMCase) and xylanase enzymes. Only one species of Micrococcus was able to produce an appreciable amount of -glucosidase. This is the first report where Micrococcus sp., isolated from termite gut, was able to produce all three enzymes (i.e. CMCase, xylanase and -glucosidase) required for degradation of cellulosic and hemicellulosic substrates. Offprint requests to: A. Varma     

Production of cyclomaltononaose (δ-cyclodextrin) by cyclodextrin glycosyltransferases from Bacillus spp. and bacterial isolates

The conversion of soluble starch to cyclomaltohexaose (α-CD), cyclomaltoheptaose (β-CD), cyclomaltooctaose (γ-CD) and cyclomaltononaose (δ-CD) by cyclodextrin glycosyltransferases (E.C. 2.4.1.19) from Bacillus spp. and bacterial isolates was studied. The results show that δ-CD was formed by all the enzymes investigated in the range of 5%–11.5% of the total amount of α-, β-, γ-, and δ-CD produced. Received: 17 February 1998 / Received revision: 18 May 1998 / Accepted: 21 May 1998     

Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

Isatin analogs as novel inhibitors of Candida spp. β-carbonic anhydrase enzymes

Enzyme inhibition data of structurally novel isatin-containing sulfonamides were determined for two carbonic anhydrases (CAs, EC 4.2.1.1) from pathogenic Candida species (CaNce103 from C. albicans and CgNce103 from C. glabrata). The compounds show K_I values in the low nanomolar range for the fungal CAs, while they have significantly higher K_I values for the human CAs. Homology models were constructed for the CaNce103 and CgNce103 and subsequently the ligands were docked into these models to rationalize their enzyme inhibitory properties.     

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal β-glycosidases

Sβgly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of Sβgly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure–function relationships in these enzymes. Received: May 20, 1999 / Accepted: January 10, 2000     

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

Production and characterization of β-1,4-glucosidase from a strain of Penicillium pinophilum

A high β-glucosidase (BGL)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer rDNA gene sequence. Under the optimal culture conditions, a maximum BGL specific activity of 3.2 U ml⁻¹ (83 U mg-protein⁻¹), one of the highest levels among BGL-producing microorganisms was obtained. An extracellular BGL was purified to homogeneity by sequential chromatography of P. pinophilum culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a Mono Q column. The relative molecular weight of P. pinophilum BGL was determined to be 120 kDa by SDS-PAGE and size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 3.5 and a temperature optimum of 32 °C. P. pinophilum BGL showed a higher activity (V_max = 1120 U mg-protein⁻¹) than most BGLs purified from other sources. The internal amino acid sequences of P. pinophilum BGL showed a significant homology with hydrolases from glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, P. pinophilum BGL is distinguished from other BGLs by its high activity.     

Production of extracellular β-mannanases by yeasts and yeast-like microorganisms

A new screening method for simultaneous detection of endo-β-1,4-mannanase and endo-β-1,4-xylanase producing microorganisms is described. Two differently dyed substrate Ostazin Brilliant Red-galactomannan and Remazol Brilliant Blue-xylan were incorporated into the same agar media. Decolorizing of one or both substrates around the cell colonies indicates secretion of the corresponding enzyme(s). The method was used to screen 449 yeasts and yeast-like microorganisms belonging to 68 different genera. The secretion of endo-β-1,4-mannanases and/or endo-β-1,4-xylanases was found within 10 genera (42 positive strains out of 261 tested). A low frequency of occurrence of endo-β-1,4-mannanases was observed within the generaCryptococcus (1 positive strain out of 15 tested),Geotrichum (1 of 6) andPichia (1 of 35). The highest frequency of occurrence of endo-β-1,4-mannanases was found within the generaStephanoascus (2 of 2) andAureobasidium (14 of 14). Strains hydrolyzing Ostazin Brilliant Red-galactomannan were cultivated in liquid media containing 1 % locust bean gum. The best producers of extracellular endo-β-1,4-mannanases were found to be the strains ofAureobasidium pullulans.     

Production of β-glucosidase by Aspergillus terreus

The production of -glucosidase by Aspergillus terreus was investigated in liquid shake cultures. Enzyme production was maximum on the 7th day of growth (2.18 U/ml) with the initial pH of the medium in the range of 4.0–5.5. Cellulose (Sigmacell Type 100) at 1.0% (wt/vol) gave maximum -glucosidase activity among the various soluble and insoluble carbon sources tested. Potassium nitrate was a suitable nitrogen source for enzyme production. Triton X-100 at 0.15% (vol/vol) increased the enzyme levels of A. terreus. The test fungal strain showed an ability to ferment glucose to ethanol.     

Production of β-carotene by aRhodotorula strain

Summary The production of -carotene by the biomass ofRhodotorula strain var.glutinis, during the stationary phase of growth and in non-proliferating conditions was assayed. When the cells were transferred to distilled water, the fraction of -carotene produced increased from 130 to 630 g per gram of dried cells.     

Production of xylooligosaccharides in SSF by Bacillus subtilis KCX006 producing β-xylosidase-free endo-xylanase and multiple xylan debranching enzymes

Xylanase and xylooligosaccharides (XOS) are employed in food and feed industries. Though xylanase production from lignocellulosic materials (LCMs) by solid-state fermentation (SSF) is well known, the XOS formed during growth is not recovered due to its conversion to xylose by β-xylosidase and subsequent bacterial metabolism. A new strain, Bacillus subtilis KCX006, was exceptionally found to synthesize β-xylosidase-free endo-xylanase and multiple xylan debranching enzymes constitutively in the presence of LCMs. Absence of β-xylosidase resulted in accumulation of XOS during growth of KCX006 on LCMs. Therefore, this strain was used for simultaneous production of xylanase and XOS from agro-residues in solid-state fermentation (SSF). Partial purification of XOS from culture supernatant using activated charcoal followed by high-performance liquid chromatography (HPLC) analysis showed xylobiose to xylotetraose formed as the major products. Among various LCM substrates, wheat bran and groundnut oil-cake supported highest xylanase and XOS production at 2158 IU/gdw and 24.92 mg/gdw, respectively. The levels of xylanase and XOS were improved by 1.5-fold (3102 IU/gdw) and 1.9-fold (48 mg/gdw), respectively, by optimization of culture conditions.     

Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine α- and β-trypsin

Several esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid and α-N-toluene-p-sulphonyl-l-homoarginine by α- and β-trypsin were compared. On the basis of values of the specificity constants (k_cat./K_m), the two enzymes display similar catalytic efficiency towards some substrates. In other cases α-trypsin is less efficient than β-trypsin. It is possible that α-trypsin possesses greater molecular flexibility than β-trypsin.     

The temperature influences the ratio of glucosidase and galactosidase activities of β-glycosidases

The selectivity for the glycon part of a donor substrate of -glycosidases from almond, a mesophilic (Kluyveromyces fragilis) and three highly thermophilic organisms (Caldocellum saccharolyticum, Sulfolobus solfataricus and Pyrococcus furiosus) was investigated at various temperatures (25–90 °C). On the basis of kinetic constants, the selectivity was calculated as the specificity constant (V _max /K _m ) ratio or V _max ratio of glucoside to galactoside donor. In the almond -glucosidase and the mesostable enzyme one enzyme activity dominated whereas the thermostable enzymes expressed both high -glucosidase and high -galactosidase activities. Surprisingly, for -glycosidases from almond, K. fragilis, and C. saccharolyticum the donor selectivity decreased as the temperature increased. In contrast, two of the highly thermostable enzymes (from S. solfataricus and P. furiosus) had constant donor selectivity as the temperature increased. The results thus showed -glycosidases of differing origins to differ markedly in their substrate specificity and in the extent to which their selectivity for the glycon part of the donor substrate is influenced by the temperature.     

Production of β-fructofuranosidase by Aspergillus japonicus in batch and fed-batch cultures

Summary A comparison of -fructofuranosidase (FFase, EC 3.2.l.26) production by Aspergillus japonicus TIT-90076 in batch and fed-batch cultures was investigated in shaken flasks. Results showed that fed-batch cultivation of A. japonicus using intermittent sucrose supply produced more FFase than batch culture, and the maximal enzyme production was 910 units ml^–1, which was about 20% higher than that in the batch cultures.     

Production of pharmaceutically important genistein and daidzein from soybean flour extract by using β-glucosidase derived from Penicillium janthinellum NCIM 1171

Genistein and daidzein are isoflavones with well-recognized biological activities. Their glycosidic forms (genistin and daidzin, respectively) are abundant in some plants. In this study, production of β-glucosidase from Penicillium janthinellum NCIM 1171 and its use in the obtaining genistein and daidzein are described. In a response surface methodology (RSM) optimized medium, levels of β-glucosidase under submerged and solid state fermentation conditions were found to be 10.2 ± 0.75 IU/mL and 121 ± 9.3 IU/g, respectively. The supernatants resulting from submerged fermentation were subjected to ion exchange and gel filtration chromatography and the enzyme was purified in a 44.4 fold manner with final recovery of 39.75 %. The β-glucosidase was deduced to be a monomeric protein with a molecular mass of 97.18 kDa. The purified protein showed 46 % sequence coverage matching with β-glucosidase derived from Penicillium sp. ABP88968. The purified enzyme was effective in producing genistein and daidzein from soybean (Glycine max) flour extract with a yield of 92.3 and 95 %, respectively. To the best of our knowledge, this is the first report on the use of a wild type strain of P. janthinellum for the production of genistein and daidzein with high productivity and purity.     

The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.