Effect of temperature and pH on phenol removal using purified Coprinus cinereus peroxidase

The removal of phenol by peroxidase-catalysed polymerization was examined using purified Coprinus cinereus peroxidase. The phenol removal efficiency increased with a decrease in the reaction temperature over the range of 0–70 °C, though only a trace of enzyme activity with 4-aminoantipyrine (4-AAP), phenol and hydrogen peroxide was found at 0 °C. The optimum pH value for phenol removal was 9.0, while the enzyme expressed maximum activity at pH 7.5 in the presence of 4-AAP, phenol and hydrogen peroxide. By measuring residual enzyme activity in the polymerizing reaction mixture, it was shown that enzyme inactivation by free radicals was more suppressed at 0 °C than at 40 °C and that the adsorption of the enzyme on the polymerized precipitate was more suppressed at pH 9.0 than that at pH 7.5.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30-01

We isolated the feather-degrading Bacillus pseudofirmus FA30-01 from the soil sample of poultry farm. The isolate completely degraded feather pieces after liquid culture at 30°C (pH 10.5) for 3 days. Strain FA30-01 is a Gram-positive, spore-forming, rod-shaped bacterium and was identified with B. pseudofirmus based on 16S rDNA analysis. The keratinase enzyme produced by strain FA30-01 was refined using ammonium sulfate precipitation, negative-ion DEAE Toyopearl exchange chromatography, and hydroxyapatite chromatography. The refinement level was 14.5-fold. The molecular weight of this enzyme was 27.5 kDa and it had an isoelectric point of 5.9. The enzyme exhibited activity at pH 5.1–11.5 and 30–80°C with azokeratin as a substrate, although the optimum pH and temperature for keratinase activity were pH 8.8–10.3 and 60°C, respectively. This enzyme is one of the serine-type proteases. Subtilisin ALP I and this enzyme had 90% homology in the N-terminal amino acid sequence. Since this enzyme differed from ALP I in molecular weight, heat resistance and isoelectric point, they are suggested to be different enzymes.     

Amylases of the Fungus Aspergillus flavipes Associated with Fucus evanescens

A promising producer of extracellular amylases, Aspergillus flavipes, was selected from 245 strains of marine fungi. Depending on the conditions of growth, this strain produced diverse amylolytic complexes. When grown on a medium containing peptone and yeast extract (pH 7.0), A. flavipes synthesized three forms of amylase, differing in pH optimum (5.5, 6.0, and 7.5). A single form of the enzyme was synthesized either in the absence of peptone from the medium or at the initial pH value of the medium, equal to 8.6. The activity of the isolated amylase forms decreased in the presence of proteolytic enzymes. New, highly stable forms of amylase (with pH optima of 5.5 and 7.5 and maximum activity at 60–80°C) were synthesized in the presence of diisopropyl fluorophosphate, an inhibitor of proteases.     

Xylanolytic activities ofSpirochaeta thermophila

Spirochetes capable of degrading xylan or cellulose have not been commonly isolated, nor have their polysaccharolytic activities been characterized.Spirochaeta thermophila strain RI 19.B1 is xylanolytic and grows well at 65°C with oatspelt (OX), birchwood (BX), corncob (CCX-A) xylans, or glucuronoxylan (MGX) as the energy source. All xylans were extensively degraded and utilized during growth. About 72–82% of the initial hexuronic acids and 57–79% of initial pentoses disappeared during growth.S. thermophila possessed xylanase, xylosidase, and arabinofuranosidase enzyme activities. Low levels of these activities were detected with growth on glucose, but high expression of these activities occurred during growth on OX. All three activities were cell-associated and were more stable in cells than cell extracts. Xylan-degrading activities were measured with cells or cell extracts exposed (60 min) to a variety of temperatures (65°–85°C) and pHs (5.0–8.0). More than 50% loss of activities occurred at temperatures above 75°C. Although pH stability was affected by buffer, the optimal range was pH 6.5–7.5. These temperature and pH profiles for xylan-degrading activities coincide with those found for the growth ofS. thermophila.     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Extracellular glucose-producing exodextranase of the yeast Lipomyces lipofer

A dextran-hydrolysing enzyme from Lipomyces lipofer IGC 4042 was purified from the supernatant of cultures grown on a mineral medium with dextran, by ultrafiltration and gel filtration on Bio Gel A-0.5 m. This preparation gave only one band by disc gel electrophoresis. Glucose was the only product of dextran hydrolysis. Optimum pH and temperature for the activity of the enzyme were pH 4.5–5.0 and 45°C, respectively. The enzyme was most stable over a pH range of 4.5–6.0, and after 2 hours at 50°C maintained over 60% of its original activity. The molecular weight was 29,000 daltons and the isoelectric point was at pH 7. K_m (45°C, pH 5) for dextran T-40 was 1.2×10^–5 M. Glucose inhibited the enzyme competitively with a K_i (45°C, pH 5) of 0.5 mM.     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Enhanced stability of cellulase-free xylanase from Chainia sp. (NCL 82.5.1)

Summary Chainia sp. (NCL 82.5.1) produces an extracellular, cellulase-free xylanase. The ready accessibility of the enzyme to cellulose pulp due to its small size and the absence of cellulase are advantageous features. The enzyme is stable at 40°C for 1h and in a pH range of 5–9 at 4°C. Improved stability of the enzyme at higher temperature and pH are desirable. Effect of a variety of compounds was studied to enhance stability. Glycerol, sorbitol, mannitol (10%) or glycine (1M) had marginal effect on thermostability. Addition of Ca⁺² or PEG (10mM) increased the half-life of the enzyme at 60°C. Cysteine (10mM) or Tween-80 (1%) showed 70% protection against thermal inactivation. Xylan (3%) offered complete protection against inactivation of the emzyme at 60°C and at pH 9.NCL Communication No. 5907     

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 ° C; pH optimum was 7–7.5; Mg⁺² optimum was 5–10 mM.The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.     

Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101

Summary An alkalophilic Bacillus sp. no. AH-101, which produced extremely thermostable alkaline protease, was isolated among 200 soil samples. The enzyme production reached its maximum level of 1500 units/ml after about 24 h in alkaline medium (pH 9.5). The enzyme was most active toward casein at pH 12–13 and stable to 10 min incubation at 60° C from pH 5–13. Calcium ions were effective in stabilizing the enzyme especially at higher temperatures. The optimum and stable temperatures were about 80° C and below about 70° C respectively in the presence of 5 mM calcium ions. The enzyme was completely inactivated by phenylmethane sulphonyl fluoride, but little affected by ethylenediaminetetraacetic acid, urea, sodium dodecylbenzenesulphonate and sodium dodecyl sulphate. The molecular weight and sedimentation constant were approximately 30 000 and 3.0S respectively, and the isoelectric point was at pH 9.2. These results indicte that no. AH-101 alkaline protease is more stable against both temperature and highly alkaline conditions than any other protease so far reported.     

Isolation and characterization of a novel intracellular glucosyltransferase from the acarbose producer <Emphasis Type="Italic">Actinoplanes</Emphasis> sp. CKD485-16

A novel intracellular glucosyltransferase (GTase) was isolated from cells of Actinoplanes sp. CKD485-16—acarbose-producing cells. The enzyme was purified by DEAE-cellulose and G75-40 Sephadex chromatography. The molecular mass of the enzyme was estimated to be 62 kDa by SDS-polyacrylamide gel electrophoresis, and its isoelectric point (pI) was pH 4.3. The N-terminal sequence of the GTase consisted of NH₂-Ser-Val-Pro-Leu-Ser-Leu-Pro-Ala-Glu-Trp. The optimum pH and temperature were 7.5 and 30°C. The enzyme was stable in a pH range of 5.5–9.0 and below 40°C. Enzymatic reactions were performed by incubating the GTase with various substrates. The GTase converted acarbose into component C, maltose into trehalose, and maltooligosaccharides into maltooligosyl trehaloses. The reactions were reversible. Various acarbose analogs were tested as inhibitors against the GTase as a means to suppress component C formation. Valienamine was the most potent, with an IC₅₀ value of 2.4×10^–3 mM and showed a competitive inhibition mode.     

Phytase production by the yeast, Pichia anomala

Pichia anomala, isolated from dried flower buds of Woodfordia fruticosa, produced a high activity of an intracellular phytase, at 68 U per g dry biomass, when grown at 20 °C for 24 h in a medium containing glucose (40 g l^–1) and beef extract (10 g l^–1) supplemented with Fe²⁺ (0.15 mM). Partially purified phytase was optimally active at 60 °C and pH 4 with a half life of 7 days at 60 °C. It retained 85% of its activity at 80 °C for 15 min. The enzyme is suitable for supplementing animal feeds to improve the availability of phosphate from phytate.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

Partial purification and characterization of guanine aminohydrolase fromtrypanosoma cruzi

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine to xanthine. A rapid procedure for the partial purification of guanine deaminase fromTrypanosoma cruzi using granulated bed electrofocusing was developed. Supernatants of cell sonicates (40,000 g) were subjected to electrofocusing with a broad range ampholyte (pH 4–9). Sections of the gel were eluted and assayed for xanthine production. Active fractions were pooled, concentrated, and again subjected to electrofocusing with a pH 5–7 range ampholyte. This procedure resulted in over 240-fold purification. The compounds 4-amino-5-imidazolecarboxamide andN ⁶-methyladenine were found to be potent competitive inhibitors of the enzyme. Their respective K_i values were 3.5×10^–6 M and 9.5×10^–6 M. Irreversible inactivation of the enzyme was observed upon incubation withp-chloromercurophenylsulfonic acid andN-ethyl-maleamide at 5.0×10^–4 M. The enzyme was labile to heat; a substantial loss of activity occurred upon incubation at 55°C for 5 min. A broad pH range of activity (pH 7.5–8.5) was observed in Tris, citrate, and phosphate buffers.     

Production and characterization of xylanase from Bacillus thermoalkalophilus grown on agricultural wastes

Summary Bacillus thermoalkalophilus isolated from termite-infested mound soils of the semi-arid zones of India had the ability to produce good amounts of xylanase(s) from cheap agricultural wastes. Of the two hemicellulosic substrates tested, bagasse was found to be the better inducer for xylanase production. Alkali treatment of bagasse and rice husk had varied effects on enzyme production. The enzyme preparation had activity optima at 60° C and 70° C and a half-life of 60 min at 65° C. The enzyme was stable for 24 h over a pH range of 4.0–6.0, while maximum activity was observed at pH 6.0–7.0. Enzyme production and activity were inhibited by the end-product of xylan hydrolysis, xylose. Offprint requests to: Ajit Varma     

A new control strategy for yeast production based on the L/A* approach

Summary We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h. Offprint requests to: J. Chaloupka     

Production of extracellular lactase fromFusarium moniliforme

Summary A strain ofFusarium moniliforme, previously used for microbial protein production, excreted lactase (-D-galactosidase, EC.3.2.1 23) when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced in both media had pH and temperature optima of 4–5 and 50–60°C respectively and was particularly suitable for processing acid whey.In the whey culture, maximum lactase yield was observed after 95 h of growth at 30°C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3 nmoles o-nitrophenyl--D-galactopyranoside sec^–1 ml^–1 of growth medium, at pH 5 and 60°C, was obtained.In the wheat bran culture, the maximum enzyme yield was obtained after 140 h of growth at 28–30°C. A marked increase in the enzyme production was observed when nitrate or phosphate was added to the growth medium. Also, the addition of certain agricultural by-products (molasses, whey) enhanced lactase production. The observed maximum yield corresponding to the hydrolysis of 182 nmoles of ONPG sec^–1 g^–1 of wheat bran, at pH 5 and 60°C, is comparable to that reported for certain microorganisms used commercially for lactase production.