Kinetics of improved production and thermostability of an intracellular β-glucosidase from a mutant-derivative of Cellulomonas biazotea

The highest productivity (20 IU l(-1) h(-1)) of beta-glucosidase by a mutant of Cellulomonas biazotea was 2.5-fold more than that of the parent organism. The enzyme had a lower activation energy (57 kJ mol(-1)) than the native enzyme (68 kJ mol(-1)). The enzyme from the mutant had enthalpy and entropy values for irreversible intactivation of 95.6 kJ mol(-1) and 60 J.mol(-1) K(-1) compared with 108 kJ mol(-1) and 86 J mol(-1) K(-1) for the native enzyme suggesting that the mutation had stabilized the enzyme.     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

Production and characterization of a highly active cellobiase from Aspergillus niger grown in solid state fermentation

Summary Aspergillus niger produced extracellular cellobiase when grown on different lignocellulosic substrates in solid state fermentation. The enzyme activity and yield were variable according to the carbon source. In Vogel’s medium, the cellobiase productivity was significantly higher on wheat bran, followed by Leptochloa fusca (kallar grass) straw augmented with corn steep liquor. Maximum yield of cellobiase/g wheat bran was significantly higher than the values reported on other potent fungi, bacteria and recombinants, harboring heterologous gene for cellobiase. This enzyme in the presence and absence of Trichoderma reesei and celluloclast, saccharified the biomass and the percentage saccharification as well as glucose yield from lignocellulosic biomass was doubled in its presence. The partially purified enzyme was thermotolerant as evidenced by melting temperature, activation energy demand for active catalysis, enthalpy and entropy of activation for reversible or irreversible thermal inactivation.     

Enhanced production and characterization of a β-glucosidase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A ₆₀₀ 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K _m and k _cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec⁻¹, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.     

Biological activity of lipopeptides from <Emphasis Type="Italic">Bacillus</Emphasis>

The lipopeptides of Bacillus are small metabolites that contain a cyclic structure formed by 7–10 amino acids (including 2–4 d-amino acids) and a beta-hydroxy fatty acid with 13–19 C atoms. These lipopeptides exhibit a variety of biological activities, including interactions with biofilms, and anti-fungal, anti-inflammatory, anti-tumor, anti-virus, and anti-platelet properties. The multiple activities of lipopeptides have stimulated significant interest in the exploitation of these lipopeptides for use as antibiotics, feed additives, anti-tumor agents, urgent thrombolytic therapeutic agents, and drug delivery systems. Understanding the natural function of these structurally diverse lipopeptides in Bacillus provides insight into microbial regulatory programs and is required for efficient development of more effective products. Currently, there is still insufficient knowledge of the direct target of these lipopeptides, and continued efforts are needed to enhance their biosynthesis efficiency for industrial applications.     

Production of single cell protein from rice polishings using Candida utilis

Microbial protein was produced from defatted rice polishings using Candida utilis in shake-flasks and a 14-l fermentor to optimize fermentation conditions before producing biomass in a 50-l fermentor. The organism supported maximum values of 0.224 h⁻¹, 0.94, 1.35, 1.75, 2.12 g l⁻¹ h⁻¹, 0.62 g cells g⁻¹ substrate utilized and 0.38 g g⁻¹ for specific growth rate, true protein productivity, crude protein productivity, cell mass productivity, substrate consumption rate, cell yield, crude protein yield, respectively in 50-l fermentor studies using optimized cultural conditions. Maximum values compared favourably or were superior to published data in literature. The biomass protein in the 50-l fermentor contained 22.3, 27.8, 19.2, 9.5, 38.12, 8.5 and 0.27% true protein, crude protein, crude fibre, ash, carbon, cellulose and RNA content, respectively. The dried biomass showed a gross metabolizable energy value of 2678 kcal kg⁻¹ and contained all essential and non-essential amino acids. Yeast biomass as animal feed may replace expensive feed ingredients currently being used in poultry feed and may improve the economics of feed produced in countries like Pakistan. This revised version was published online in August 2006 with corrections to the Cover Date.     

Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.     

Kinetics and thermodynamics of the native and mutated extracellular endo-glucanases from Cellulomonas biazotea

The mutation had dramatic effect on the kinetic and thermodynamic parameters inferring thermostability of endo-glucanase from Cellulomonas biazotea mutant 51 SM(r). The denaturation activation energies of native and mutated enzymes were 73.3 and 68.8 kJ/mol respectively. They showed compensation effect at 55 degrees C. Both enthalpy and entropy values of irreversible thermal inactivation for mutated enzyme were decreased suggesting that the mutation partly stabilized the enzyme.     

Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger

Carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 was purified by a combination of ammonium sulphate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography on FPLC with 9-folds increase in specific activity. Native and subunit molecular weights were found to be 36 kDa each. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). Similarly, the enzyme was modified by EDC in the presence of ethylenediamine dihydrochloride plus cellobiose for 75 min (EDAM75). The neutralization (GAM15 and GAM75) and reversal (EDAM75) of negative charges of carboxyl groups of CMCase had profound effect on the specificity constant (k(cat)/K(m)), pH optima, pK(a)'s of the active-site residues and thermodynamic parameters of activation. The specificity constants of native, GAM15, GAM75, and EDAM75 were 143, 340, 804, and 48, respectively. The enthalpy of activation (DeltaH(#)) of Carboxymethylcellulose (CMC) hydrolysis of native (50 and 15 kJ mol(-1)) and GAM15 (41 and 16 kJ mol(-1)) were biphasic whereas those of GAM75 (43 kJ mol(-1)) and EDAM75 (41 k J mol(-1)) were monophasic. Similarly, the entropy of activation (DeltaS(#)) of CMC hydrolysis of native (-61 and -173 J mol(-1) K(-1)) and GAM15 (-91 and -171 J mol(-1) K(-1)) were biphasic whereas those of GAM75 (-82 J mol(-1) K(-1)) and EDAM75 (-106 J mol(-1) K(-1)) were monophasic. The pH optima/pK(a)'s of both acidic and basic limbs of charge neutralized CMCases increased compared with those of native enzyme. The CMCase modification in the presence of glycinamide and absence of cellobiose at different pH's periodically activated and inhibited the enzyme activity indicating conformational changes. We believe that the alteration of the surface charges resulted in gross movement of loops that surround the catalytic pocket, thereby inducing changes in the vicinity of active site residues with concomitant alteration in kinetic and thermodynamic properties of the modified CMCases.     

Solid-state fermentation-supported enhanced production of α-galactosidase by a deoxyglucose-resistant mutant of <Emphasis Type="Italic">Aspergillus niger</Emphasis> and thermostabilization of the production process

The aim of the present investigation was to comparatively evaluate the behaviour of A. niger and its derepressed mutant in production of α-galactosidase in submerged (SmF) and solid state fermentation (SSF) using basal Vogel’s medium or corn steep liquor as nitrogen source and observe the response of latter source under both cultural techniques under different temperature regimes, and determine if SSF can be exploited in a wide range of temperature expected to vary in this fermentation system. All studies were performed in both systems under pre-optimized cultural conditions. Higher melting temperature and negative values of entropy of activation in SSF indicated that the genetic system of both organisms was thermodynamically resistant in the presence of corn steep liquor but sensitive to inactivation in the presence of Vogel’s nitrogen sources in submerged fermentation. This was reflected as the organisms demanded higher magnitudes of energy for product formation in the presence of ammonium salts. Studies on influence of corn steep liquor revealed that it had stabilizing effect too in both fermentation systems but the mutant strain was more stable in both fermentation systems. Because of these properties, the mutant organism may be exploited for bulk production of α-galactosidase in SSF under condition where temperature may fluctuate during fermentation.