Temperature and carbon source affect the production and secretion of a thermostable β-xylosidase by Aspergillus fumigatus

The effect of the temperature of growth and carbon source on the production and secretion of β-xylosidase (EC 3.2.1.37) by the thermotolerant fungi Aspergillus fumigatus was studied in submerged cultures. In cultures developed at optimal temperature (30 °C), the enzyme was predominantly cell-bound, while in cultures developed at higher temperature (42 °C), the β-xylosidase activity was predominantly found in the cell-free filtrates. The use of corn cob powder instead of xylan as substrate increased considerably the secretion of enzyme. The highest level of extracellular β-xylosidase (45 U/ml or 360 U/mg protein) was obtained in 3% corn cob cultures grown at 42 °C for 72 h. The partially purified enzyme was active and stable at high temperatures. The presence of high titres of β-xylosidase activity in association with xylanase in the culture filtrates enhanced the efficiency of the pulp hydrolysis process.     

Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon, Halobacterium sp. SP1(1)

Aims:  Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach.
Methods and Results:  The significant factors influencing the protease production as screened by Plackett–Burman method were identified as soybean flour and FeCl₃. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l⁻¹): NaCl, 250; KCl, 2; MgSO₄, 10; tri-Na-citrate, 1·5; soybean flour, 10 and FeCl₃, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml⁻¹ of protease.
Conclusions:  Soybean flour and FeCl₃ were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9-fold increase in extracellular protease production.
Significance and Impact of the Study:  The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.     

Comprehensive studies on optimization of cellulase and xylanase production by a local indigenous fungus strain via solid state fermentation using oil palm frond as substrate

The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 10⁶ spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.     

Production and optimization of process parameters for alkaline protease production by a newly isolated Bacillus sp. under solid state fermentation

Production of alkaline protease employing the laboratory isolate, Bacillus sp. under solid state fermentation (SSF) was optimized. The effect of wheat bran and lentil husk was examined. Wheat bran showed highest enzyme production. The appropriate incubation time, inoculum size, moisture level and buffer solution level were determined. Maximum yields of 429.041 and 168.640 U g⁻¹ were achieved by employing wheat bran and lentil husk as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10 with 30 and 40% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20 and 25% and 0.5:1 for wheat bran and lentil husk, respectively.     

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii

Aspergillus tamarii expresses an extracellular alkaline protease that we show to be effective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30 °C for 96 h using a 5% inoculum. The crude enzyme was isolated, purified and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

2,3-丁二醇生产菌株生产能力的比较和培养基优化

对5株克雷伯氏肺炎杆菌 (包括两株乳酸途径被敲除的工程菌株) 发酵生产2,3-丁二醇能力进行了比较,其中K. pneumonia HR521 LDH (乳酸合成途径中ldhA基因被敲除) 具有最佳的发酵性能。通过正交试验优化了其发酵培养基的主要组分,优化后的培养基组成为：葡萄糖 90 g/L,(NH4)2HPO4 3 g/L,玉米浆 (CLSP) 6 g/L,乙酸钠 5 g/L,KCl 0.4 g/L,MgSO4 0.1 g/L,FeSO4·7H2O 0.02 g/L,MnSO4 0.01 g/L。在优化后的发酵培养基中进行摇瓶发酵,24 h发酵乙偶姻和2,3-丁二醇的终浓度为37.46 g/L,比未优化前增加了10 g/L,2,3-丁二醇得率达到了理论得率的90.53％,生产强度1.56 g/(L·h),检测不到副产物乳酸的生成,利于后提取工艺的进行和工业生产的应用。     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

Application of a statistical design to the optimization of parameters and culture medium for alpha-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product)

The production optimization of alpha-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a Box-Behnken design under the response surface methodology. The screening of nineteen nutrients for their influence on alpha-amylase production was achieved using a Plackett-Burman design. KH(2)PO(4), urea, glycerol, (NH(4))(2)SO(4), CoCl(2), casein hydrolysate, soybean meal hydrolysate, MgSO(4) were selected based on their positive influence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the alpha-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced alpha-amylase yield of 72.7%.     

Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process

The biochemical conversion of cellulosic biomass to ethanol, a promising alternative fuel, can be carried out efficiently and economically using the simultaneous saccharification and fermentation (SSF) process. The SSF integrates the enzymatic hydrolysis of cellulose to glucose, catalyzed by the synergistic action of cellulase and beta-glucosidase, with the fermentative synthesis of ethanol. Because the enzymatic step determines the ethanol. Because the enzymatic step determines the availability of glucose to the ethanologenic fermentation, the kinetic of cellulose hydrolysis by cellulase and beta-glucosidase and the susceptibility of the two enzymes to inhibition by hydrolysis and fermentation products are of significant importance to the SSF performance and were investigated under realistic SSF conditions. A previously developed SSF mathematical model was used to conceptualize the depolymerization of cellulose. The model was regressed to the collected data to determine the values of the enzyme parameters and was found to satisfactorily predict the kinetics of cellulose hydrolysis. Cellobiose and glucose were identified as the strongest inhibitors of cellulase and beta-glucosidase, respectively. Experimental and modeling results are presented in light of the impact of enzymatic hydrolysis on fuel ethanol production. (c) 1993 Wiley & Sons, Inc.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Optimization of culture parameters for extracellular protease production from a newly isolated Pseudomonas sp. using response surface and artificial neural network models

Radial basis function (RBF) artificial neural network (ANN) and response surface methodology (RSM) were used to build a predictive model of the combined effects of independent variables (pH, temperature, inoculum volume) for extracellular protease production from a newly isolated Pseudomonas sp. The optimum operating conditions obtained from the quadratic form of the RSM and ANN models were pH 7.6, temperature 38 °C, and inoculum volume of 1.5 with 58.5 U/ml of predicted protease activity within 24 h of incubation. The normalized percentage mean squared error obtained from ANN and RSM models were 0.05 and 0.1%, respectively. The results demonstrated an higher prediction accuracy of ANN compared to RSM. This superiority of ANN over other multi factorial approaches could make this estimation technique a very helpful tool for fermentation monitoring and control.     

Botryosphaeran, a new substrate for the production of β-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the β-1,3;1,6-d-glucan type produced by B. rhodina) as sole carbon source with the objective of producing β-glucanases of the 1,3-type. Conditions for β-1,3-glucanase production by T. harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/l of EPS. Good agreement was obtained between the experimental values of β-1,3-glucanase activity and the corresponding values predicted by the mathematical model. The crude β-1,3-glucanase preparations were active towards a number of different β-1,3-glucans and β-glucosides. The mycelium of B. rhodina also proved to be a good substrate for β-1,3-glucanase production by both fungal species.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Homology modeling and molecular dynamics study of West Nile virus NS3 protease: a molecular basis for the catalytic activity increased by the NS2B cofactor

The West Nile virus (WNV) NS3 serine protease, which plays an important role in assembly of infective virion, is an attractive target for anti-WNV drug development. Cofactors NS2B and NS4A increase the catalytic activity of NS3 in dengue virus and Hepatitis C virus, respectively. Recent studies on the WNV-NS3 characterize the catalytically active form of NS3 by tethering the 40-residue cofactor NS2B. It is suggested that NS2B is essential for the NS3 activity in WNV, while there is no information of the WNV-NS3-related crystal structure. To understand the role of NS2B/substrate in the NS3 catalytic activity, we built a series of models: WNV-NS3 and WNV-NS3-NS2B and WNV-NS3-NS2B-substrate using homology modeling and molecular modeling techniques. Molecular dynamics (MD) simulations were performed for 2.75 ns on each model, to investigate the structural stabilization and catalytic triad motion of the WNV NS3 protease with and without NS2B/substrate. The simulations show that the NS3 rearrangement occurs upon the NS2B binding, resulting in the stable D75-OD1...H51-NH hydrogen bonding. After the substrate binds to the NS3-NS2B active site, the NS3 protease becomes more stable, and the catalytic triad is formed. These results provide a structural basis for the activation and stabilization of the enzyme by its cofactor and substrate.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

MalphaNP acid, a powerful chiral molecular tool for preparation of enantiopure alcohols by resolution and determination of their absolute configurations by the (1)H NMR anisotropy method

A novel methodology using a chiral molecular tool of MalphaNP acid (1), 2-methoxy-2-(1-naphthyl)propionic acid, useful for preparation of enantiopure secondary alcohols and determination of their absolute configurations by the (1)H NMR anisotropy method was developed; racemic MalphaNP acid (1) was enantioresolved with (-)-menthol, and the enantiopure MalphaNP acid (S)-(+)-(1) obtained was allowed to react with racemic alcohol, yielding a mixture of diastereomeric esters, which was clearly separated by HPLC on silica gel. By applying the sector rule of (1)H NMR anisotropy effect, the absolute configuration of the first-eluted MalphaNP ester was unambiguously determined. Solvolysis or reduction of the first-eluted MalphaNP esters yielded enantiopure alcohols.     

The Full-Length, Cytoplasmic C-Terminus of the β2-Adrenergic Receptor Expressed in E. coli Acts as a Substrate for Phosphorylation by Protein Kinase A, Insulin Receptor Tyrosine Kinase, GRK2, but Not Protein Kinase C and Suppresses Desensitization when Expressed in Vivo

The ability of the cytoplasmic, full-length C-terminus of the β2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS–PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the “activated” conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.