Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.165, #3-4)

Large-Scale Production of the Immunomodulator c-di-GMP from GMP and ATP by an Enzymatic Cascade by Volker Spehr; Ralf Warrass; Klaus Höcherl; Thomas Ilg (pp. 761-775).

(3′–5′)-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a “one-pot” three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1β, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.

Keywords: C-di-GMP; Diguanylate cyclase; Enzymatic cascade; Immunomodulation; Biotechnological production

Investigation of the Effective Action Distance Between Hematopoietic Stem/Progenitor Cells and Human Adipose-Derived Stem Cells During Their In Vitro Co-culture by Kedong Song; Hai Wang; Hong Wang; Ling Wang; Mo Qiao; Shuang Wu; Tianqing Liu (pp. 776-784).

The in vitro suitable action distance between umbilical cord blood-derived hematopoietic stem/progenitor cells and its feeder cell, human adipose-derived stem cells, during their co-culture, was investigated through a novel transwell co-culture protocol, in which the distance between the two culture chambers where each cell type is growing can be adjusted from 10 to 450 μm. The total cell number was determined with a hemacytometer, and the cell morphology was observed under an inverted microscope each day. After 7 days of co-culture, the fold-expansion, surface antigen expression of CD34⁺ and CFU-GM assay of the hematopoietic mononuclear cells (MNCs) were analyzed. The results showed that there was an optimal communication distance at around 350 μm between both types of stem cells during their in vitro co-culture. By using this distance, the UCB-MNCs and CD34⁺ cells were expanded by 15.1 ± 0.2 and 5.0 ± 0.1-fold, respectively. It can therefore be concluded that the optimal action distance between stem cells and their supportive cells, when cultured together for 7 days, is of around 350 μm.

Keywords: Adipose-derived stem cells; Hematopoietic stem/progenitor cells; Transwell; Co-culture; Expansion

Isolation, Characterization, and Catalytic Properties of a Novel Lipase Which Is Activated in Ionic Liquids and Organic Solvents by Neda Akbari; Somayeh Daneshjoo; Jafer Akbari; Khosro Khajeh (pp. 785-794).

A novel extracellular lipase with organic solvent tolerance was isolated from a local Pseudomonas species. The lipase gene was cloned and expressed in Escherichia coli as a heterologous host and purified by affinity chromatography. The activity of purified lipase was investigated in the presence of imidazolium-based ionic liquids (ILs) such as EMIM[Cl], BMIM[Cl], and HMIM[Cl]. It has been found that the activity of treated lipase with ILs was higher than untreated control in the hydrolysis reaction. Also, the results indicated that the enzymatic activity strongly depends on IL concentration in reaction media. The best concentration of the IL was 30%, 45%, and 50% (v/v) for HMIM[Cl], BMIM[Cl], and EMIM[Cl], respectively. Additionally, the enzyme exhibited excellent stability in the presence of 25% of n-hexane, toluene, acetone, and t-butanol. The optimum values of pH and temperature were determined 10 and 55 °C, respectively. The K _m and V _max values were calculated 0.4 mM and 1.92 U/ml, respectively, using p-nitrophenyl palmitate as substrate. With respect to the biochemical properties of the newly isolated lipase such as high-level stability and noticeable activity in the presence of organic solvents and ionic liquids, the newly isolated lipase seems to be a good candidate for environmental and industrial processes carried out in non-aqueous media.

Keywords: Lipase; Biochemical characterization; Organic solvent; Ionic liquid; Pseudomonas sp.

Enhanced Activity and Enantioselectivity of a Hyperthermophilic Esterase from Archaeon Aeropyrum pernix K1 by Acetone Treatment by Fangdi Cong; Kezhi Xing; Renjun Gao; Shugui Cao; Guirong Zhang (pp. 795-801).

To improve the activity and enantioselectivity of hyperthermophilic archaeon Aeropyrum pernix K1 esterase (APE1547) and its mutants, they were purified by acetone-treated method. It was found that the acetone treatment not only caused APE1547 and its mutants to display higher activity and enantioselectivity but also saved more than 90% of time spent in purifying them by Ni-chelating column. In hydrolysis of p-nitrophenyl caprylate, the acetone-treated APE1547 and mutant A containing the following substitutions R11G, L36P, V225A, I551L, and A564T showed 5.7- and 6.9-fold active increase, respectively. In the resolution of 2-octanol acetate, the acetone-treated mutant A had a 9-fold enantioselective increase relative to that purified by Ni-chelating column. In addition, the impact of pH, temperature, and chemical reagents on activity of APE1547 and mutant A was discussed in this paper.

Keywords: Catalytic activity; Enantioselectivity; Hyperthermophilic esterase; 2-Octanol acetate; Acetone treatment

Analysis of the Amino Acids of Soy Globulins by AOT Reverse Micelles and Aqueous Buffer by Xiaoyan Zhao; Jun Chen; Zhifang Lu; Xiangqing Ling; Peng Deng; Qingjun Zhu; Fangling Du (pp. 802-813).

The 7S and 11S globulins from soybean proteins using reverse micelle and aqueous buffer extraction methods were characterized by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscope (SEM), and their amino acid compositions were also evaluated. SDS-PAGE did not show electrophoretic differences between 7S and 11S globulin subunits with two extraction methods. SEM analysis showed that the AOT reverse micelle processing of 7S and 11S globulins induced a reduction of droplet size. Some individual amino acid contents of 7S and 11S globulins using two extraction methods were different, some were similar. In all the samples, the glutamic acid, aspartic acid, and leucine were the dominant amino acids while the cystine and methionine were the first-limiting amino acids. The proportion of essential amino acids to the total amino acids (E/T) of the 7S globulin from aqueous buffer and reverse micelles was similar. While significant differences were obtained in the proportion of E/T of the 11S globulin.

Keywords: Reverse micelles; Soybean; 7S and 11S globulins; Amino acids

Evaluation of Ethanol Production from Corncob Using Scheffersomyces (Pichia) stipitis CBS 6054 by Volumetric Scale-up by Jae-Won Lee; J. Y. Zhu; Danilo Scordia; Thomas W. Jeffries (pp. 814-822).

In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric acid. At same pretreatment condition, the highest glucose yield obtained from pretreated biomass by enzymatic hydrolysis was about 76%, based on a glucan content of 37/100 g. In hydrolysate obtained by pretreatment, glucose concentration was low, while xylose concentration was significantly increased above 0.368% of sulfuric acid. At 1.656% of sulfuric acid, xylose and glucose concentration was highest. In subsequent, fermentation with hydrolysate, maximal ethanol yield was attained after 24 h with 0.368% of sulfuric acid. The fermentation efficiency of hydrolysate obtained by enzymatic hydrolysis reached a maximum of 75% at an acid charge of 0.368%.

Keywords: Pretreatment; Enzymatic hydrolysis; Corncob; Ethanol

An Improved Procedure for the Purification of Catalytically Active Alkane Hydroxylase from Pseudomonas putida GPo1 by Meng Xie; Hernan Alonso; Anna Roujeinikova (pp. 823-831).

Bacterial alkane hydroxylases are of high interest for bioremediation applications as they allow some bacteria to grow in oil-contaminated environments. Furthermore, they have tremendous biotechnological potential as they catalyse the stereo- and regio-specific hydroxylation of chemically inert alkanes, which can then be used in the synthesis of pharmaceuticals and other high-cost chemicals. Despite their potential, progress on the detailed characterization of these systems has so far been slow mainly due to the lack of a robust procedure to purify its membrane protein component, monooxygenase AlkB, in a stable and active form. This study reports a new method for isolating milligramme amounts of recombinant Pseudomonas putida GPo1 AlkB in a folded, catalytically active form to purity levels above 90%. AlkB solubilised and purified in the detergent lauryldimethylamine oxide was demonstrated to be active in catalysing the epoxidation reaction of 1-octene with an estimated K _m value of 0.2 mM.

Keywords: Biocatalysis; Alkane hydroxylase; Biomodification of hydrocarbons; Alkane biodegradation

Reduction of Enzyme Dosage by Oxygen Delignification and Mechanical Refining for Enzymatic Hydrolysis of Green Liquor-Pretreated Hardwood by Bon-Wook Koo; Trevor H. Treasure; Hasan Jameel; Richard B. Phillips; Hou-min Chang; Sunkyu Park (pp. 832-844).

In this study, a strategy to reduce enzyme dosage is evaluated by applying two post-treatments, oxygen delignification and mechanical refining. The sugar conversion for GL12 substrates was increased from 51.5% to 77.9% with post-treatments at the enzyme dosage of 10 FPU. When the amount of enzyme was reduced to 5 FPU with post-treatments, the conversion of 71.8% was obtained, which was significant higher than the conversion without any post-treatment using 10 FPU (51.5%). This clearly demonstrates the benefit of post-treatments that allows more than 50% of enzyme reduction at the same level of enzymatic conversion. Enzyme-accessible surface area and pore volume were evaluated by Simons' staining and DSC thermoporometry methods, and strong correlations were found with the sugar conversion.

Keywords: Green liquor pretreatment; Post-treatment; Mechanical refining; Oxygen delignification; Reduction of enzyme dosage; Enzyme accessibility

Improvement of l-Arginine Production by Overexpression of a Bifunctional Ornithine Acetyltransferase in Corynebacterium crenatum by Wenfang Dou; Meijuan Xu; Dongmei Cai; Xiaomei Zhang; Zhiming Rao; Zhenghong Xu (pp. 845-855).

Ornithine acetyltransferase (EC 2.3.1.35; OATase) gene (argJ) from the l-arginine-producing mutant Corynebacterium crenatum SYPA5-5 was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). Analysis of the argJ sequence revealed that the argJ coded a polypeptide of 388 amino acids with a calculated molecular weight of 39.7 kDa. In this study, the function of the OATase (argJ) of C. crenatum SYPA5-5 has been identified as a conserved ATML sequence for the autolysis of the protein to α- and β-subunits. When the argJ regions corresponding to the α- and β-subunits were cloned and expressed separately in E. coli BL21, OATase activities were abolished. At the same time, a functional study revealed that OATase from C. crenatum SYPA5-5 was a bifunctional enzyme with the functions of acetylglutamate synthase (EC 2.3.1.1, NAGS) and acetylornithine deacetylase (EC 3.5.1.16, AOase) activities. In order to investigate the effects of the overexpression of the argJ gene on l-arginine production, the argJ gene was inserted into pJCtac to yield the recombinant shuttle plasmid pJCtac-argJ and then transformed into C. crenatum SYPA5-5. The results showed that the engineered strains could not only express more OATase (90.9%) but also increase the production of l-arginine significantly (16.8%).

Keywords: l-Arginine; Corynebacterium crenatum ; argJ; Ornithine acetyltransferases,; Overexpression

Searching of Predictors to Predict pH Optimum of Cellulases by Shaomin Yan; Guang Wu (pp. 856-869).

The optimal working conditions for enzymes are very much elegant, and their determination is often through experimental approach, which generally is costly and time-consuming. Therefore, it is important to develop methods to use as simple as possible information to predict the optimal working condition for enzymes. Cellulase is a very important enzyme widely used in industries. In this study, we attempted to use a 20–1 feedforward backpropagation neural network to screen 24 amino acid properties related to the primary structure of cellulases as predictors to predict the pH optimum in cellulases. The results show that some predictors can predict the pH, especially amino acid distribution probability.

Keywords: Cellulase; Enzyme; pH optimum; Prediction

Scale-up of Ethanol Production from Winter Barley by the EDGE (Enhanced Dry Grind Enzymatic) Process in Fermentors up to 300 l by Nhuan P. Nghiem; Frank Taylor; David B. Johnston; Jay K. Shetty; Kevin B. Hicks (pp. 870-882).

A fermentation process, which was designated the enhanced dry grind enzymatic (EDGE) process, has recently been developed for barley ethanol production. In the EDGE process, in addition to the enzymes normally required for starch hydrolysis, commercial β-glucanases were used to hydrolyze (1,3)(1,4)-β-d-glucans to smaller molecules, thus reducing the viscosity of the mash to levels sufficiently low to allow transport and mixing in commercial equipment. Another enzyme, a developmental β-glucosidase, then was used to hydrolyze the resulting oligomers to glucose, which subsequently was fermented to produce additional ethanol. The EDGE process was developed with Thoroughbred, a winter hulled barley, using a shake flask model. To move toward commercialization, it is necessary to prove that the developed process would be applicable to other barley varieties and also to demonstrate its scalability. Experiments were performed in 7.5, 70, and 300-l fermentors using Thoroughbred and Eve, a winter hull-less barley. It was shown that the process was scalable for both barley varieties. Low levels of glucose throughout the course of the fermentations demonstrated the high efficiency of the simultaneous saccharification and fermentation process. Final ethanol concentrations of 14% (v/v) were achieved for initial total solids of 28.5–30% (w/w), which gave an ethanol yield of 83–87% of the theoretical values. The distillers dried grains with solubles co-products contained very low levels of β-glucans and thus were suitable for use in feed formulations for all animal species.

Keywords: Ethanol fermentation; Winter barley; β-Glucans; Process scale-up; Biorefinery

Effects of Propionic Acid and pH on Ethanol Fermentation by Saccharomyces cerevisiae in Cassava Mash by Cheng-Ming Zhang; Li Jiang; Zhong-Gui Mao; Jian-Hua Zhang; Lei Tang (pp. 883-891).

The effects of propionic acid on ethanol and glycerol production by Saccharomyces cerevisiae in cassava mash were examined along with the influence of pH (4.0, 5.0, and 6.0) and of dissolved solids content (22%, 25%, and 27%). Inhibition by propionic acid increased as solids content increased and medium pH declined. Complete inhibition of ethanol fermentation was observed in mashes at pH 4.0 (60 mM propionic acid for 22% solids and 45 mM for 25% and 27%). Glycerol production linearly decreased with increased undissociated propionic acid concentration in all mashes at all pH levels, which partly contributed to increased final ethanol production when propionic acid concentration in mashes was low (≤30 mM).

Keywords: Cassava; Ethanol; Glycerol; Propionic acid; S. cerevisiae

Molecular Characterization of N-Terminal Pro-sequence of Keratinase Ker P from Pseudomonas aeruginosa: Identification of Region with Chaperone Activity by Richa Sharma; N. Apurva Ratan Murty; Rani Gupta (pp. 892-901).

In silico analysis of keratinase Ker P from Pseudomonas aeruginosa revealed that its full gene of 1,497 bp constituted of a 72-bp signal sequence along with a long 520 bp pro-sequence and 905 bp core region. Position specific multiple sequence alignment of Ker P protein with other distant proteases revealed high variability within their N-terminal regions while the core protein was considerably conserved. Ker P (F1) and its four N-terminal truncations (F2-F5) lacking 72, 177, 405, 507 bp, respectively, were cloned and constitutively expressed as extracellular protein in pEZZ-18 secretory vector with Escherichia coli HB101 as the expression host. Ker P F1, Ker P F2, Ker P F3 and Ker P F4 products were active whereas no keratinolytic activity was obtained in Ker P F5. Further analysis revealed that only 187 bp pro-sequence region is required for correct folding of the protein into its active conformation and, thus, has chaperone-like activity. Further, comparative biochemical characterization revealed that the full-length keratinase Ker P F1 was catalytically more efficient than the truncated forms. Among the truncated enzymes, keratinase Ker P F4 exhibited better thermostability than Ker P F2 with a t_1/2 of >1 h at 60 °C. It also had a higher V _max and K _m on casein as compared with Ker P F2. However, no significant variation was observed with respect to kinetics on synthetic substrates.

Keywords: Keratinase; N-terminal truncation; Pseudomonas aeruginosa ; Pro-sequence; pEZZ 18

Anticoagulant, Antiherpetic and Antibacterial Activities of Sulphated Polysaccharide from Indian Medicinal Plant Tridax procumbens L. (Asteraceae) by Shabeena Yousuf Naqash; R. A. Nazeer (pp. 902-912).

The sulphated polysaccharide from the widespread Tridax procumbens plant was studied for the anticoagulant, antiherpetic and antibacterial activity. The anticoagulant activity was determined by the activated partial thromboplastin time assay. The sulphated polysaccharide from T. procumbens represented potent anticoagulant reaching the efficacy to heparin and chondroitin sulphate. Moreover, the sulphated polysaccharide extracted from T. procumbens was found non-toxic on Vero cell lines up to the concentration of 200 μg/ml. Sulphated polysaccharide exhibited detectable antiviral effect towards HSV-1 with IC₅₀ value 100–150 μg/ml. Furthermore, sulphated polysaccharide from T. procumbens was highly inhibitory against the bacterial strains Vibrio alginolyticus and Vibrio harveyi isolated from oil sardine.

Keywords: Tridax procumbens ; Sulphated polysaccharide; aPTT; HSV-I

Induction of a High-Yield Lovastatin Mutant of Aspergillus terreus by ¹²C⁶⁺ Heavy-Ion Beam Irradiation and the Influence of Culture Conditions on Lovastatin Production Under Submerged Fermentation by Shi-Weng Li; Mei Li; Hong-Ping Song; Jia-Li Feng; Xi-Sheng Tai (pp. 913-925).

Heavy-ion beams, possessing a wide mutation spectrum and increased mutation frequency, have been used effectively as a breeding method. In this study, the heavy-ion beams generated by the Heavy-Ion Research Facility in Lanzhou were used to mutagenize Aspergillus terreus CA99 for screening high-yield lovastatin strains. Furthermore, the main growth conditions as well as the influences of carbon and nitrogen sources on the growth and the lovastatin production of the mutant and the original strains were investigated comparatively. The spores of A. terreus CA99 were irradiated by 15, 20, 25, and 30 Gy of 80 MeV/u ¹²C⁶⁺ heavy-ion beams. Based on the lovastatin contents in the fermentation broth, a strain designated as A. terreus Z15-7 has been selected from the clone irradiated by the heavy-ion beam. When compared with the original strain, the content of lovastatin in the fermentation broth of A. terreus Z15-7 increased 4-fold. Moreover, A. terreus Z15-7 efficiently used the carbon and nitrogen sources for the growth and production of lovastatin when compared to the original strain. The maximum yield of lovastatin, 916.7 μg/ml, was obtained as A. terreus Z15-7 was submerged cultured in the chemically defined medium supplemented with 3% glycerol as a carbon source, 1% corn meal as an organic nitrogen source, and 0.2% sodium nitrate as an inorganic nitrogen source at 30 °C in the shake flask. The result shows that heavy-ion beam irradiation is an effective method for the mutation breeding of lovastatin production of A. terreus.

Keywords: Aspergillus terreus ; Heavy-ion beam; Mutation breeding; Lovastatin

Co-Expression of an Organic Solvent-Tolerant Lipase and its Cognate Foldase of Pseudomonas aeruginosa CS-2 and the Application of the Immobilized Recombinant Lipase by Ren Peng; Jinping Lin; Dongzhi Wei (pp. 926-937).

The genes of CS-2 lipase and its cognate foldase were cloned from Pseudomonas aeruginosa CS-2. A stop codon was not found in the lipase gene. The amino acid sequence deduced from the lipase gene from P. aeruginosa CS-2 showed 97.8%, 71.3%, and 71.2% identity with lipases from P. aeruginosa LST-03, P seudomonas mendocina ymp, and Pseudomonas stutzeri A1501, respectively. The co-expression of CS-2 lipase and its cognate foldase of P. aeruginosa CS-2 in E scherichia coli BL21 (DE3) resulted in the formation of a soluble lipase. The recombinant lipase and foldase were purified to homogeneity using nickel affinity chromatography and about 10.2-fold with 40.9% recovery was achieved for the purification of the recombinant lipase. The molecular masses of the lipase and the foldase were estimated to be 35.7 and 38.3 kDa in SDS-PAGE, respectively. The recombinant lipase showed stability in the presence of some organic solvents. The recombinant CS-2 lipase was immobilized and subsequently used for the synthesis of butyl acetate in heptane. The conversion of substrate decreased from 98.2% to 87.4% after 5 cycles in reuse of the immobilized lipase.

Keywords: Lipase; Foldase; Pseudomonas aeruginosa ; Butyl acetate

C–S Targeted Biodegradation of Dibenzothiophene by Stenotrophomonas sp. NISOC-04 by Moslem Papizadeh; Mohammad Roayaei Ardakani; Hossein Motamedi; Iraj Rasouli; Mohammad Zarei (pp. 938-948).

Crude oil-contaminated soil samples were gathered across Khuzestan oilfields (National Iranian South Oil Company, NISOC) consequently experienced a screening procedure for isolating C–S targeted dibenzothiophene-biodegrading microorganisms with previously optimized techniques. Among the isolates, a bacterial strain was selected due to its capability of biodegrading dibenzothiophene in a C–S targeted manner in aqueous phases and medium mostly consisting of separately biphasic water–gasoline. The 16S rDNA of the isolate was amplified using eubacterial-specific primers and then sequenced. Based on sequence data analysis, the microorganism, designated NISOC-04, clustered most closely with the members of the genus Stenotrophomonas. Gas chromatography indicated that Stenotrophomonas sp. NISOC-04 utilizes 82% of starting 0.8 mM dibenzothiophene within a 48-h-long exponential growth phase. Growth curve analysis revealed the inability of Stenotrophomonas sp. NISOC-04 to utilize dibenzothiophene (DBT) as the exclusive carbon or carbon/sulfur source. Gibbs’ assay showed no 2-hydroxy biphenyl accumulation, but HPLC confirmed the presence of 2-hydroxy biphenyl as the final product of DBT desulfurization. Under sulfur starvation, Stenotrophomonas sp. NISOC-04 produced a huge biomass with untraceable sulfur and utilized atmospheric insignificant sulfur levels.

Keywords: Biodesulfurization; Dibenzothiophene; Mineralization; Stenotrophomonas ; C–S targeted biodegradation

Expression and Characterization of a Novel Lipase from Aspergillus fumigatus with High Specific Activity by Jiao-Jiao Shangguan; Yu-Qiang Liu; Fu-Jun Wang; Jian Zhao; Li-Qiang Fan; Su-Xia Li; Jian-He Xu (pp. 949-962).

A novel lipase gene from Aspergillus fumigatus, afl1-1, was cloned and expressed with a molecular mass of 38 kDa in Escherichia coli for the first time. The recombinant lipase had a preference for short carbon chain p-nitrophenyl esters, especially toward C2 p-nitrophenyl ester and exhibited potent hydrolysis activity that had not been observed. The optimum pH and temperature of this new enzyme were 8.5 and 65 °C, respectively. The recombinant lipase (AFL1-1) is an alkaline enzyme which was stable in the pH range 6.0∼8.5 for 16 h (at 4 °C) and at 30∼50 °C for 1 h. It is an intracellular enzyme which was purified approximately 8.47-fold with an overall yield of 86.1% by single-step Ni-NTA affinity purification, with a very high specific activity of approximately 1.00 × 10³ U mg⁻¹ on a standard substrate of p-nitrophenyl acetate. The Michaelis–Menten kinetic parameters V _max and K _m of the lipase were 1.37 mM mg⁻¹ min⁻¹ and 14.0 mM, respectively. Ca²⁺ and other metal ions could not activate the lipase. According to the homology analysis and site-directed mutagenesis assay, the catalytic triad of the recombinant lipase was identified as Ser-165, Asp-260, and His-290 residues.

Keywords: Aspergillus fumigatus ; Recombinant lipase; Expression; Characterization; Active site

Isolation, Identification, and Culture Optimization of a Novel Glycinonitrile-Hydrolyzing Fungus—Fusarium oxysporum H3 by Jin-Song Gong; Zhen-Ming Lu; Jing-Song Shi; Wen-Fang Dou; Hong-Yu Xu; Zhe-Min Zhou; Zheng-Hong Xu (pp. 963-977).

Microbial transformation of glycinonitrile into glycine by nitrile hydrolase is of considerable interest to green chemistry. A novel fungus with high nitrile hydrolase was newly isolated from soil samples and identified as Fusarium oxysporum H3 through 18S ribosomal DNA, 28S ribosomal DNA, and the internal transcribed spacer sequence analysis, together with morphology characteristics. After primary optimization of culture conditions including pH, temperature, carbon/nitrogen sources, inducers, and metal ions, the enzyme activity was greatly increased from 326 to 4,313 U/L. The preferred carbon/nitrogen sources, inducer, and metal ions were glucose and yeast extract, caprolactam, and Cu²⁺, Mn²⁺, and Fe²⁺, respectively. The maximum enzyme formation was obtained when F. oxysporum H3 was cultivated at 30 °C for 54 h with the initial pH of 7.2. There is scanty report about the optimization of nitrile hydrolase production from nitrile-converting fungus.

Keywords: Fusarium oxysporum ; Glycinonitrile; Identification; Nitrile hydrolase; Optimization

Differential Proteins of the Optic Ganglion in Octopus vulgaris Under Methanol Stress Revealed Using Proteomics by Lin Huang; Qing-Yu Huang; Hai-Bin Chen; Fu-Sheng Huang; He-Qing Huang (pp. 978-988).

An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0–8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

Keywords: Methanol; Biomarkers; Proteomics; Optic ganglia; Octopus vulgaris

Improving the Thermostability of a Methyl Parathion Hydrolase by Adding the Ionic Bond on Protein Surface by Yidan Su; Jian Tian; Ping Wang; Xiaoyu Chu; Guoan Liu; Ningfeng Wu; Yunliu Fan (pp. 989-997).

The thermostability of the methyl parathion hydrolase (MPH_OCH) from Ochrobactrum sp. M231 was improved using site-directed mutagenesis. Two prolines (Pro76 and Pro78) located on the protein surface were selected for mutations after inspection of the sequence alignment of MPH_OCH and OPHC2, a thermostable organophosphorus hydrolase from Pseudomonas pseudoalcaligenes C2-1. The temperature of the double-point mutant (P76D/P78K) at which the mutant lost 50% of its activity (T50) was approximately 68 °C, which is higher than that of WT enzyme (64 °C), P76D (67 °C), and P78K (59 °C). Structural analysis of P76D/P78K indicated that the substituted residues (Asp76 and Lys78) could generate an ionic bond and increase the structural electrostatic energy, which could then increase the stability of the protein. These results also suggest that the thermal stability of proteins could be improved by adding the ionic bond on protein surface.

Keywords: Methyl parathion hydrolase; Site-directed mutagenesis; Thermostability; Ionic bond

Dissolution and Delignification of Bamboo Biomass Using Amino Acid-Based Ionic Liquid by Nawshad Muhammad; Zakaria Man; Mohamad Azmi Bustam; M. I. Abdul Mutalib; Cecilia D. Wilfred; Sikander Rafiq (pp. 998-1009).

In the present work, the dissolution of bamboo biomass was tested using a number of ionic liquids synthesized in laboratory. It was observed that one of the synthesized amino acid-based ionic liquids, namely 1-ethyl-3-methylimidazolium glycinate, was capable of dissolving the biomass completely. The dissolved biomass was then regenerated using a reconstitute solvent (acetone/water) and was characterized using Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy. The results were compared to preconditioned bamboo biomass. The regenerated biomass was found to have a more homogenous macrostructure, which indicates that the crystalline form and structure of its cellulose has changed from type Ι to type ΙΙ during the dissolution and regeneration process.

Keywords: Bamboo biomass; Amino acid ionic liquid; Dissolution; Regeneration; Cellulose crystallinity

Plasma-Assisted Pretreatment of Wheat Straw for Ethanol Production by Nadja Schultz-Jensen; Zsófia Kádár; Anna Belinda Thomsen; Henrik Bindslev; Frank Leipold (pp. 1010-1023).

The potential of wheat straw for ethanol production after pretreatment with O₃ generated in a plasma at atmospheric pressure and room temperature followed by fermentation was investigated. We found that cellulose and hemicellulose remained unaltered after ozonisation and a subsequent washing step, while lignin was degraded up to 95% by O₃. The loss of biomass after washing could be explained by the amount of lignin degraded. The washing water of pretreated samples (0–7 h) was analyzed for potential fermentation inhibitors. Approximately 30 lignin degradation products and a number of simple carboxylic acids and phenolic compounds were found, e.g., vanillic acid, acetic acid, and formic acid. Some components had the highest concentration at the beginning of the ozonisation process (0.5, 1 h), e.g., 4-hydroxybenzladehyde, while the concentration of others increased during the entire pretreatment (0–7 h), e.g., oxalic acid and acetovanillon. Interestingly, washing had no effect on the ethanol production with pretreatment times up to 1 h. Washing improved the glucose availability with pretreatment times of more than 2 h. One hour of ozonisation was found to be optimal for the use of washed and unwashed wheat straw for ethanol production (maximum ethanol yield, 52%). O₃ cost estimations were made for the production of ethanol at standard conditions.

Keywords: Pretreatment method; Wheat straw; Lignin degradation products; Fermentation inhibitors; Enzymatic hydrolysis; Simultaneous saccharification and fermentation

Relatively High-Substrate Consistency Hydrolysis of Steam-Pretreated Sweet Sorghum Bagasse at Relatively Low Cellulase Loading by Fei Shen; Yuehua Zhong; Jack N. Saddler; Ronghou Liu (pp. 1024-1036).

Sweet sorghum bagasse (SSB) was steam pretreated in the conditions of 190 °C for 5 min to assess its amenability to the pretreatment and enzymatic hydrolysis. Results showed that pretreatment conditions were robust enough to pretreat SSB with maximum of 87% glucan and 72% xylan recovery. Subsequent enzymatic hydrolysis showed that the pretreated SSB at 2% substrate consistency resulted in maximum of 70% glucan–glucose conversion. Increasing substrate consistency from 2% to 16% led to a significant reduction in glucan conversion. However, the decrease ratio of glucan–glucose conversion was the minimum when the consistency increased from 2% to 12%. When the pretreated SSB consistency of 12% was applied for hydrolysis, increase in cellulase loading from 7.5 up to 20 filter paper units (FPU)/g glucan resulted only in 14% increase in glucan–glucose conversion compared to 20% increase with cellulase loading varying from 2.5 to 7.5 FPU/g glucan. More than 10 cellobiase units (CBU)/g glucan β-glucosidase supplementation had no noticeable improvement on glucan–glucose conversion. Additionally, supplementation of xylanase was found to significantly increase glucan–glucose conversion from 50% to 80% with the substrate consistency of 12%, when the cellulase and β-glucosidase loadings were at relatively low enzyme loadings (7.5 FPU/g and 10 CBU/g glucan). It appeared that residual xylan played a critical role in hindering the ease of hydrolysis of SSB. A proper xylanase addition was suggested to achieve a high hydrolysis yield at relatively high substrate consistency with relatively low enzyme loadings.

Keywords: Sweet sorghum bagasse; Steam pretreatment; Relatively high substrate consistency; Enzymatic hydrolysis; Xylanase addition

In silico Analysis of Molecular Mechanisms of Galanthus nivalis Agglutinin-Related Lectin-Induced Cancer Cell Death from Carbohydrate-Binding Motif Evolution Hypothesis by Qi-jia Yu; Zi-yue Li; Shun Yao; Miao Ming; Shu-ya Wang; Bo Liu; Jin-ku Bao (pp. 1037-1046).

Galanthus nivalis agglutinin-related lectins, a superfamily of strictly mannose-binding-specific lectins widespread amongst monotyledonous plants, have drawn a rising attention for their remarkable anti-proliferative and apoptosis-inducing activities toward various types of cancer cells; however, the precise molecular mechanisms by which they induce tumor cell apoptosis are still only rudimentarily understood. Herein, we found that the three conserved motifs “QXDXNXVXY,” the mannose-specific binding sites, could mutate at one or more amino acid sites, which might be a driving force for the sequential evolution and thus ultimately leading to the complete disappearance of the three conserved motifs. In addition, we found that the motif evolution could result in the diversification of sugar-binding types that G. nivalis agglutinin-related lectins could bind from specific mannose receptors to more types of sugar-containing receptors in cancer cells. Subsequently, we indicated that some sugar-containing receptors such as TNFR1, EGFR, Hsp90, and Hsp70 could block downstream anti-apoptotic or survival signaling pathways, which, in turn, resulted in tumor cell apoptosis. Taken together, our hypothesis that carbohydrate-binding motif evolution may impact the G. nivalis agglutinin-related lectin-induced survival or anti-apoptotic pathways would provide a new perspective for further elucidating the intricate relationships between the carbohydrate-binding specificities and complex molecular mechanisms by which G. nivalis agglutinin-related lectins induce cancer cell death.

Keywords: Galanthus nivalis agglutinin-related lectin; Evolution; Cancer; Apoptosis; Carbohydrate-binding motif

Biochemical Characterization of Two Truncated Forms of Amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 to Identify Its Enzymatically Active Region by Fu-Pang Lin; Hsiu-Yen Ma; Hui-Ju Lin; Shiu-Mei Liu; Wen-Shyong Tzou (pp. 1047-1056).

The enzymatically active region of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 (TsaNTOU1Apu) was identified by truncation mutagenesis. Two truncated TsaNTOU1Apu enzymes, TsaNTOU1ApuM957 and TsaNTOU1ApuK885, were selected and characterized. Both TsaNTOU1ApuM957 and TsaNTOU1ApuK885 showed similar specific activities toward various substrates. The overall catalytic efficiency (k _cat/apparent K _m) for the soluble starch or pullulan substrate, however, was 20–25% lower in TsaNTOU1ApuK885 than in TsaNTOU1ApuM957. Both truncated enzymes exhibited similar thermostability and substrate-binding ability against the raw starch. The fluorescence and circular dichroism spectrometry studies indicated that TsaNTOU1ApuK885 retained an active folding conformation similar to that of TsaNTOU1ApuM957. These results indicate that a large part of the TsaNTOU1Apu, such as the C-terminal carbohydrate-binding module family 20, the second fibronectin type III, and a portion of the first FnIII motifs, could be removed without causing a serious aberrant structural change or a dramatic decrease in hydrolysis of soluble starch and pullulan.

Keywords: Thermoanaerobacterium saccharolyticum NTOU1; Amylopullulanase; Truncation mutagenesis; Circular dichroism

Cellulase Production by Aspergillus japonicus URM5620 Using Waste from Castor Bean (Ricinus communis L.) Under Solid-State Fermentation by Polyanna Nunes Herculano; Tatiana Souza Porto; Keila Aparecida Moreira; Gustavo A. S. Pinto; Cristina Maria Souza-Motta; Ana Lúcia F. Porto (pp. 1057-1067).

The activity of β-glucosidase (βG), total cellulase (FPase) and endoglucanase (CMCase), produced by Aspergillus japonicus URM5620, was studied on solid-state fermentation using castor bean meal as substrate. The effect of the substrate amount, initial moisture, pH, and temperature on cellulase production was studied using a full factorial design (2⁴). The maximum βG, FPase, and CMCase activity was 88.3, 953.4, and 191.6 U/g dry substrate, respectively. The best enzyme activities for all three enzymes were obtained at the same conditions with 5.0 g of substrate, initial moisture 15% at 25 °C and pH 6.0 with 120 h of fermentation. The optimum activity for FPase and CMCase was found at pH 3.0 at an optimum temperature of 50 °C for FPase and of 55 °C for CMCase. The cellulases were stable in the range of pH 3.0–10.0 at 50 °C temperature. The enzyme production optimization demonstrated clearly the impact of the process parameters on the yield of the cellulolytic enzymes.

Keywords: Aspergillus japonicus ; Cellulolytic activities; Castor bean meal; Solid-state fermentation; Optimum activity; Stability

Enhanced Production of Citric Acid in Yarrowia lipolytica by Triton X-100 by Maryam Mirbagheri; Iraj Nahvi; Giti Emtiazi; Farshad Darvishi (pp. 1068-1074).

Various chemical surfactants could affect permeability of yeast cells. In this study, effects of the surfactant addition upon yeast cells permeability and citric acid (CA) production by Yarrowia lipolytica strains DSM 3286 and M7 were investigated. The addition of Triton X-100 increased 1.4–1.8-fold of the maximum CA quantity achieved for both strains, with final CA concentrations ranging between 75–85 g/l that correspond to CA conversion yields per unit of glucose consumed of ~0.80–0.84 g/g. Scanning electron micrographs of yeast cells showed that the cells treated with Triton X-100 had altered cell structure and were smaller and narrower compared with the non-treated ones. The results showed that Triton X-100 could be used in order to increase the efficiency of CA production by Y. lipolytica strains.

Keywords: Yarrowia lipolytica ; Citric acid; Triton X-100; Surfactant; Permeabilization

Protective Effect of Withania somnifera and Cardiospermum halicacabum Extracts Against Collagenolytic Degradation of Collagen by Krishnamoorthy Ganesan; Praveen Kumar Sehgal; Asit Baran Mandal; Sadulla Sayeed (pp. 1075-1091).

The irreversible destruction of extracellular matrix (ECM) such as cartilage, tendon, and bone that comprise synovial joints is the hallmark of both rheumatoid arthritis and osteoarthritis by over-expression of matrix metalloproteinase (MMP)-collagenases. We report herein the detailed study on the inhibitory effects of Withania somnifera extract (WSE) and Cardiospermum halicacabum extract (CHE) on Clostridium histolyticum collagenase (ChC) activity against the degradation of the ECM component of bovine Achilles tendon type I collagen by hydroxyproline assay method. Interaction of WSE and CHE with ChC exhibited 71% and 88% inhibition, respectively, to the collagenolytic activity of ChC against collagen degradation, and the inhibition was found to be concentration-dependent. The inhibition kinetics of ChC by both the extracts has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala. Both WSE and CHE are provided competitive and mixed type inhibition on ChC activity, respectively. Circular dichroism studies of ChC on treatment with WSE and CHE revealed changes in the secondary structure of collagenase. These results suggest that the WSE and CHE facilitated collagen stabilization through collagenase inhibition.

Keywords: Extracellular matrix; Matrix metalloproteinases; Collagen; Collagenase; Collagenase inhibition kinetics

Proteomic Analysis of Differential Protein Expression of Achilles Tendon in a Rabbit Model by Two-Dimensional Polyacrylamide Gel Electrophoresis at 21 Days Postoperation by Jiasharete Jielile; Ainuer Jialili; Gulnur Sabirhazi; Nuerai Shawutali; Darebai Redati; Jiangtao Chen; Bin Tang; Jingping Bai; Kayrat Aldyarhan (pp. 1092-1106).

Postoperative early kinesitherapy has been advocated as an optimal method for treating Achilles tendon rupture. However, an insight into the rationale of how early kinesitherapy contributes to healing of Achilles tendon remains to be achieved, and research in the area of proteomic analysis of Achilles tendon has so far been lacking. Forty-two rabbits were randomized into control group, immobilization group, and early motion group, and received postoperative cast immobilization and early motion treatments. Achilles tendon samples were prepared 21 days following microsurgery, and the proteins were separated with two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were first recognized by PDQuest software, and then identified using peptide mass fingerprinting, tandem mass spectrometry, and database searching. A total of 463 ± 12, 511 ± 39, and 513 ± 80 protein spots were successfully detected in the two-dimensional polyacrylamide gels for the Achilles tendon samples of rabbits in the control group, immobilization group, and early motion group, respectively. There were 15, 8, and 9 unique proteins in these three groups, respectively, and some differentially expressed proteins were also identified in each group. It was indicated that some of the differentially expressed proteins were involved in various metabolism pathways and may play an important role in healing of Achilles tendon rupture. Postoperative early kinesitherapy resulted in differentially expressed proteins in ruptured Achilles tendon compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to healing of Achilles tendon rupture through a mechanobiological mechanism due to the application of postoperative early kinesitherapy.

Keywords: Achilles tendon rupture; “Pa” bone suture; Postoperative early kinesitherapy; Cast immobilization; Centrifugal stress; Mechanobiology; Two-dimensional electrophoresis

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.165, #3-4)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.165, #3-4)