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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.141, #2-3)

Cloning, expression, and sequence analysis of diaminopropionate ammonia lyase gene from a nonvirulentsalmonella typhimurium PU011 by K. R. Rupesh; K. R. Padma; P. Saravanan; S. Jayachandran (pp. 161-174).

Background: Seeds ofLathyrus sativus, a legume plant, contain 3-oxalyl and 2,3-dioxalyl DAP (O-DAP), neurotoxins which when consumed causes Neurolathyrism or Osteolathyrism, in humans, affecting nervous system and bone formation respectively. Some microorganisms viz virulent and non-virulentSalmonella typhimurium, Salmonella typhi and Pseudomonad have been shown to detoxifyL-α,β-diaminopropionate (DAP), the immediate precursor of O-DAP. Result: The gene coding for diaminopropionate ammonia lyase (DAPAL) which detoxifies DAP was cloned from nonvirulentS. typhimurium PU011 intoEscherichia coli DH5α and the nucleotides sequenced (1212 bp). Whereas the specific enzyme activity of DAPAL obtained from recombinantE. coli PU018 was 0.346 U/mg, the specific activity of the enzyme from nonvirulentS. typhimurium PU011 was 0.351 U/mg. The DAPAL corresponding to 43 kDa protein was found both in nonvirulentS. typhimurium PU011 andE. coli PU018. The Km value was found to be 0.740 mM and 0.680 mM forS. typhimurium PU011 and 0.741 mM and 0.683 mM forE. coli PU018 when grown in minimal medium (MM+DAP) andL. sativus seed extracts respectively, indicating that both of them were capable of utilizing the neurotoxins present inL. sativus seeds. The biomass, enzyme production and the effect of pH and temperature on DAPAL enzyme activity from both non-virulentS. typhimurium PU011 andE. coli PU018 were found to be similar. Conclusion: The recombinantE. coli PU018 as well as non-virulentS. typhimurium PU011 are as good as pathogenicS. typhimurium in detoxifying DAP, the immediate precursor of O-DAP present inL. sativus seeds.

Keywords: Diaminopropionate ammonia lyase; Salmonella typhimurium, Lathyrus sativus ; diaminopropionic acid; neurolathyrism

Cloning, overexpression, purification, and characterization of receptor-interacting protein 3 truncation inEscherichia coli by Mi Suk Jeong; Jeong Soon Park; Se Bok Jang (pp. 175-186).

To facilitate structural studies of receptor-interacting protein 3 (RIP3), we developed a large-scale expression system of a glutathione-S-transferase (GST) fused with an 82 amino acid RIP3 protein inEscherichia coli. RIP3 truncation was subcloned into the pGEX-4T-1 vector and overexpressed in BL21(DE3)RIL cells. The soluble RIP3 protein was successfully purified to homogeneity using GST tag, an anion-exchange column, and gel filtration chromatography. The purity, identity, and conformation of the RIP3 protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, and fluorescence spectroscopic studies. RIP3 showed dominance of the α-helix structure and temperature-dependent conformational change.

Keywords: Subcloning; overexpression; purification; characterization; receptor-interacting protein 3

Optimization of keratinase production and enzyme activity using response surface methodology with streptomyces sp7 by Radhika Tatineni; Kiran Kumar Doddapaneni; Ravi Chandra Potumarthi; Lakshmi Narasu Mangamoori (pp. 187-201).

A two-step response surface methodology (RSM) study was conducted for the optimization of keratinase production and enzyme activity from poultry feather byStreptomyces sp7. Initially different combinations of salts were screened for maximal production of keratinase at a constant pH of 6.5 and feather meal concentration of 5 g/L. A combination of K₂HPO₄, KH₂PO₄, and NaCl gave a maximum yield of keratinase (70.9 U/mL) production. In the first step of the RSM study, the selected five variables (feather meal, K₂HPO₄, KH₂PO₄, NaCl, and pH) were optimized by a 2⁵ full-factorial rotatable central composite design (CCD) that resulted in 95 U/mL of keratinase production. The results of analysis of variance and regression of a second-order model showed that the linear effects of feather meal concentration (p<0.005) and NaCl (p<0.029) and the interactive effects of all variables were more significant and that values of the quadratic effects of feather meal (p<1.72e-5), K₂HPO₄ (p<4.731e-6), KH₂PO₄ (p<1.01e-10), and pH (p 7.63e-7) were more significant than the linear and interactive effects of the process variables. In the second step, a 2³ rotatable full-factorial CCD and response surface analysis were used for the selection of optimal process parameters (pH, temperature, and rpm) for keratinase enzyme activity. These optima were pH 11.0, 45°C, and 300 rpm.

Keywords: Poultry feather; keratinase; response surface methodology; central composite design

High-level expression of acidic partner-mediated antimicrobial peptide from tandem genes inEscherichia coli by Yun-Qi Wang; Ji-Ye Cai (pp. 203-213).

A novel strategy for constructing multiple joined genes of acidic partner-mediated antimicrobial peptide is described. This strategy allows the expression of antimicrobial peptide byEscherichia coli in a stable form and with high yield. Cecropin A (1–8)-melittin (1–10) (CAME) hybrid peptide was selected as a model of antimicrobial peptide. An acidic fragment from magainin intervening sequence was fused to the antimicrobial peptide as a partner to neutralize the lethal effects on the host cells. Multiple copies of the fusion peptide gene were tandemly linked and cloned into the expression vector pET21a. Multimers were expressed at high levels, reaching up to 36% of total cell proteins, and expression levels were proportional to the degree of multimerization. The fusion proteins were mainly expressed as inclusion bodies, probably owing to cysteine residues in the multimers. The target CAME peptide was obtained by cleaving the multimers with cyanogen bromide and purified by cation-exchange chromatography. Recombinant CAME peptide showed strong antimicrobial activities against both Gram-negative and -positive bacteria. These results might provide an efficient solution for high-level expression of various kinds of antimicrobial peptides that are toxic to the host.

Keywords: Antimicrobial peptide; expression; tandem genes; fusion protein; inclusion bodies

Cell immobilization and xylitol production using sugarcane bagasse as raw material by Silvio S. Silva; Solange I. Mussatto; Júlio C. Santos; Diego T. Santos; Juliana Polizel (pp. 215-227).

Sugarcane bagasse pretreated by three different procedures (with 2% [v/v] polyethyleneimine (PEI), with 2% [w/v] NaOH, or with a sequence of NaOH and PEI) was used as cell immobilization carrier for xylitol production byCandida guilliermondii yeast. Fermentations using these pretreated carriers were performed in semidefined medium and in a hydrolysate medium produced from sugarcane bagasse hemicellulose. Sugarcane bagasse pretreated with NaOH was the best carrier obtained with respect to immobilization efficiency, because it was able to immobilize a major quantity of cells (0.30 g of cells/g of bagasse). Fermentation in semidefined medium using the NaOH-pretreated carrier attained a high efficiency of xylose-to-xylitol bioconversion (96% of the theoretical value). From hydrolysate medium, the bioconversion efficiency was lower (63%), probably owing to the presence of other substances in the medium that caused an inadequate mass transfer to the cells. In this fermentation medium, better results with relation to xylitol production were obtained by using PEI-pretreated carrier (xylose-to-xylitol bioconversion of 81% of the theoretical and volumetric productivity of 0.43 g/[L·h]). The results showed that sugarcane bagasse is a low-cost material with great potential for use as cell immobilization carrier in the fermentative process for xylitol production.

Keywords: Sugarcane bagasse; immobilized cells; hemicellulosic hydrolysate; fermentation; xylitol

Statistical optimization of conditions for protease production fromBacillus sp. and its scale-up in a bioreactor by Saurabh Saran; Jasmine Isar; Rajendra Kumar Saxena (pp. 229-239).

A statistical approach, response surface methodology (RSM), was used to study the production of extracellular protease fromBacillus sp., which has properties of immense industrial importance. The most influential parameters for protease production obtained through the method of testing the parameters one at a time were starch, soybean meal, CaCl₂, agitation rate, and inoculum density. This method resulted in the production of 2543 U/mL of protease in 48 h fromBacillus sp. Based on these results, face-centered central composite design falling under RSM was employed to further enhance protease activity. The interactive effect of the most influential parameters resulted in a 1.50-fold increase in protease production, yielding 3746 U/mL in 48 h. Analysis of variance showed the adequacy of the model and verification experiments confirmed its validity. On subsequent scale-up in a 30-L bioreactor using conditions optimized through RSM, 3978 U/mL of protease was produced in 18 h. This clearly indicated that the model remained valid even on a large scale. RSM is a quick process for optimization of a large number of variables and provides profound insight into the interactive effect of various parameters involved in protease production.

Keywords: Bacillus sp.; fermentation; protease; response surface methodology; face-centered central composite design

Protective effects of a freeze-dried extract of vegetables and fruits on the hydroxyl radical-mediated oxidative damage of DNA and decrease of erythrocytes deformability by Hsiao-Ning Wang; Tsan-Zon Liu; Ya-Lei Chen; David Shiuan (pp. 241-249).

The protective effects of a freeze-dried extracts of vegetables and fruits (BauYuan; BY) on the hydroxyl radical-mediated DNA strand breakages and the structural integrity of human red blood cells (RBCs) were investigated. First, the supercoiled plasmid (pEGFP-C1) DNA was subjected to oxidation damage by an ascorbate-fortified Fenton reaction and the protective effects were analyzed by agarose gel electrophoresis. In the absence of BY extracts, exposure of the high-throughput ·OH-generating system (Fe²⁺ concentration >1.0 µM) caused a complete fragmentation of DNA. Supplementation of BY extract (1 mg/mL) to the plasmid DNA prior to the exposure could prevent it significantly. In contrast, as the plasmid exposed to a low-grade ·OH-generating system (Fe²⁺<0.1 µM), the BY extract (1 mg/mL) provided an almost complete protection. Next, the cell deformabilities were measured to assess the protection effects of various BY extracts on human erythrocytes exposed to the oxidative insults. We found that both the aqueous extract and the organic solvent-derived extracts could strongly protect human RBCs from the reactive oxygen species (ROS)-mediated decrease in the deformability indices. The results implicated that the BY extracts could effectively protect the cell membrane integrity via scavenging ROS which enabling RBCs to maintain a balance of water content and surface area to prevent the drop of cell deformability.

Keywords: Freeze-dried extract of vegetables and fruits; hydroxyl radical; oxidative damage; DNA; protective effects; deformability profile

Kinetic characterization of extracellular α-amylase from a derepressed mutant ofBacillus licheniformis by Ikram-ul-Haq; Hamad Ashraf; Sikander Ali; M. A. Qadeer (pp. 251-264).

Three strains ofBacillus licheniformis were isolated and screened for α-amylase production by solid-state fermentation. Of these, IS-2 gave relatively higher enzyme production (32±2.3 U/[g·min]) and was selected for improvement after treatment withN-methylN-nitroN-nitroso guanidine (NG) or nitrous acid (NA) to enhance its hydrolytic potential. Among the mutant variants, NA-14 gave higher enzyme production (98±1.6 U/[g·min]), and hence, was selected for kinetic and thermal characterization. M1 as a moistening agent (pH 7.0, optimized) supported 2.65-fold improved amylolytic activity by the derepressed mutant 72 h after inoculation. The values of product yield coefficient (Y _p/x=1833.3 U/g) and specific rate constant (q _p=25.46 U/[g·h]) with starch were severalfold improved over those from other carbon sources and the other cultures. The purified enzyme from NA-14 was most active at 40°C; however, the activity remained almost constant up to 44°C. The NA-induced random mutagenesis substantially improved the enthalpy (ΔH _D=94.5±11 kJ/mol) and entropy of activation (ΔS=−284±22 J/[mol·K]) for α-amylase activity and substrate binding for starch hydrolysis. The results of this study (117.8±5.5 U/[g·min]) revealed a concomitant improvement in the endogenous metabolism of the mutant culture for α-amylase production.

Keywords: Bacillus licheniformis ; α-amylase; mutant; solid-state fermentation; wheat bran; thermal characterization

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis by Bariş Binay; Nevin Gül Karagüler (pp. 265-272).

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺.The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.

Keywords: NAD⁺-dependent lactate dehydrogenase; substrate inhibition; protein engineering; biocatalysis; chiral hydroxyacids

Kinetic and thermodynamic study of a chemically modified highly active xylanase fromScopulariopsis sp by Ahmed Jawaard Afzal; Saleem Ahmed Bokhari; Khawar Sohail Siddiqui (pp. 273-297).

The amino groups of purified least acidic xylanase (LAX) isomer and carboxyl groups of purified highly acidic xylanase (HAX) isomer fromScopulariopsis sp. were chemically modified, resulting in charge neutralization and reversal. Modification of the second amino group was accompanied by the complete loss of enzyme activity in both the absence and presence of xylose. Multiple alignments of family 10 and 11 xylanases revealed that there is a pair of fully conserved Lys residues only in family 10 members. Xylanase structures from family 10 members showed that one of the conserved Lys residues is found near the active-site cleft that makes an H-bond with the substrate. The LAX and HAX isoenzymes in which one amino and three to four carboxyl groups were modified were subjected to kinetic and thermodynamic characterization. There were no differences in pH optima between the native and modified HAX, but there was a broadening of pH optimum toward the alkaline range for charge-neutralized LAX and a double pH optimum for charge-reversed LAX. TheV _max/K _m of both modified LAX and HAX decreased relative to the native species. The thermodynamics of xylan hydrolysis showed that the decrease in the catalytic activity of modified LAX enzymes was entropically driven. When compared with native enzyme, the thermostabilities of modified LAX enzymes increased in the presence and decreased in the absence of substrate. The thermodynamics of kinetic stability for modified LAX enzymes revealed that this increase in thermolability was owing to the decrease in ΔH# with a concomitant increase in ΔS# compared with native LAX. The thermostabilities of all the modified HAX species decreased except that of charge-neutralized HAX, whose half-life significantly increased in 50% (v/v) aqueous dioxan. These results suggest that the altered properties of the modified enzymes were a result of the conformational changes brought about by chemical modification.

Keywords: Fungus; family 10 xylanases; chemical modification; thermostability; structure-function-stability relationship

Microbial growth on pall rings by Åsa Tynell; Gunnar Börjesson; Margareta Persson (pp. 299-319).

Biogas is upgraded using an absorption with water-wash technique by 11 of a total of 14 upgrading plants in Sweden. However, problems with microbial growth on the pall rings in the absorption column, and in one case in the desorption column, have a negative impact on the upgrading of raw gas to vehicle gas. Five of the nine biogas plants studied here have experienced problems with microbial growth. The objectives of this study were to identify such microbial growth and to determine possible factors for its control, in order to provide recommendations for process management. A questionnaire was sent out and visits were made to the upgrading plants to collect information about the upgrading process. Phospholipid fatty acid (PLFA) analysis was performed to determine microbial biomass and community structure in samples from four upgrading plants. In samples from two of the plants, methane-oxidizing bacteria (type I methanotrophs) were indicated, while samples from one of the other plants showed biomarkers indicating actinomycetes. Factors affecting development of microbial growth were found to be water quality and the pH and temperature of the process water. Plants that used wastewater in the upgrading process experienced far more problems than those using clean water of drinking quality.

Keywords: Biogas upgrading; wastewater; actinomycetes; methanotrophs; phospholipid fatty acids

Expression and intein-mediated purification of novel staphylokinase SakSTAR with reduced immunogenicity and antiplatelet and antithrombin activation by Rafal Kochanowski; Roman Kotlowski; Piotr Szweda (pp. 321-333).

In an effort to combine the benefits of fibrinolytics, such as staphylokinase (Sak), with those of thrombin inhibitors for the prevention of vessel reocclusion after vascular injury, we produced chimeric protein with plasminogen activator and thrombin-inhibiting properties. This fusion protein was a construct consisting of Sak (SakSTAR) lengthened about 36 amino acids from the C-terminus end of hirudin. We inserted 16 point mutations into the sequence of the gene encoding SakSTAR for reduced antibody binding from 50% to about 17% and inserted two RGD sequences for antiplatelet activity. The inhibition rate of platelet aggregation was 27%. Moreover, we proposed an efficient method of expression and purification in which we used 16 mg/L of anEscherichia coli strain of this novel fusion protein and retained full biologic activities toward plasminogen and thrombin.

Keywords: Staphylokinase; plasminogen activator; intein-mediated purification; hirudin; thrombin

Effect of a nonmetabolizable analog of fructose-1,6-bisphosphate on glycolysis and ethanol production in strains ofSaccharomyces cerevisiae andEscherichia coli by Nhuan P. Nghiem; Timothy M. Cofer (pp. 335-347).

Fructose-1,6-bisphosphate (F-1,6-P2) is an allosteric activator of two key enzymes of glycolysis: phosphofructokinase and pyruvate kinase. Regulation of glycolysis in a wild-typeSaccharomyces cerevisiae and a recombinantEscherichia coli by a dead-end structural analog of F-1,6-P2 was studied. 2,5-Anhydromannitol (2,5-AM), a structural analog of β-d-fructose, was used. On being taken up by the cells, 2,5-AM was converted into its monophosphate and diphosphate by the enzymes of the glycolytic pathway. The final product, 2,5-anhydromannitol-1,6-bisphosphate, could not be metabolized further and, therefore, accumulated inside the cells. Glucose and fructose were used as substrates. It was found that 2,5-AM at concentrations of 1 mM or less did not have any effect on either substrate consumption or ethanol production. At concentrations of 2,5-AM of 2.5 mM or greater, significant inhibition of both glucose and fructose was observed, with fructose inhibition much more severe. We discuss the possible mechanisms of glycolysis inhibition by 2,5-AM at high concentrations and the regulation of glycolysis by this compound.

Keywords: Glycolysis regulation; Saccharomyces cerevisiae ; Escherichia coli ; β-d-fructose structural analog; 2,5-anhydromannitol; fructose-1, 6-bisphosphate

Differential display identifies genes in chinese hamster ovary cells sensitive to elevated ammonium by Peifeng Chen; Sarah W. Harcum (pp. 349-359).

Ammonium is a toxic waste product that has been reported to negatively inhibit cell growth and recombinant glycosylation in Chinese hamster ovary (CHO) cells; however, the effect of this toxicity on intracellular gene expression has received only limited investigation. We used a differential display method to identify genes in CHO cells that were affected by ammonium stress. Eight genes whose mRNA levels significantly changed in response to elevated ammonium were isolated and identified. Five of the genes were identified as having lower expression under the ammonium stress, whereas three genes were identified as having higher expression. Sequence homology with other mammalian organisms was used to attribute function to these newly identified genes. The identified ammonium-sensitive genes were grouped into three broad functional groups: cellular processes, energy metabolism, and genetic-information processing. The three cellular process-related genes had lower expression (anaphase-promoting complex subunit 5, eukaryotic initiation factor 5A II, KIAA1091 protein). The two energy-related genes had higher expression under ammonium stress (adenosine triphosphate synthase subunit C and mitofusin 1). Both of the genetic information-processing genes (endoplasmic reticulum [ER]-resident protein ERdj5 and structure-specific recognition protein 1) had lower expression under the ammonium stress, whereas the 26S proteasome subunit adenosine triphosphatase 3 gene had higher expression. These preliminary results indicate that ammonium stress lowers expression of genes controlling cell cycle, protein folding, and quality and raises genes that control energy metabolism and degradation. Our findings demonstrate the usefulness of mRNA differential-display techniques for the detection of CHO cell genes affected by ammonium stress.

Keywords: Gene expression; bioreactor stress; ammonium; differential display; Cricetulus griseus

Purification and characterization of an alkaline protease prot 1 frombotrytis cinerea by Ferid Abidi; Ferid Limam; M. Nejib Marzouki (pp. 361-376).

Alkaline thiol protease named Prot 1 was isolated from a culture filtrate ofBotrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a suitable and high thermostability. The optimal pH and temperature for activity were 9.0–10.0 and 60°C, respectively. This protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50°C over 120 min; it maintained 50% of activity after 60 min of heating at 60°C. Furthermore, the protease retained almost complete activity after 4 wk storage at 25°C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and, thus, the protease shows remarkable properties as a biodetergent catalyst.

Keywords: Alkaline protease; Botrytis cinerea ; purification; characterization; detergent enzyme

Screening and optimization of media constituents for enhancing lipolytic activity by a soil microorganism using statistically designed experiments by M. Ali Haider; K. Pakshirajan (pp. 377-390).

Soil contaminated with vegetable cooking oil was used in the isolation of a lipase-producing microorganism. The effectiveness of two different statistical design techniques in the screening and optimization of media constituents for enhancing the lipolytic activity of the soil microorganism was determined. The media constituents for lipase production by the isolated soil microorganism were screened using a Plackett-Burman design. Oil, magnesium sulfate, and ferrous sulfate were found to influence lipolytic activity at 24 and 72 h of culture with very high confidence levels. Whereas oil and ferrous sulfate showed a positive effect, magnesium sulfate indicated a negative effect on the lipolytic activity. A central composite design (CCD) followed by response surface methodology was used in optimizing these media constituents for enhancing the lipolytic activity. The regression model obtained for 72 h of lipolytic activity was found to be the best fit, withR ²=0.97, compared with the other model. An optimum combination at 9.3 mL/L of oil, 0.311 g/L of magnesium sulfate, and 0.007 g/L of ferrous sulfate in the media gave a maximum measured lipolytic activity of 7.1 U/mL at 72 h of culture. This increase in lipolytic activity was found to be 10.25% higher than the maximum experimentally observed value in the CCD.

Keywords: Lipases; soil microorganism; media optimization; Plackett-Burman design; response surface methodology; central composite design

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.141, #2-3)