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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #3)

Characterization of a Thermostable Family 1 Glycosyl Hydrolase Enzyme from Putranjiva roxburghii Seeds by Girijesh Kumar Patel; Bibekananda Kar; Ashwani Kumar Sharma (pp. 523-535).

A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic studies showed that enzyme hydrolyzed p-nitrophenyl β-d-glucopyranoside (pNP-Glc) with higher efficiency (K _cat/K _m = 2.27 × 10⁴ M⁻¹ s⁻¹) as compared to p-nitrophenyl β-d-galactopyranoside (pNP-Gal; K _cat/K _m = 1.15 × 10⁴ M⁻¹ s⁻¹). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range 30–65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min⁻¹. Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity and higher catalytic efficiency can be utilized for different commercial applications.

Keywords: Putranjiva roxburghii ; Euphorbiaceae ; β-glucosidase; β-galactosidase; Kinetic studies; CD studies

Production and Characteristics of the Whole-Cell Lipase from Organic Solvent Tolerant Burkholderia sp. ZYB002 by Zheng-Yu Shu; Ji-Guang Wu; Lan-Xing Cheng; De Chen; Yong-Mei Jiang; Xin Li; Jian-Zhong Huang (pp. 536-548).

The thermostable and organic solvent tolerant whole-cell lipase (WCL) was produced by Burkholderia sp. ZYB002 with broad spectrum organic solvent tolerance. The production medium of the WCL was primarily optimized, which resulted in the maximum activity of 22.8 U/mL and the 5.1-fold increase of the WCL yield. The optimized culture medium was as follows (% w/v or v/v): soybean meal 2, soybean oil 0.5, manganese sulfate 0.1, K₂HPO₄ 0.1, olive oil 0.5, initial pH 6.0, inoculum density 2, liquid volume 35 mL in 250-mL Erlenmeyer flask, and incubation time 24 h. The biochemical characterization of the WCL from Burkholderia sp. ZYB002 was determined, and the results showed that the optimal pH and temperature for lipolytic activity of the WCL was 8.0 and 65°C, respectively. The WCL was stable at temperature up to 70°C for 1 h and retained 79.2% of its original activity. The WCL was highly stable in the pH range from 3.0 to 8.5 for 6 h. Ca²⁺, K⁺, Na⁺, NO ₃ ⁻ , etc. ions stimulated its lipolytic activity, whereas Zn²⁺ ion caused inhibition effect. The WCL was also relatively stable in n-butanol at a final concentration of 50% (v/v) for 24 h. However, the WCL was strongly inhibited in Triton X-100 at a final concentration of 10% (v/v). The WCL with thermal resistance and organic solvent tolerance showed its great potential in various green industrial chemical processes.

Keywords: Burkholderia sp. ZYB002; Whole cell lipase; Organic solvent tolerance; Production; Enzyme characteristics

Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR by Yan Liao; Min Zeng; Zhen-fang Wu; Hui Chen; Hong-ning Wang; Qi Wu; Zhi Shan; Xue-yi Han (pp. 549-562).

The mutant acid phytase (phyA m) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NP^ep-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyA ep gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower k _m value than that of PP-NP^m-8 (P < 0.05). In addition, the overall catalytic efficiency (k _cat/k _m) of the mutant one was 84% higher (P < 0.05) than that of PP-NP^m-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.

Keywords: Phytase; phyA m gene; Directed evolution; Error-prone PCR; Enzyme activity

A Laccase of Fomes durissimus MTCC-1173 and Its Role in the Conversion of Methylbenzene to Benzaldehyde by R. Sahay; R. S. S. Yadav; Sudha Yadava; K. D. S. Yadav (pp. 563-575).

A laccase has been purified from the liquid culture growth medium containing bagasse particles of Fomes durissimus. The method involved concentration of the culture filtrate by ultrafiltration and anion exchange chromatography on diethyl aminoethyl cellulose. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis both gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the purified laccase determined from SDS-PAGE analysis was 75 kDa. Using 2,6-dimethoxyphenol as the substrate, the determined K _m and k _cat values of the laccase are 182 μM and 0.35 s⁻¹, respectively, giving a k _cat/K _m value of 1.92 × 10³ M⁻¹ s⁻¹. The pH and temperature optimum were 4.0 and 35 °C, respectively. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover, it transformed methylbenzene to benzaldehyde in the absence of mediator molecules, property exhibited by yellow laccases.

Keywords: Yellow laccases; Fomes durissimus ; Metalloenzymes; Lignolytic fungi; Lignolytic enzymes; 2, 6-Dimethoxyphenol

Soybean Hulls Pretreated Using Thermo-Mechanical Extrusion—Hydrolysis Efficiency, Fermentation Inhibitors, and Ethanol Yield by Juhyun Yoo; Sajid Alavi; Praveen Vadlani; Keith C. Behnke (pp. 576-589).

Soybean hulls were subjected to thermo-mechanical extrusion pretreatment at various in-barrel moisture contents and screw speeds. Extrusion degraded the lignocellulosic structure and enhanced enzymatic hydrolysis of soybean hulls, with up to 155% increase in glucose yield as compared to untreated substrate. Greater glucose yields were observed at higher in-barrel moistures (45% and 50%) and lower screw speed (280 and 350 rpm). Maximum 74% cellulose to glucose conversion resulted from using a two-enzyme cocktail consisting of cellulase and β-glucosidase. Conversion increased to 87% when a three-enzyme cocktail having a cell wall degrading enzyme complex was used for hydrolysis. Fermentation inhibitors, such as furfural, 5-(hydroxymethyl)-2-furaldehyde (HMF), and acetic acid, were found in the extrusion pretreated soybean hulls and hydrolysate. However, their concentrations were below the known thresholds for inhibition. Fermentation of hydrolysate by Saccharomyces cerevisiae led to high yields of ethanol, with concentration ranging from 13.04 to 15.44 g/L.

Keywords: Extrusion; Pretreatment; Soybean hulls; Hydrolysis; Inhibitors; Ethanol

Construction of a Genetically Engineered Microorganism that Simultaneously Degrades Organochlorine and Organophosphate Pesticides by Jijian Yang; Ruihua Liu; Wenli Song; Yao Yang; Feng Cui; Chuanling Qiao (pp. 590-598).

Field contamination with pesticide mixtures of organophosphates (OPs) and organochlorines (OCs) is becoming global issues to be solved urgently. The strategy of utilizing engineered microorganisms that have an ability to simultaneously degrade OPs and OCs has increasingly received great interest. In this work, an OP degradation gene (mpd) and an OC degradation gene (linA) were simultaneously introduced into Escherichia coli by using two compatible plasmids, resulting in strains with both OP degradation and OC degradation capabilities. To overcome the potential substrate uptake limitation, MPH was displayed on the cell surface of Escherichia coli using the N- and C-terminal domains of ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC–MPH was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, both LinA and green fluorescent protein (GFP) were functionally co-expressed in the MPH-displaying Escherichia coli. The engineered Escherichia coli degraded OPs as well as OCs rapidly, and it can be easily monitored by GFP fluorescence.

Keywords: Organophosphate; Organochlorine; Co-contamination; Green fluorescent protein; Biodegradation

Enhanced β-Galactosidase Production from Whey Powder by a Mutant of the Psychrotolerant Yeast Guehomyces pullulans 17–1 for Hydrolysis of Lactose by Jin-Li Xu; Jun Zhao; Ling-Fei Wang; Huai-Yong Sun; Chun-Li Song; Zhen-Ming Chi (pp. 599-611).

In order to isolate β-galactosidase overproducers of the psychrotolerant yeast Guehomyces pullulans 17–1, its cells were mutated by using nitrosoguanidine (NTG). One mutant (NTG-133) with enhanced β-galactosidase production was obtained. The mutant grown in the production medium with 30.0 g/l lactose and 2.0 g/l glucose could produce more β-galactosidase than the same mutant grown in the production medium with only 30.0 g/l lactose while β-galactosidase production by its wild type was sensitive to the presence of glucose in the medium. It was found that 40.0 g/l of the whey powder was the most suitable for β-galactosidase production by the mutant. After optimization of the medium and cultivation conditions, the mutant could produce 29.2 U/ml of total β-galactosidase activity within 132 h at the flask level while the mutant could produce 48.1 U/ml of total β-galactosidase activity within 144 h in 2-l fermentor. Over 77.1% of lactose in the whey powder (5.0% w/v) was hydrolyzed in the presence of the β-galactosidase activity of 280 U/g of lactose within 9 h while over 77.0% of lactose in the whey was hydrolyzed in the presence of β-galactosidase activity of 280 U/g of lactose within 6 h. This was the first time to show that the β-galactosidase produced by the psychrotolerant yeast could be used for hydrolysis of lactose in the whey powder and whey.

Keywords: Guehomyces pullulans ; β-Galactosidase; Whey; Whey powder; The psychrotolerant yeast

Tandem Multimer Expression and Preparation of Hypoglycemic Peptide MC6 from Momordica charantia in Escherichia coli by Fu-Jun Wang; Huai-Lei Song; Xiao-Meng Wang; Wen-Ji Zhang; Bi-Lian Wang; Jian Zhao; Zhi-Bi Hu (pp. 612-619).

Tandem repeat multimers of Momordica charantia (MC) peptide MC6 were designed and the recombinant plasmid containing 10 copies of MC6 gene was constructed to improve the expression level of MC6 in Escherichia coli. Under the selected conditions of cultivation and induction, the expression level of recombinant TrxA–MC6₁₀ protein was above 25% of total bacteria protein. This fusion protein was purified and cleaved with HCl (13%, w/v). Either the un-cleaved or cleaved recombinant proteins was analyzed pharmacological activity by alloxan-induced diabetic mice and only the cleaved products of the recombinant protein showed significant hypoglycemic effects. The study provides a convenient and economical method for the large-scale production of anti-diabetic medicines for pharmaceutical applications.

Keywords: Momordica charantia ; Hypoglycemic peptide MC6; Multimer; Isocaudamer

Lactobacillus plantarum AS1 Isolated from South Indian Fermented Food Kallappam Suppress 1,2-Dimethyl Hydrazine (DMH)-Induced Colorectal Cancer in Male Wistar Rats by R. Satish Kumar; P. Kanmani; N. Yuvaraj; K. A. Paari; V. Pattukumar; C. Thirunavukkarasu; V. Arul (pp. 620-631).

The relationship between antioxidant and anticancer properties of probiotic bacterium strain Lactobacillus plantarum AS1 (AS1) in colon cancer induced by 1,2-dimethylhydrazine (DMH) has been studied. In this study, an increased level of lipid peroxide (LPO) products and increased activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione-S transferase) and marker enzymes (alkaline phosphatase and acid phosphatase) in colon and plasma of cancer-bearing animals have been observed. AS1 was supplemented either before initiation or during initiation and selection/promotion phases of colon carcinogenesis and was found to be effective in altering lipid peroxidation and antioxidant enzyme activities and marker enzymes to a statistically significant level measured either in the colon and in the plasma. These alterations inclined towards normal in a time-dependent manner on AS1 supplementation. The mean tumor volume diameter and total number of tumors were found to be statistically decreased in AS1 pre- and post-treated rats. Furthermore, histopathological examination shows remarkable difference between control and treated groups. The in vitro antioxidant assay shows that AS1 has promising antioxidant property. These results demonstrate that AS1 strain can modulate the development of DMH-induced rat colon carcinogenesis through an antioxidant-dependent mechanism.

Keywords: Lactobacillus plantarum AS1; Colorectal cancer; 1,2-Dimethylhydrazine; Probiotics

Effect of Extracorporeal Shockwave Treatment on the Melanogenic Activity of Cultured Melanocytes by Faye Huang; Hsi-Kung Kuo; Ching-Hua Hsieh; Pei-Chang Wu; Yi-Chan Wu; Ching-Jen Wang (pp. 632-639).

In addition to the traditional lithotripsy treatment, extracorporeal shockwaves (ESWs) have been shown to be effective in the treatment of certain musculoskeletal disorders and in enhancing skin flap neovascularization. However, relatively little is known about its effect on melanocytes. To investigate its effect on the melanogenic activity of cultured melanocytes, mouse B16F10 melanocytes were treated with defocused ESWs of different energies (15, 21, and 27 kV) and at different doses (300 and 600 impulses). Cell viability was measured 1 and 24 h after treatment. Melanin content was measured and compared against a standard curve generated with fungal melanin. Cellular tyrosinase activity was calculated with the 3,4-dihydroxyphenylalanine (DOPA) oxidase assay. The results demonstrated that ESW treatment reduced cell viability. Our results also indicated that the overall decrease in cell viability lasted for 6 days. After ESW treatment with 300 or 600 impulses at 21 kV, no significant change in melanin content or tyrosinase activity of the B16F10 melanocytes was noted as compared to those of the control. The present study suggests that ESW treatment does not alter the melanogenic activity of the cultured melanocytes.

Keywords: Extracorporeal shockwave; Melanocytes; Cell viability; Melanin content

Expression, Purification, and Antibacterial Activity of Bovine Lactoferrampin–Lactoferricin in Pichia pastoris by Xiang-Shan Tang; Zhi-Ru Tang; Sheng-Ping Wang; Ze-Meng Feng; Dong Zhou; Tie-Jun Li; Yu-Long Yin (pp. 640-651).

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N₁ domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA–LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA–LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA–LFA–LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA–LFC peptide was purified via two-step column chromatography and identified by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA–LFC. The fermentation supernate and the purified LFA–LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA–LFC in P. pastoris and may have potential practical applications in microbial peptide production.

Keywords: Bovine lactoferricin; Bovine lactoferampin; Antimicrobial peptides; Antibacterial activity; Pichia pastoris ; Pigs; Nutrition; Amino acids

Cloning of Thermostable Cellulase Genes of Clostridium thermocellum and Their Secretive Expression in Bacillus subtilis by Jian-Ming Liu; Xiu-Juan Xin; Chun-Xiu Li; Jian-He Xu; Jie Bao (pp. 652-662).

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.

Keywords: Clostridium thermocellum ; Bacillus subtilis ; pP43JM2; Cellulase genes; Synergy

Purification and Biochemical Characterization of a Highly Thermostable Xylanase from Actinomadura sp. Strain Cpt20 Isolated from Poultry Compost by Zina Taibi; Boudjemaa Saoudi; Mokhtar Boudelaa; Héla Trigui; Hafedh Belghith; Ali Gargouri; Ali Ladjama (pp. 663-679).

An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption–ionization time-of-flight mass spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase activity were pH 10 and 80 °C, respectively. This xylanase was stable within a pH range of 5–10 and up to a temperature of 90 °C. It showed high thermostability at 60 °C for 5 days and half-life times at 90 °C and 100 °C were 2 and 1 h, respectively. The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan. This enzyme obeyed the Michaelis–Menten kinetics, with the K _m and k _cat values being 1.55 mg soluble oat-spelt xylan/ml and 388 min⁻¹, respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn²⁺, Ca²⁺, and Cu²⁺, it was, strongly inhibited by Hg²⁺, Zn²⁺, and Ba²⁺. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.

Keywords: Actinomadura ; Xylan; Xylanase; Purification; MALDI-TOF MS; Thermostability

Analysis of the Production Process of Optically Pure d-Lactic Acid from Raw Glycerol Using Engineered Escherichia coli Strains by John A. Posada; Carlos A. Cardona; Ramon Gonzalez (pp. 680-699).

Glycerol has become an ideal feedstock for producing fuels and chemicals. Here, five technological schemes for optically pure d-lactic acid production from raw glycerol were designed, simulated, and economically assessed based on five fermentative scenarios using engineered Escherichia coli strains. Fermentative scenarios considered different qualities of glycerol (pure, 98 wt.%, and crude, 85 wt.%) with concentrations ranging from 20 to 60 g/l in the fermentation media, and two fermentation stages were also analyzed. Raw glycerol (60 wt.%) was considered as the feedstock feeding the production process in all cases; then a purification process of raw glycerol up to the required quality was required. Simulation processes were carried out using Aspen Plus, while economic assessments were performed using Aspen Icarus Process Evaluator. D-Lactic acid recovery and purification processes were based on reactive extraction with tri-n-octylamine using dichloromethane as active extractant agent. The use of raw glycerol represents only between 2.4% and 7.8% of the total production costs. Also, the total production costs obtained of D-lactic acid in all cases were lower than its sale price indicating that these processes are potentially profitable. Thus, the best configuration process requires the use of crude glycerol diluted at 40 g/l with total glycerol consumption and with D-lactic acid recovering by reactive extraction. The lowest obtained total production cost was 1.015 US$/kg with a sale price/production cost ratio of 1.53.

Keywords: D-Lactic acid production; Engineered Escherichia coli ; Glycerol bioconversion; Process assessment; Process design; Process simulation

Interspecies Interaction of Signal Peptide PapR Secreted by Bacillus cereus and Its Effect on Production of Antimicrobial Peptide by In-Cheol Yeo; Nam Keun Lee; Chang-Jun Cha; Young Tae Hahm (pp. 700-710).

This study was carried out to investigate the interspecies interaction of PapR peptide secreted by Bacillus cereus on production of BSAP-254, an antimicrobial peptide produced by Bacillus subtilis SC-8 isolated from the Korean fermented soybean paste and exhibited narrow antagonistic activity against the B. cereus group, but not against other foodborne pathogens. PapR is a signal peptide that activates PlcR, which is a pleiotropic regulator controlling the expression of various virulence factors in B. cereus. When B. subtilis SC-8 was co-cultured with B. cereus, it completely inhibited the growth of B. cereus within 12 h, and the rate of BSAP-254 production was increased 34.2% at 12 h. Furthermore, 5 μM of synthetic PapR peptide added to the culture of B. subtilis SC-8 increased the rate of BSAP-254 production up to 59.7%. The growth of B. subtilis SC-8, however, was not significantly different with or without the addition of PapR. When B. cereus papR mutant was co-cultured with B. subtilis SC-8, the growth of the mutant was not inhibited and the rate of BSAP-254 production was decreased by 45%.

Keywords: Bacillus cereus group; PapR; Bacillus subtilis ; Antimicrobial peptide; Interspecies interaction

Chemometric Analysis with Near-Infrared Spectroscopy for Chemically Pretreated Erianthus toward Efficient Bioethanol Production by Yoshiki Horikawa; Tomoya Imai; Rie Takada; Takashi Watanabe; Keiji Takabe; Yoshinori Kobayashi; Junji Sugiyama (pp. 711-721).

In this paper, we report the combination of a near-infrared (NIR) spectroscopic method with multivariate analysis in order to develop a calibration model of the saccharification ratio of chemically pretreated Erianthus. The regression models clearly depend on the NIR spectral regions, and the information of CH and aromatic framework vibrations contributed most effectively to the alkaline dataset. From interpretations of the regression coefficient, lignin and cellulose were negatively and positively correlated with the saccharification ratio, respectively, and this result was supported by the data from wet chemical analysis. A more complex dataset was obtained from varied chemical pretreatments; here, the saccharification ratio was either small or had no linear correlation with each structural monocomponent. These results enabled the successful construction of the PLS regression model. NIR spectroscopy can be a rapid screening method for the saccharification ratio, and furthermore, can provide information of the key factors influencing the realization of more efficient enzymatic accessibility.

Keywords: Near-infrared (NIR) spectroscopy; Partial least-squares (PLS) regression; Regression coefficient; Saccharification ratio

Heterologous Expression of a Nelumbo nucifera Phytochelatin Synthase Gene Enhances Cadmium Tolerance in Arabidopsis thaliana by Zhaolei Liu; Chunsun Gu; Fadi Chen; Dongyin Yang; Kunwei Wu; Sumei Chen; Jiafu Jiang; Zhen Zhang (pp. 722-734).

Phytochelatin synthase (PCS) is a key enzyme involved in the synthesis of phytochelatins, which are thought to play important roles in heavy metal detoxification. The sacred lotus (Nelumbo nucifera), one of the most popular ornamental species, has been shown to be a potential phytoremediator of heavy metal polluted water. However, the phytochelatin synthase gene in N. nucifera has not been identified yet. Here, we report the isolation and function characterization of a N. nucifera homologue of phytochelatin synthase. The sequence obtained shares a high degree of similarity with PCSs from other plant species and was named as Nelumbo nucifera phytochelatin synthase1 (NnPCS1). By using quantitative RT-PCR, we found that the expression of NnPCS1 in leaves of N. nucifera was dramatically increased in response to Cadmium (Cd) treatment. We further showed that, when exposed to Cd stress, Arabidopsis transgenic plants heterologous expressing NnPCS1 accumulated more Cd when compared with wild type. These results suggest that NnPCS1 involved in the response of N. nucifera to Cd stress and may represent a useful target gene for the phytoremediation of Cd-polluted water.

Keywords: Nelumbo nucifera ; Phytochelatin synthase; Phytoremediation; Cadmium stress; Arabidopsis

Further Stabilization of Leu¹⁵⁵ Mutant Thermolysins by Mutation of an Autodegradation Site by Yoshiki Matsumiya; Naoko Murata; Kuniyo Inouye; Motoki Kubo (pp. 735-743).

The autodegradation-resistant mutant thermolysins (TLNs), L155A (Leu¹⁵⁵ to Ala) and L155S (Leu¹⁵⁵ to Ser), were previously constructed by site-directed mutagenesis to enhance thermostability. These mutations suppressed autodegradation at position 154–155, resulting in increased thermostability. However, a new autodegradation site became apparent in these mutant TLNs, at position 155–156. In this study, further stabilization of the mutant TLNs to suppress this new autodegradation was attempted by the substitution of Ile¹⁵⁶ to Asp and Val (L155A-I156N, L155A-I156V, L155S-I156N, and L155S-I156V). SDS–PAGE analysis showed that the autodegradation at 155–156 of all double-mutant TLNs was suppressed. Thermostability at 80 °C was enhanced in all double-mutant TLNs (half-life at 80 °C: WT, 18.3 min; L155A, 25.0 min; L155S, 24.0 min; L155A-I156N, 60.8 min; L155A-I156V, 62.4 min; L155S-I156N, 93.3 min; and L155S-I156V, 40.0 min), and k _cat/K _m values were: WT, 220; L155A, 240; L155S, 123; L155A-I156N, 62; L155A-I156V, 760; L155S-I156N, 240; and L155S-I156V, 520 min⁻¹ mM⁻¹.

Keywords: Thermolysin; Autodegradation; Thermostability; Protease; Bacillus

Recycling of Cellulosic Fibers by Enzymatic Process by K. M. Shojaei; F. Dadashian; M. Montazer (pp. 744-752).

In this research, enzymatic treatment as an environmental friendly process has been used for recycling process of old cellulosic wastes such as cotton, viscose, and lyocell. Cellulase hydrolyses cellulosic chains and shortens cellulosic fibers. This study investigates to detect the optimum enzyme concentration and time of treatments for suitable changes of length and weight loss. The main purposes of this article are shortening of cellulosic fibers and evaluating of enzymatic treatment in different kind of cellulosic fibers. According to the data of experiments, with the increase of enzyme concentration and the treatment time, the length and weight loss percentage of the cellulosic fibers has been decreased. The length and weight loss percentage of treated viscose is more than that of lyocell and cotton fibers. Optimized condition, reaction time, and enzyme concentration have been determined by mean length of treated cellulosic samples. Suitable longitudinal distribution of fiber for papermaking industries is in the range of 0 to 4 mm. Optimum enzyme concentration and treatment time for recycling cotton, lyocell, and viscose fibers are 2% and 48 h for cotton and lyocell and 0.5% and 48 h for viscose, respectively. According to the data of experiment, the length of treated fibers is appropriate for its usage as a raw material in papermaking industries.

Keywords: Recycling; Cellulosic fiber; Cellulose; Enzyme; Weight loss; Waste; Fiber shortening

Identification of Potential Markers Related to Neoadjuvant Chemotherapy Sensitivity of Breast Cancer by SELDI-TOF MS by Kun Zhang; Kai Yuan; Hongyan Wu; Qing Li; Yulong Wang; Shouhua Chen; Lili Zhang; He Gu; Rongzhan Fu (pp. 753-763).

Neoadjuvant chemotherapy (NACT) is known to be beneficial for patients with locally advanced breast cancer. However, there is still no unified standard on the evaluation of NACT. To identify the potential markers related to NACT sensitivity of breast cancer, in the present study, we examined the protein spectrum of breast cancer tissues before and after NACT using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Totally, 87 protein samples were extracted from tissues of breast cancer, with 30 from patients before NACT, 30 from patients after NACT, and 27 from patients without any treatment. To eliminate confounding factors a couple of tissue samples from the same patient were mixed. SELDI-TOF MS analysis demonstrated that the intensities of eight different protein peaks, i.e., 26,055.46, 17,898.94, 8,949.50, 11,652.02, 11,053.48, 38,546.56, 5,825.89, and 22,250.63 Da, were higher in samples after NACT than those before NACT. Although further experiments are needed to prove the reliability of the proteins identified in this study, our results will help the establishment of protein model based on drug resistance-related protein peaks to screen whether a patient is suitable for adopting NACT and to improve cancer treatment.

Keywords: Breast cancer; Tissue; Neoadjuvant chemotherapy; Biomarker; Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

A Hydrogen Peroxide Biosensor Based on Direct Electrochemistry of Hemoglobin in Palladium Nanoparticles/Graphene–Chitosan Nanocomposite Film by Aili Sun; Qinglin Sheng; Jianbin Zheng (pp. 764-773).

Thermally two-dimensional lattice graphene (GR) and biocompatibility chitosan (CS) act as a suitable support for the deposition of palladium nanoparticles (PdNPs). A novel hydrogen peroxide (H₂O₂) biosensor based on immobilization of hemoglobin (Hb) in thin film of CS containing GR and PdNPs was developed. The surface morphologies of a set of representative membranes were characterized by means of scanning electron microscopy and showed that the PdNPs are of a sphere shape and an average diameter of 50 nm. Under the optimal conditions, the immobilized Hb showed fast and excellent electrocatalytic activity to H₂O₂ with a small Michaelis–Menten constant of 16 μmol L⁻¹, a linear range from 2.0 × 10⁻⁶ to 1.1 × 10⁻³ mol L⁻¹, and a detection limit of 6.6 × 10⁻⁷ mol L⁻¹. The biosensor also exhibited other advantages, good reproducibility, and long-term stability, and PdNPs/GR–CS nanocomposites film would be a promising material in the preparation of third generation biosensor.

Keywords: Direct electrochemistry; Nanocomposite; Hemoglobin; Hydrogen peroxide; Graphene

Identification and Molecular Characterization of a Novel DyP-Type Peroxidase from Pseudomonas aeruginosa PKE117 by Jing Li; Chen Liu; BaoZhen Li; HongLi Yuan; JinShui Yang; BeiWen Zheng (pp. 774-785).

A new DyP-type peroxidase from Pseudomonas aeruginosa PKE117 was identified and characterized. The dypPa was first identified via sequence analysis and then cloned in Escherichia coli. Subsequently, the recombinant protein DyPPa was expressed and purified. Its DNA sequence analysis revealed an open reading frame of 897 bp, encoding a protein monomer of 299 amino acid residues with isoelectric point 4.62. According to SDS-PAGE analysis and FPLC result, DyPPa mainly existed as homodimer (64 kDa). DyPPa displayed typical heme absorbance of Soret band, with an Rz value of 1.18. Inductively coupled plasma-atomic absorption spectrum data also indicated DyPPa contained iron. Multiple amino acid sequence alignment of DyPPa with other members of the DyP-type peroxidases family showed the presence of conserved D139, H210, and R227 amino acids and GXXDG motifs, which were commonly shared by the DyP-type peroxidase family. Although the primary structure homology between DyPPa and other family members was very low, their secondary and tertiary structure displayed high homology, which explained the high decolorizing activity of DyPPa. Specifically, DyPPa displayed a good thermal stability and maximal activity on Reactive blue 5 under pH 3.5. Therefore, it was proposed that DyPPa, with a wide range of substrate specificity, was a novel member of the DyP-type peroxidases family.

Keywords: DyP-type peroxidase; Pseudomonas aeruginosa ; Cloning; Expression; Characterization

Chemiluminescence Response of Murine Macrophages on Multilayer Microfluidic Chips by Linfen Yu; Qun Li; Hongwei Gai; Zhanhui Wang (pp. 786-795).

We have demonstrated an integrated platform for microfluidics and chemiluminescence (CL) detection that is capable of parallel cell culture, convenient liquid manipulation, and sensitive chemiluminescent detection. Luminol-dependent CL responses induced by three different stimuli, phytohemagglutinin (PHA), concanavalin A (ConA), and lipopolysaccharides (LPS), which can evoke a CL response in macrophages, were evaluated on this microfluidic chip. We studied the dose-dependent effect of these three stimuli on CL response in murine macrophages. PHA produced the highest CL response compared to LPS and ConA. The CL intensity produced by PHA was 6.85 and four times higher than that by LPS and ConA, respectively, at the low concentration of 100 μg/ml. We have found microfluidic based CL to be a very useful screening tool, which is less laborious and more sensitive. This microfluidic system is disposable and capable of rapid device prototyping; it may prove to be very useful in clinical and pharmaceutical applications.

Keywords: Microfluidic chip; Macrophage; Cell chemiluminescence

Effects of Different Substrate Composition on Biosynthesis of Polyhydroxybutyrate-co-hydroxyvalerate by Recombinant Escherichia coli by Chih-Ching Chien; Hsiu-Hsiung Li; Po-Chi Soo; Shan-Yu Chen; Yu-Hong Wei; Wen-Ming Chen (pp. 796-804).

Cupriavidus necator is well known for its ability to accumulate polyhydroxybutyrate (PHB). When supplemented with propionic acid (or sodium propionate) in the growth medium, the bacterium is also able to synthesize polyhydroxybutyrate-co-hydroxyvalerate (PHBV). In order to increase the fraction of 3-hydroxyvalerate (3HV) in PHBV, we cloned the propionate permease gene prpP from C. necator and the propionyl-CoA synthase gene prpE from Cupriavidus taiwanensis and transformed into an Escherichia coli containing phaCAB operon of C. necator. The effects on PHBV accumulation in cells co-expressed with phaCAB and prpE or prpP in the media contained mixed carbon sources (glucose and sodium propionate) were evaluated. The HV fraction in PHBV increased when prpE or prpP was overexpressed in the cells. Concentrations of yeast extracts could also affect the fraction of HV. In addition, when glucose was replaced by sodium pyruvate, sodium succinate, or sodium gluconate, only PHB were detected in the recombinant strains.

Keywords: Polyhydroxybutyrate; Polyhydroxybutyrate-co-hydroxyvalerate; PHBV; Propionate; Cupriavidus

Metabolic Engineering of Nocardia sp. CS682 for Enhanced Production of Nargenicin A₁ by Sushila Maharjan; Dinesh Koju; Hei Chan Lee; Jin Cheol Yoo; Jae Kyung Sohng (pp. 805-817).

A number of secondary metabolites having therapeutic importance have been reported from the genus Nocardia. One of the polyketide antibiotic compounds isolated from Nocardia is nargenicin A₁. Recently, nargenicin A₁ has been isolated from Nocardia sp. CS682, a new Nocardia strain isolated from soil in Jeonnam, South Korea. It possesses strong antibacterial activity against methicillin-resistant Staphylococcus aureus. In this study, we applied a metabolic engineering approach based on recombinant DNA technology in order to boost the production of nargenicin A₁ from Nocardia sp. CS682. Initially, we optimized the transformation of this new strain by electroporation method. Heterologous expression of S-adenosylmethionine synthetase (MetK1-sp) in Nocardia sp. CS682 enhanced the production of nargenicin A₁ by about 2.8 times due to transcriptional activation of biosynthetic genes as revealed by reverse transcription polymerase chain reaction analysis. Similarly, expression of acetyl-CoA carboxylase genes improved nargenicin A₁ production by about 3.8 times in Nocardia sp. ACC18 compared to that in Nocardia sp. CS682 and Nocardia sp. NV18 by increasing precursor pool. Thus, enhanced production of nargenicin A₁ from Nocardia sp. CS682 can be achieved by expression of transcriptional activator genes and precursor genes from Streptomyces strains.

Keywords: Acetyl-CoA carboxylase; Nargenicin A₁ ; Nocardia sp. CS682; S-adenosylmethionine synthetase; Methicillin-resistant Staphylococcus aureus

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #3)