Applied Biochemistry and Biotechnology (v.178, #8)

Biocatalytic Resolution of Rac-α-Ethyl-2-Oxo-Pyrrolidineacetic Acid Methyl Ester by Immobilized Recombinant Bacillus cereus Esterase by Jian-yong Zheng; Yin-yan Liu; Wei-feng Luo; Ren-chao Zheng; Xiang-xian Ying; Zhao Wang (1471-1480).
A new esterase-producing strain (Bacillus cereus WZZ001) which exhibiting high hydrolytic activity and excellent enantioselectivity on rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester (R, S-1) has been isolated from soil sample by our laboratory. In this study, the stereoselective hydrolysis of (R, S-1) was performed using the recombinant Bacillus cereus esterase which expressed in Escherichia coli BL21 (DE3). Under the optimized conditions of pH 8.0, 35 °C, and concentration of substrate 400 mM, a successful enzymatic resolution was achieved with an e.e. s of 99.5 % and conversion of 49 %. Immobilization considerably increased the reusability of the recombinant esterase; the immobilized enzyme showed excellent reusability during 6 cycles of repeated 2 h reactions at 35 °C. Thereby, it makes the recombinant B. cereus esterase a usable biocatalyst for industrial application.
Keywords: Esterase; Bacillus cereus ; Enantioselective hydrolysis; Recombinant; α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester

The Stability Enhancement of Nitrile Hydratase from Bordetella petrii by Swapping the C-terminal Domain of β subunit by Weifeng Sun; Longbao Zhu; Xianggui Chen; Lunjie Wu; Zhemin Zhou; Yi Liu (1481-1487).
The thermal stability of most nitrile hydratases (NHase) is poor, which has been enhanced to some extent by molecular modifications in several specific regions of the C-terminal domain (C-domain) of β subunit of NHase. Since the C-domain could be present as a naturally separate domain in a few NHases, the whole C-domain is proposed to be related to the NHase stability. The chimeric NHase (SBpNHase) from the thermal-sensitive BpNHase (NHase from Bordetella petrii) and the relatively thermal-stable PtNHase (NHase from Pseudonocardia thermophila) was constructed by swapping the corresponding C-domains. After 30 min incubation at 50 °C, the original BpNHase nearly lost its activity, while the SBpNHase retained 50 % residual activity, compared with the melting temperature (Tm) (50 °C) of the original BpNHase, that of the SBpNHase was 55 °C. The SBpNHase with higher thermal stability would be useful for the thermal stability enhancement of NHase and for the understanding of the relationship between the stability of NHase and its structure.
Keywords: Bordetella petrii ; NHase; Pseudonocardia thermophila ; Enzyme thermal stability

Electrospun Polyvinyl Alcohol/ Pluronic F127 Blended Nanofibers Containing Titanium Dioxide for Antibacterial Wound Dressing by M. R. El-Aassar; G. F. El fawal; Nehal M. El-Deeb; H. Shokry Hassan; Xiumei Mo (1488-1502).
In this study, an antibacterial electrospun nanofibers for wound dressing application was successfully prepared from polyvinyl alcohol (PVA), Pluronic F127 (Plur), polyethyleneimine (PEI) blend solution with titanium dioxide nanoparticles (TiO2 NPs). PVA–Plur–PEI nanofibers containing various ratios of TiO2 NPs were obtained. The formation and presence of TiO2 in the PVA–Plu–PEI/ TiO2 composite was confirmed by X-ray diffraction (XRD). Transmission electron microscopy (TEM), Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), mechanical measurement, and antibacterial activity were undertaken in order to characterize the PVA–Plur–PEI/TiO2 nanofiber morphology and properties. The PVA–Plu–PEI nanofibers had a mean diameter of 220 nm, and PVA–Plur–PEI/TiO2 nanofibers had 255 nm. Moreover, the antimicrobial properties of the composite were studied by zone inhibition against Gram-negative bacteria, and the result indicates high antibacterial activity. Results of this antibacterial testing suggest that PVA–Plur–PEI/TiO2 nanofiber may be effective in topical antibacterial treatment in wound care; thus, they are very promising in the application of wound dressings.
Keywords: Nanofibers; Blend; TiO2 ; Antibacterial; Wound dressings

Zymomonas mobilis immobilized in doped calcium alginate (Ca-alginate) was successfully employed for the production of ethanol in an immobilized cell reactor. Polyethylene oxide and F127 dimethacrylate were evaluated as potential dopants for Ca-alginate beads to decrease lag time and increase initial ethanol yield. The influence of the type and concentration of the dopant on the effectiveness of the microbe immobilized in Ca-alginate beads to produce ethanol was studied, and results were compared to the widely used 2 % Ca-alginate with no dopants, which acted as control. Immobilized cell reactors that were operated using beads doped with 0.25 % polyethylene oxide (PEO) reached an ethanol yield of ∼70 % in 24 h, which was significantly higher than an ethanol yield of 25 % obtained for the control reactor operated using undoped Ca-alginate beads. This study shows that the use of water-soluble dopants can potentially reduce the lag phase and thus improve the initial production yield of immobilized cell reactors, likely due to an increase in porosity and diffusion rate of the doped beads.
Keywords: Ca-alginate; Immobilized cell reactor; PEO; FDMA

Directed Evolution and Mutant Characterization of Nitrilase from Rhodococcus rhodochrous tg1-A6 by Hui Luo; Jinwei Ma; Yanhong Chang; Huimin Yu; Zhongyao Shen (1510-1521).
In this paper, a molecularly directed evolution-based approach was applied to modify the nitrilase from Rhodococcus rhodochrous tg1-A6 for improving properties in catalyzing nitriles. In the process of error-prone polymerase chain reaction (PCR) with the wild-type nitrilase gene acting as the template, a library of the randomly mutated nitrilase gene was constructed. Since the pH value of catalyzing solution decreased when glycolonitrile was used as the substrate of nitrilase, a high-throughput strategy based on the color change of a pH-sensitive indicator was established for rapid screening of the mutated nitrilase. After three rounds of random mutation and screening about 5000 clones, a variant (Mut3) with 5.3-fold activity of the wild-type counterpart was obtained. Five amino acid substitutions (D27E, N97K, L246F, D108E, and S111R) were found in the variant Mut3. The properties of three mutated enzymes obtained in the three-round mutation were investigated. In the conversion of glycolonitrile, the variant (Mut2) accumulated the highest concentration of glycolic acid at 10.6 g l−1, a much higher value than the wild type (3.2 g l−1).
Keywords: Nitrilase; Directed evolution; Glycolonitrile; High-throughput screening; Glycolic acid

Effect of Natural Mineral on Methane Production and Process Stability During Semi-Continuous Mono-Digestion of Maize Straw by A. González-Suárez; I. Pereda-Reyes; E. Pozzi; A. José da Silva; D. Oliva-Merencio; M. Zaiat (1522-1533).
The effect of natural mineral on the mono-digestion of maize straw was evaluated in continuously stirred tank reactors (CSTRs) at 38 °C. Different strategies of mineral addition were studied. The organic loading rate (OLR) was varied from 0.5 to 2.5 g volatile solid (VS) L−1 d−1. A daily addition of 1 g mineral L−1 in reactor 2 (R2) diminished the methane production by about 11 % with respect to the initial phase. However, after a gradual addition of mineral, an average methane yield of 257 NmL CH4 g VS−1 was reached and the methane production was enhanced by 30 % with regard to R1. An increase in the frequency of mineral addition did not enhance the methane production. The archaeal community was more sensitive to the mineral than the bacterial population whose similarity stayed high between R1 and R2. Significant difference in methane yield was found for both reactors throughout the operation.
Keywords: Mono-digestion; Mineral; Maize straw; Methane yield; Archaeal community; Bacterial population

The aim of this work is to study the freezing process of water molecules surrounding Antarctic Gram-negative bacterium Marinomonas primoryensis antifreeze protein (MpAFP) and the MpAFP interactions to the surface of ice crystals under various marine environments (at different NaCl concentrations of 0.3, 0.6, and 0.8 mol/l). Our result indicates that activating temperature region of MpAFPs reduced as NaCl concentration increased. Specifically, MpAFP was activated and functioned at 0.6 mol/l with temperatures equal or larger 278 K, and at 0.8 mol/l with temperatures equal or larger 270 K. Additionally, MpAFP was inhibited by ice crystal network from 268 to 274 K and solid–liquid hybrid from 276 to 282 K at 0.3 mol/l concentration. Our results shed lights on structural dynamics of MpAFP among different marine environments.
Keywords: MARTINI force field; MpAFPs; Coarse-grained simulation; Free energy landscape

In Silico Characterization of the Binding Affinity of Dendrimers to Penicillin-Binding Proteins (PBPs): Can PBPs be Potential Targets for Antibacterial Dendrimers? by Shaimaa Ahmed; Suresh B. Vepuri; Muthusamy Ramesh; Rahul Kalhapure; Nadia Suleman; Thirumala Govender (1546-1566).
We have shown that novel silver salts of poly (propyl ether) imine (PETIM) dendron and dendrimers developed in our group exhibit preferential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus. This led us to examine whether molecular modeling methods could be used to identify the key structural design principles for a bioactive lead molecule, explore the mechanism of binding with biological targets, and explain their preferential antibacterial activity. The current article reports the conformational landscape as well as mechanism of binding of generation 1 PETIM dendron and dendrimers to penicillin-binding proteins (PBPs) in order to understand the antibacterial activity profiles of their silver salts. Molecular dynamics at different simulation protocols and conformational analysis were performed to elaborate on the conformational features of the studied dendrimers, as well as to create the initial structure for further binding studies. The results showed that for all compounds, there were no significant conformational changes due to variation in simulation conditions. Molecular docking calculations were performed to investigate the binding theme between the studied dendrimers and PBPs. Interestingly, in significant accordance with the experimental data, dendron and dendrimer with aliphatic cores were found to show higher activity against S. aureus than the dendrimer with an aromatic core. The latter showed higher activity against MRSA. The findings from this computational and molecular modeling report together with the experimental results serve as a road map toward designing more potent antibacterial dendrimers against resistant bacterial strains.
Keywords: Dendrimers; Molecular dynamics; Antibacterial resistance; Penicillin-binding proteins; Binding analysis

Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses by Linzhu Ren; Zhiyuan Peng; Xinrong Chen; Hongsheng Ouyang (1567-1585).
Cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. The first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. During infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. Using subgenomic replicon, which are genetically engineered viral RNA molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. The second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. The third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. This review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades.
Keywords: Positive-sense single strand RNA virus ((+) ssRNA virus); Reporter system; Recombinant virus; Replicon; Protease; Replicase

Production of Rebaudioside A from Stevioside Catalyzed by the Engineered Saccharomyces cerevisiae by Yan Li; Yangyang Li; Yu Wang; Liangliang Chen; Ming Yan; Kequan Chen; Lin Xu; Pingkai Ouyang (1586-1598).
Rebaudioside A has superior taste quality among the steviol glycosides extracted from Stevia rebaudiana leaves. Given its high purity as a general-purpose sweetener, rebaudioside A has received significant attention and has been widely applied in food and beverages in recent decades. Stevioside is one of the major steviol glycosides and can be converted to rebaudioside A by the uridine-diphosphate dependent glucosyltransferase UGT76G1 in S. rebaudiana. To explore the applicability of and limits in producing rebaudioside A from stevioside through whole-cell biocatalysis, the engineered Saccharomyces cerevisiae expressing UGT76G1, using a newly constructed constitutive expression vector, was used as the whole-cell biocatalyst. Citrate was added to the reaction mixture to allow metabolic regulation when glucose was fed to provide the activated sugar donor UDP-glucose for glycosylation of stevioside in vivo. In an evaluation of the whole-cell reaction parameters involving cell permeability, temperature, pH, citrate and Mg2+ concentrations, and glucose feeding, production of 1160.5 mg/L rebaudioside A from 2 g/L stevioside was achieved after 48 h without supplementation of extracellular UDP-glucose.
Keywords: Rebaudioside A; Saccharomyces cerevisiae ; Stevioside; UDP-glucosyltransferase; Whole-cell biocatalyst

Fermented Brown Rice Extract Causes Apoptotic Death of Human Acute Lymphoblastic Leukemia Cells via Death Receptor Pathway by Yukiko Horie; Hideyuki Nemoto; Mari Itoh; Hiroaki Kosaka; Kyoji Morita (1599-1611).
Mixture of brown rice and rice bran fermented with Aspergillus oryzae, designated as FBRA, has been reported to reveal anti-carcinogenic and anti-inflammatory effects in rodents. Then, to test its potential anti-cancer activity, the aqueous extract was prepared from FBRA powder, and the effect of this extract on human acute lymphoblastic leukemia Jurkat cells was directly examined. The exposure to FBRA extract reduced the cell viability in a concentration- and time-dependent manner. The reduction of the cell viability was accompanied by the DNA fragmentation, and partially restored by treatment with pan-caspase inhibitor. Further studies showed that FBRA extract induced the cleavage of caspase-8, -9, and -3, and decreased Bcl-2 protein expression. Moreover, the expression of tBid, DR5, and Fas proteins was enhanced by FBRA extract, and the pretreatment with caspase-8 inhibitor, but not caspase-9 inhibitor, restored the reduction of the cell viability induced by FBRA extract. These findings suggested that FBRA extract could induce the apoptotic death of human acute lymphoblastic leukemia cells probably through mainly the death receptor-mediated pathway and supplementarily through the tBid-mediated mitochondrial pathway, proposing the possibility that FBRA was a potential functional food beneficial to patients with hematological cancer.
Keywords: Fermented brown rice; Apoptotic cell death; Non-mitochondrial pathway; Death receptors; Jurkat cells; Rice bran

Enzymatic Hydrophobic Modification of Jute Fibers via Grafting to Reinforce Composites by Ruirui Liu; Aixue Dong; Xuerong Fan; Yuanyuan Yu; Jiugang Yuan; Ping Wang; Qiang Wang; Artur Cavaco-Paulo (1612-1629).
Horseradish peroxidase (HRP)/H2O2 system catalyzes the free-radical polymerization of aromatic compounds such as lignins and gallate esters. In this work, dodecyl gallate (DG) was grafted onto the surfaces of lignin-rich jute fabrics by HRP-mediated oxidative polymerization with an aim to enhance the hydrophobicity of the fibers. The DG-grafted jute fibers and reaction products of their model compounds were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results clearly indicated the grafting of DG to the jute fiber by HRP. Furthermore, the hydrophobicity of jute fabrics was determined by measuring the wetting time and static contact angle. Compared to the control sample, the wetting time and static contact angle of the grated fabrics changed from ~1 s to 1 h and from ~0° to 123.68°, respectively. This clearly proved that the hydrophobicity of jute fabrics improved considerably. Conditions of the HRP-catalyzed DG-grafting reactions were optimized in terms of the DG content of modified jute fabrics. Moreover, the results of breaking strength and elongation of DG-grafted jute/polypropylene (PP) composites demonstrated improved reinforcement of the composite due to enzymatic hydrophobic modification of jute fibers.
Keywords: Enzyme biocatalysis; Hydrophobicity; Lignin; Composite; Heterogeneous biocatalysis; Horseradish peroxidase

The present study describes grafting of zinc oxide (ZnO) nanoparticles with polyethyleneimine (PEI) followed by modification with glutraldehyde used as the bridge for binding the enzyme to support. The prepared nanocomposites were then characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, and transmission electron microscopy, utilized for synthesis of geranyl acetate in n-hexane. Among all the three prepared nanocomposites (ZnO + PEI, ZnO + PEI + SAA, ZnO + PEI + GLU), Candida rugosa lipase immobilized on ZnO-PEI-GLU was found to be best for higher ester synthesis. The operating conditions that maximized geranyl acetate resulted in the highest yield of 94 % in 6 h, molar ratio of 0.1:0.4 M (geraniol/vinyl acetate) in the presence of n-hexane as reaction medium. Various kinetic parameters such as V max, K i(G), K m(G), and K m(VA) were determined using nonlinear regression analysis for order bi–bi mechanism. The kinetic study showed that reaction followed order bi–bi mechanism with inhibition by geraniol. Activation energy (E a ) was found to be lower for immobilized lipase (12.31 kJ mol−1) than crude lipase (19.04 kJ mol−1) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 2.23-fold increased catalytic activity than crude lipase and recycled 20 times. The studies revealed in this work showed a promising perspective of using low-cost nanobiocatalysts to overcome the well-known drawbacks of the chemical-catalyzed route.
Keywords: Geranyl acetate; Polyethyleneimine; Immobilization; Biosynthesis; Lipase

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.
Keywords: Cell walls; Fluorescence assay; High-throughput; Polysaccharide endotransglycosylases; Transglycosylation

Production, Purification, and Biochemical Characterization of Thermostable Metallo-Protease from Novel Bacillus alkalitelluris TWI3 Isolated from Tannery Waste by Marimuthu Anandharaj; Balayogan Sivasankari; Nagarajan Siddharthan; Rizwana Parveen Rani; Subramaniyan Sivakumar (1666-1686).
Protease enzymes in tannery industries have enormous applications. Seeking a potential candidate for efficient protease production has emerged in recent years. In our study, we sought to isolate proteolytic bacteria from tannery waste dumping site in Tamilnadu, India. Novel proteolytic Bacillus alkalitelluris TWI3 was isolated and tested for protease production. Maximum protease production was achieved using lactose and skim milk as a carbon and nitrogen source, respectively, and optimum growth temperature was found to be 40 °C at pH 8. Protease enzyme was purified using ammonium sulfate precipitation method and anion exchange chromatography. Diethylaminoethanol (DEAE) column chromatography and Sephadex G-100 chromatography yielded an overall 4.92-fold and 7.19-fold purification, respectively. The 42.6-kDa TWI3 protease was characterized as alkaline metallo-protease and stable up to 60 °C and pH 10. Ca2+, Mn2+, and Mg2+ ions activated the protease, while Hg2+, Cu2+, Zn2+, and Fe2+ greatly inhibited it. Ethylenediaminetetraacetic acid (EDTA) inhibited TWI3 protease and was activated by Ca2+, which confirmed that TWI3 protease is a metallo-protease. Moreover, this protease is capable of dehairing goat skin and also removed several cloth stains, which makes it more suitable for various biotechnological applications.
Keywords: Protease; Bacillus alkalitelluris ; Tannery; Dehairing; Destaining