Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.170, #2)

MS Analysis and Molecular Characterization of Botrytis cinerea Protease Prot-2. Use in Bioactive Peptides Production by Ferid Abidi; Nayssene Aissaoui; Jean-Charles Gaudin; Jean-Marc Chobert; Thomas Haertlé; Mohamed Nejib Marzouki (pp. 231-247).

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8 %) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74 %) and with chymotrypsin from Metarhizium anisopliae (71 %). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.

Keywords: Protease Prot-2; Botrytis cinerea ; Antimicrobial activity; Fish protein hydrolysates; Proteomics; Trypsin/chymotrypsin

Impact of Nitrate Dose on Toluene Degradation under Denitrifying Condition by Dong-Ju Kim; Mee-Rye Park; Dae-Soon Lim; Jae-Woo Choi (pp. 248-256).

In this study, we investigated the impact of nitrate dose on toluene degradation by Pseudomonas putida to elucidate the upper limit of nitrate concentration and whether an optimum ratio of nitrate to toluene concentration exists. Batch microcosm studies were conducted in order to monitor toluene degradation for various ratios (2–20) of nitrate to toluene with nitrate concentrations ranging from 0 to 700 mg L⁻¹ for a given toluene concentration of 50 and 25 mg L⁻¹ during 4-day (short term) and 14-day (long term) incubation time, respectively. The short-term study revealed that nitrate concentration of 500 mg L⁻¹ was toxic to bacteria and the optimum concentration was 300 mg L⁻¹ yielding the highest toluene degradation rate (0.083 mg L⁻¹ h⁻¹). In the batch study of long term, toluene degradation was limited to 6 days after which the nitrate at 50 mg L⁻¹ was depleted, indicating that nitrate was a necessary electron acceptor. For both batch studies, an optimum ratio of 6 was found yielding the highest toluene degradation rate. This indicates that an appropriate nitrate dose is essential for efficient degradation of toluene when bioremediation of groundwater contaminated with toluene is under consideration.

Keywords: Toluene; Degradation; Nitrate dose; Optimum ratio; Pseudomonas putida

RAPD Markers Associated with Salt Tolerance in Soybean Genotypes Under Salt Stress by Faheema Khan; Khalid Rehman Hakeem; Tariq O. Siddiqi; Altaf Ahmad (pp. 257-272).

In order to investigate the influence of genetic background on salt tolerance in soybean (Glycine max), ten soybean genotypes (Pusa-20, Pusa-40, Pusa-37, Pusa-16, Pusa-24, Pusa-22, BRAGG, PK-416, PK-1042, and DS-9712) released in India, were selected and grown hydroponically. The 10-day-old seedlings were subjected to 0, 25, 50, 75, 100, 125, and 150 mM NaCl for 15 days. Plant growth, leaf osmotic adjustment, and random amplified polymorphic DNA (RAPD) analysis were studied. In comparison to control plants, the plant growth in all genotypes was decreased by salt stress, respectively. Salt stress decreased leaf osmotic potential in all genotypes; however, the maximum reduction was observed in genotype Pusa-24 followed by PK-416 and Pusa-20, while minimum reduction was shown by genotype Pusa-37, followed by BRAGG and PK-1042. Pusa-16, Pusa-22, Pusa-40, and DS-9712 were able to tolerate NaCl treatment up to the level of 75 Mm. The difference in osmotic adjustment between all the genotypes was correlated with the concentrations of ion examined such as Na⁺ and the leaf proline concentration. These results suggest that the genotypic variation for salt tolerance can be partially accounted by plant physiological measures. Twenty RAPD primers revealed high polymorphism and genetic variation among ten soybean genotypes studied. The closer varieties in the cluster behaved similarly in their response to salinity tolerance. Intra-clustering within the two clusters precisely grouped the ten genotypes in sub-cluster as expected from their physiological findings. Our study shows that RAPD technique is a sensitive, precise, and efficient tool for genomic analysis in soybean genotypes.

Keywords: RAPD; Molecular markers; Soybean; Salt stress; Genotypes

Stabilisation of Recombinant Aequorin by Polyols: Activity, Thermostability and Limited Proteolysis by Mehdi Zeinoddini; Khosro Khajeh; Saman Hosseinkhani; Ali Reza Saeedinia; Seyed-Mortaza Robatjazi (pp. 273-280).

The photoprotein aequorin is a calcium-dependent bioluminescent enzyme which is most widely used in biotechnology processes, but this protein is susceptible to aggregation and proteolysis degradation. Various additives such as polyols are known to enhance the stability of proteins and protect them in native folded and functional state. In this work, for study of aequorin stability, the histidine-tagged apoaequorin was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. Kinetics of light emission of purified aequorin upon addition of Ca²⁺ showed a linear dependency on aequorin concentration. Furthermore, the effect of some stabilisers, such as glycerol, glucose, lactose, terehalose, sucrose and sorbitol on thermostability of recombinant aequorin was measured. Results indicate that the recombinant aequorin is very stable in phosphate buffer including 30 mM sorbitol, since after heat shock of 30 min at different temperatures, a slight decrease in activity was observed. However, flexibility and exposure of tryptophan residues of aequorin to the solvent, in the presence and absence of stabilisers, with respect to fluorescence quenching by acrylamide, indicated identical characterisation. In addition, according to limited proteolysis of aequorin demonstrating that this enzyme is sensitive to proteases as in the presence of 2 ng/ml of protease, aequorin was completely digested. In conclusion, sorbitol increases stability of aequorin with high photoactivity and not effect for flexibility and limited proteolysis of this photoprotein.

Keywords: Aequorin; Expression; Stability; Proteolysis

Computational Approach for Identification of Anopheles gambiae miRNA Involved in Modulation of Host Immune Response by Krishnaraj Thirugnanasambantham; Villianur Ibrahim Hairul-Islam; Subramanian Saravanan; Subramaniyan Subasri; Ariraman Subastri (pp. 281-291).

MicroRNAs (miRNAs) are small, noncoding RNAs that play key roles in regulating gene expression in animals, plants, and viruses, which involves in biological processes including development, cancer, immunity, and host–microorganism interactions. In this present study, we have used the computational approach to identify potent miRNAs involved in Anopheles gambiae immune response. Analysis of 217,261 A. gambiae ESTs and further study of RNA folding revealed six new miRNAs. The minimum free energy of the predicted miRNAs ranged from −27.2 to −62.63 kcal/mol with an average of −49.38 kcal/mol. While its A + U % ranges from 50 to 65 % with an average value of 57.37 %. Phylogenetic analysis of the predicted miRNAs revealed that aga-miR-277 was evolutionary highly conserved with more similarity with other mosquito species. Observing further the target identification of the predicted miRNA, it was noticed that the aga-miR-2304 and aga-miR-2390 are involved in modulation of immune response by targeting the gene encoding suppressin and protein prophenoloxidase. Further detailed studies of these miRNAs will help in revealing its function in modulation of A. gambiae immune response with respect to its parasite.

Keywords: miRNA; A. gambiae ; Immune response; miRNA target; EST sequence

Production of Enzymes by Paenibacillus chitinolyticus and Paenibacillus ehimensis to Obtain Chitooligosaccharides by Nathália Kelly de Araújo; Cristiane Fernandes de Assis; Everaldo Silvino dos Santos; Gorete Ribeiro de Macedo; Louise Fonseca de Farias; Humberto Arimatéia Jr.; Matheus de Freitas Fernandes Pedrosa; Maria Giovana Binder Pagnoncelli (pp. 292-300).

Obtaining oligosaccharides from chitosan has been the focus of several studies in the pharmaceutical, chemical, food, and medical areas, due to their functional properties. Here, we evaluated the production potential of biologically functional chitooligosaccharides using enzymes extracts produced by Paenibacillus chitinolyticus and Paenibacillus ehimensis. After 48 h of fermentation, these microorganisms were able to produce chitosanases, which generated oligomers with a degree of polymerization between dimers and hexamers. The maximum conversion of chitosan to oligomers was 99.2 %, achieved after 12 h incubation of chitosan with enzymes produced by P. ehimensis. The chitooligosaccharides generated were capable of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical, reaching a maximum scavenging rate of 61 and 39 % when produced with P. ehimensis and P. chitinolyticus enzymes, respectively. The use of these enzymes in the crude form could facilitate their use in industrial applications.

Keywords: Chitosan; Paenibacillus chitinolyticus ; Paenibacillus ehimensis ; Chitooligosaccharides; Antioxidant

Exon Array Analysis of Alternative Splicing of Genes in SOD1G93A Transgenic Mice by Ming Hu; Yansu Guo; Huifang Chen; Weisong Duan; Chunyan Li (pp. 301-319).

Alternative splicing is a common strategy for creating functional diversities of proteins. While conventional identification of splice variants generally targets individual genes in amyotrophic lateral sclerosis, we present a novel exon-centric array that allows genome-wide identification of splice variants and concurrently provides analysis of gene expression. Compare 1 was asymptomatic SOD1G93A transgenic mice with nontransgenic littermates; compare 2 was symptomatic with asymptomatic transgenic mice. RT-PCR was performed to validate. Pathway and GO analysis were performed on abnormal genes. These findings could guide us to demonstrated the potential influence of mutant human CuZn-SOD1 and of splicing regulation in pathological processes.

Keywords: Alternative splicing; Exon array; Gene expression; Pathway analysis; GO analysis; Amyotrophic lateral sclerosis

Molecular Cloning and Overexpression of an Endo-β-1,4-xylanase Gene from Aspergillus niger in Industrial Saccharomyces cerevisiae YS2 Strain by Baoyu Tian; Yan Xu; Wanling Cai; Qingeng Huang; Yuanyuan Gao; Xin Li; Jianzhong Huang (pp. 320-328).

The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to overexpress the xylanase by constructing a homology-driven integration vector. The total mRNA from a xylanase-producing strain of Aspergillus niger IME-216 was extracted and used as the template for the production of endo-β-1,4-xylanase cDNA by reverse transcription. The fusion fragment containing the phosphoglycerate kinase promoter, α-factor signal peptide, xylanase gene encoding the mature peptide, and CYC1 terminator was first generated by overlap extension polymerase chain reaction. Then, the vector pUPX was constructed by inserting the fusion fragment into the S. cerevisiae plasmid pUG6. Then, A 2.2-kb rDNA sequence was further cloned and attached to the SalI-digested pUPX to obtain the integration plasmid pUPXR. The pUPXR was linearized by KpnI, transformed into the industrial strain S. cerevisiae YS2 using the lithium acetate method and integrated into the S. cerevisiae chromosome. The maximum yield of the recombinant xylanase produced by the engineered S. cerevisiae strain YS2_2 was 74.8 U per microliter, which was about 1.5-fold higher than the original 50 U per microliter by Aspergillus niger IME-216 strain under the flask culture at 28 °C for 72 h. The findings of our study can be used for further development of industrial S. cerevisiae strain for producing interested enzymes, or improving the achievement of metabolism, for example, simultaneous fermentation of glucose and xylose to producing bioethanol.

Keywords: Aspergillus niger ; Endo-β-1,4-xylanase; Bioethanol; Saccharomyces cerevisiae ; Integration expression

Distribution of Hydrocarbon-Degrading Bacteria in the Soil Environment and Their Contribution to Bioremediation by Yuki Fukuhara; Sachie Horii; Toshihide Matsuno; Yoshiki Matsumiya; Masaki Mukai; Motoki Kubo (pp. 329-339).

A real-time PCR quantification method for indigenous hydrocarbon-degrading bacteria (HDB) carrying the alkB gene in the soil environment was developed to investigate their distribution in soil. The detection limit of indigenous HDB by the method was 1 × 10⁶ cells/g-soil. The indigenous HDB were widely distributed throughout the soil environment and ranged from 3.7 × 10⁷ to 5.0 × 10⁸ cells/g-soil, and the ratio to total bacteria was 0.1–4.3 %. The dynamics of total bacteria, indigenous HDB, and Rhodococcus erythropolis NDKK6 (carrying alkB R2) during bioremediation were analyzed. During bioremediation with an inorganic nutrient treatment, the numbers of these bacteria were slightly increased. The numbers of HDB (both indigenous bacteria and strain NDKK6) were gradually decreased from the middle stage of bioremediation. Meanwhile, the numbers of these bacteria were highly increased and were maintained during bioremediation with an organic nutrient. The organic treatment led to activation of not only the soil bacteria but also the HDB, so an efficient bioremediation was carried out.

Keywords: Biostimulation; Bioaugmentation; Indigenous hydrocarbon-degrading bacteria; Alkane hydroxylase gene; Rhodococcus

Characterization of β-Galactosidase Isoforms from Bacillus circulans and Their Contribution to GOS Production by Anja Warmerdam; Ekaraj Paudel; Wanqing Jia; Remko M. Boom; Anja E. M. Janssen (pp. 340-358).

A β-galactosidase preparation from Bacillus circulans consists of four isoforms called β-gal-A, β-gal-B, β-gal-C, and β-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms may be relevant for GOS production, which is typically done at high substrate concentrations. At initial lactose concentrations in between 0.44 % and 0.68 % (w/w), β-gal-A showed the least oligosaccharide formation, followed by β-gal-B and β-gal-C; most oligosaccharides were formed by β-gal-D. The differences in behavior were confirmed by studying the thermodynamics of lactose conversion with isothermal titration calorimetry since especially β-gal-A showed a different profile than the other isoforms. Also during the conversion of allolactose and 4-galactosyllactose at 0.44 % and 0.61 % (w/w), respectively, β-gal-A and β-gal-D showed clear differences. In contrast to above findings, the selectivity of the isoforms did hardly differ at an initial lactose concentration of 30 % (w/w), except for a slightly higher production of galactose with β-gal-A. These differences were hypothesized to be related to the different accessibility of the active sites of the isoforms for different-sized reactants. The initial GOS formation rates of the isoforms indicate that β-gal-A and β-gal-B are the best isoforms for GOS production at high lactose concentrations.

Keywords: β-Galactosidases; Bacillus circulans ; Galacto-oligosaccharide production; Substrate concentration; Isothermal titration calorimetry

Identification and Functional Characterization of an α-Amylase with Broad Temperature and pH Stability from Paenibacillus sp. by Thangamani Rajesh; Yong Hyun Kim; Yong-Keun Choi; Jong Min Jeon; Hyun Joong Kim; Sung-Hee Park; Hyung-Yeon Park; Kwon-Young Choi; Hyungsup Kim; Hyung Joo Kim; Sang Hyun Lee; Yung-Hun Yang (pp. 359-369).

Amylases are important industrial enzymes that have been applied widely in the food, detergent, and pulp industries and fermentation processes. In the present study, a gene encoding an alpha-amylase from the genomic DNA library of Paenibacillus sp. was identified and characterized. The amylase gene designated amy1 was shown to consist of 1,980 bp and shared sequence identity towards α-amylase genes from other Bacillus sp. The deduced amino acid sequence for Amy1 indicated 80 % sequence identity with other Bacillus strains. Heterologous expression of recombinant Amy1 in Escherichia coli BL21(DE3) facilitated the recovery of this protein in soluble form. Enzyme kinetic data revealed Amy1 to have a K _m of 23.83 mg/mL and K _cat of 48.74 min⁻¹ and K _cat /K _m of 2 min⁻¹ mg⁻¹ mL⁻¹ for starch. The activity of this protein was found to be enhanced by Mn²⁺, and furthermore, Amy1 remained active at a broad pH range (4–10) and temperature (30–90 °C). The ability of Amy1 to act on food waste under broad temperature and pH conditions, together with its ability to produce simple sugars, shows many advantages for further application in the food industry.

Keywords: Paenibacillus sp.; α-Amylase; Thermostability; pH stability; Starch hydrolysis

The Kinetics Behavior of the Reduction of Formaldehyde Catalyzed by Alcohol Dehydrogenase (ADH) and Partial Uncompetitive Substrate Inhibition by NADH by Nuan Wen; Wenfang Liu; Yanhui Hou; Zhiping Zhao (pp. 370-380).

Alcohol dehydrogenase (ADH) catalyzes the final step in the biosynthesis of methanol from CO₂. Here, we report the steady-state kinetics for ADH, using a homogeneous enzyme preparation with formaldehyde as the substrate and nicotinamide adenine dinucleotide (NADH) as the cofactor. When changing NADH concentrations with the fixed concentrations of HCHO (more or less than NADH), kinetic studies revealed a particular zigzag phenomenon for the first time. Increasing formaldehyde concentration can weaken substrate inhibition and improve catalytic efficiency. The kinetic mechanism of ADH was analyzed using the secondary fitting method. The double reciprocal plots (1/v∼1/[HCHO] and 1/[NADH]) strongly demonstrated that the substrate inhibition by NADH was uncompetitive versus formaldehyde and partial. In the direction of formaldehyde reduction, ADH has an ordered kinetic mechanism with formaldehyde adding to enzyme first and product methanol released last. The second reactant NADH can combine with the enzyme–methanol complex and then methanol dissociates from it at a slower rate than from enzyme–methanol. The reaction velocity depends on the relative rates of the alternative pathways. The addition of NADH also accelerates the releasing of methanol. As a result, substrate inhibition and activation occurred intermittently, and the zigzag double reciprocal plot (1/v∼1/[NADH]) was obtained.

Keywords: ADH; NADH; Formaldehyde; Enzyme kinetics; Substrate inhibition

Isolation of Biphenyl and Polychlorinated Biphenyl-Degrading Bacteria and Their Degradation Pathway by Young-Cheol Chang; Kazunori Takada; DuBok Choi; Tadashi Toyama; Ken Sawada; Shintaro Kikuchi (pp. 381-398).

Four strains of biphenyl-degrading bacteria were isolated from a sewage and identified from the Rhodococcus genus (SK-1, SK-3, and SK-4) and Aquamicrobium genus (SK-2) by 16S rRNA sequence. Among these strains, strain SK-2 was most suitable for biphenyl degradation. When 0.65, 1.3, 2.6, or 3.9 mM of biphenyl was used, the biphenyl was completely degraded within 24 and 96 h of culture, respectively. However, in the case of 6.5 and 9.75 mM of biphenyl, the biphenyl degradation yields were about 80 % and 46.7 % after 120 h of culture, respectively. The isolated strains could degrade a broad spectrum of aromatic compounds including high-chlorinated polychlorinated biphenyl (PCB) congeners in the presence of biphenyl. In addition, strain SK-2 could utilize PCB congeners containing one to six chlorine substituents such as 2,2′,4,4′,5,5′-hexachlorobiphenyl. The PCB utilization rate by the strain SK-2 was increased compared to that of other PCB congener-utilizing bacteria. The four isolates metabolized 4-chlorobiphenyl to 4-chlorobenzoic acid and 2-hydroxy-6-oxo-6-(4′-chlorophenyl)-hexa-2,4-dienoic acid. These results suggest the isolated strains might be good candidates for the bioremediation of PCB-contaminated soil, especially high-saline soils.

Keywords: Biphenyl; Polychlorinated biphenyl (PCB); Biphenyl-degrading mechanism; Rhodococcus genus; Aquamicrobium genus

Cell Debris Self-Immobilized Thermophilic Lipase: a Biocatalyst for Synthesizing Aliphatic Polyesters by Yang Sun; Yan Yang; Chenhui Wang; Jiaming Liu; Wei Shi; Xiaobo Zhu; Laijin Lu; Quanshun Li (pp. 399-405).

The paper explored the catalytic activity of a cell debris self-immobilized thermophilic lipase for polyester synthesis, using the ring-opening polymerization of ε-caprolactone as model. Effects of biocatalyst concentration, temperature, and reaction medium on monomer conversion and product molecular weight were systematically evaluated. The biocatalyst displayed high catalytic activity at high temperatures (70–90 °C), with 100 % monomer conversion. High monomer conversion values (>90 %) were achieved in both hydrophobic and hydrophilic solvents, and also in solvent-free system, with the exception of dichloromethane. Poly(ε-caprolactone) was obtained in 100 % monomer conversion, with a number-average molecular weight of 1,680 g/mol and a polydispersity index of 1.35 in cyclohexane at 70 °C for 72 h. Furthermore, the biocatalyst exhibited excellent operational stability, with monomer conversion values exceeding 90 % over the course of 15 batch reactions.

Keywords: Cell debris; Immobilization; Thermophilic lipase; Ring-opening polymerization; ε-Caprolactone

Culture and Differentiation of Rat Neural Stem/Progenitor Cells in a Three-Dimensional Collagen Scaffold by Dan Ge; Kedong Song; Shui Guan; Yanli Qi; Bo Guan; Wenfang Li; Junshan Liu; Xuehu Ma; Tianqing Liu; Zhanfeng Cui (pp. 406-419).

A stable and fast method for constructing a neural-like tissue from rat neural stem/progenitor cells (rNS/PCs) based on three-dimensional (3D) collagen gel is described. First step, the collagen-embedded rNS/PCs expanded with the medium consisting of DMEM/F12/RPMI1640 (1:1:1) supplemented with EGF and bFGF was used to expand the cells in gel in 96-well plates until the average diameter of cell clusters was about 50–100 μm with the cell density higher than 10⁷ cells/mL. In the second step, the initial medium was replaced with NB/B-27 supplemented with bFGF and BDNF. The results show that cells in collagen presented neural-like morphology and maintained live cell rate around 82 % in neural network pattern at least for 42 days under static conditions. The cell–collagen constructs were detected by immunofluorescence and immunohistochemistry test after 42 days of culture, part of cells still maintained the character of rNS/PCs, and others differentiated into neurons, astrocytes, and oligodendrocytes. Our 3D neural-like tissue construct was similar to the neural tissue in morphology and cell compositions. They thus have a potential to be used for drug screening, detection of environment toxins, and replacement therapy.

Keywords: Neural stem/progenitor cells; Collagen gel; Three-dimensional culture; Neural-like tissue; Neural network

A Novel Multifunctional α-Amylase from the Thermophilic Fungus Malbranchea cinnamomea: Biochemical Characterization and Three-Dimensional Structure by Peng Han; Peng Zhou; Songqing Hu; Shaoqing Yang; Qiaojuan Yan; Zhengqiang Jiang (pp. 420-435).

A novel α-amylase (McAmyA) from the thermophilic fungus, Malbranchea cinnamomea was purified, characterized and crystallized in the present study. McAmyA was purified to apparent homogeneity with a molecular mass of 60.3 kDa on SDS-PAGE. The enzyme exhibited maximal activity at pH 6.5 and was stable within pH 5.0–10.0. It was most active at 65 °C and was stable up to 50 °C. McAmyA was capable of hydrolyzing amylose, starch, amylopectin, pullulan, cyclodextrins and maltooligosaccharides. The full-length cDNA of an α-amylase gene (McAmyA) from the strain was cloned. McAmyA consisted of a 1,476-bp open reading frame encoding 492 amino acids. It displayed the highest amino acid sequence homology (less than 60 %) with the reported α-amylases. The crystal structure of McAmyA was solved at a resolution of 2.25 Å (PDB code 3VM7). The overall structure of McAmyA reveals three domains with ten α helices and 14 β strands, and the putative catalytic residues are positioned at domain A with somewhat different secondary structural circumstances compared with typical α-amylases.

Keywords: α-Amylase; Crystal structure; Malbranchea cinnamomea ; Property; Thermophilic fungi

Role of Met93 and Thr96 in the Lid Hinge Region of Rhizopus chinensis Lipase by Shan-shan Zhu; Ming Li; Xiaowei Yu; Yan Xu (pp. 436-447).

We engineered Rhizopus chinensis lipase to study its critical amino acid role in catalytic properties. Based on the amino acid sequence and three-dimensional model of the lipase, residues located in its lid hinge region (Met93 and Thr96) were replaced with corresponding amino acid residues (Ile93 and Asn96) found in the lid hinge region of Rhizopus oryzae lipase. The substitutions in the lid hinge region affected not only substrate specificity but also the thermostability of the lipase. Both lipases preferred p-nitrophenyl laurate and glyceryl trilaurate (C12). However, the variant S4-3O showed a slight decline in activity toward long-chain fatty acid (C16–C18). When enzymes activities decreased by half, the temperature of the variant (45 °C) was 22 °C lower than the parent (67 °C), probably substantially destabilized the structure of the lid region. The interfacial kinetic analysis of S4-3O suggested that the lower catalytic efficiency was due to a higher K _m* value. According to the lipase structure investigated, Ile93Met played a role of narrowing the size of the hydrophobic patch, which affected the substrate binding affinity, and Asn96Thr destabilized the structure of the lipase by disrupting the H-bond interaction in the lid region.

Keywords: Rhizopus chinensis lipase; Lid; Hinge; Substrate specificity; Thermostability

Comparison of Yarrowia lipolytica and Pichia pastoris Cellular Response to Different Agents of Oxidative Stress by Marlene Lopes; Manuel Mota; Isabel Belo (pp. 448-458).

Yeast cells exposed to adverse conditions employ a number of defense mechanisms in order to respond effectively to the stress effects of reactive oxygen species. In this work, the cellular response of Yarrowia lipolytica and Pichia pastoris to the exposure to the ROS-inducing agents’ paraquat, hydrogen peroxide, and increased air pressure was analyzed. Yeast cells at exponential phase were exposed for 3 h to 1 mM paraquat, to 50 mM H₂O₂, or to increased air pressure of 3 or 5 bar. For both strains, the cellular viability loss and lipid peroxidation was lower for the cells exposed to increased air pressure than for those exposed to chemical oxidants. The glutathione induction occurred only in Y. lipolytica strain and reached the highest level as a response to PQ exposure. In general, antioxidant enzymes were more expressed in Y. lipolytica than in P. pastoris. The enzyme superoxide dismutase was induced in both strains under all the oxidant conditions but was dependent on the cellular growth phase, being undetectable in non-growing cells, whereas glutathione reductase was more induced in those conditions. Hydrogen peroxide was the most efficient inducer of catalase. Both yeast cultures underwent no cellular growth inhibition with increased air pressure, indicating that these yeast species were able to adapt to the oxidative stressful environment.

Keywords: Oxidative stress; Antioxidant enzymes; Increased air pressure; Yarrowia lipolytica ; Pichia pastoris

In Vitro Culture, Determination, and Directed Differentiation of Adult Adipose-Derived Stem Cells Towards Cardiomyocyte-Like Cells Induced by Angiotensin II by Kedong Song; Zhaomin Wang; Wenfang Li; Chao Zhang; Mayasari Lim; Tianqing Liu (pp. 459-470).

The in vitro basic biological characteristics and directed differentiation potential towards cardiomyocytes of adult adipose-derived stem cells (ADSCs) induced by angiotensin II were both investigated. ADSCs were isolated from adult adipose tissue and cultured in vitro, and were subsequently induced into adipocytes, chondrocytes, and osteoblasts for assays of multipotential differentiation. The morphological characteristics of ADSCs were observed under an inverted microscope in bright field and phase-contrast ways and a confocal laser scanning microscopy. Moreover, the directional differentiation potential was observed by Oil Red, alkaline phosphatase, von Kossa, and toluidine blue stainings, respectively. The expressions of CD34, CD44, CD45, CD105, and HLA-DR were also detected via flow cytometry. Following to this, ADSCs were induced by angiotensin II and basic fibroblast growth factor for the purpose of directional differentiation towards cardiomyocyte-like cells, and the cells treated with 5-azacytidine were regarded as the control. The results showed that the isolated and cultured ADSCs presented a typical morphology of fusiform shape and also expressed CD44, CD105, but not CD34, CD45, and HLA-DR with assays of flow cytometry. The multi-differentiations to adipocytes, chondrocytes, and osteoblasts confirmed that the isolated cells maintained the stem characteristics generating from adipose tissues. After 4 weeks of induction by angiotensin II, the cells expressed myosin heavy chain, troponin I, and connexin43 by immunocytochemistry staining, but without beating of the cells. This current study indicated that ADSCs possessed the characteristics of mesenchymal stem cells and angiotensin II could induce ADSCs into cardiomyocyte-like cells.

Keywords: Adipose-derived stem cells; Cardiomyocyte-like cells; Tissue engineering; Cell differentiation; Angiotensin II; 5-Azacytidine

Erratum to: The Metabolic Advantage of Choline Lactate in Growth Media: An Experimental Analysis with Staphylococcus lentus by Sudharshan Sekar; Surianarayanan Mahadevan; Perinkulam Ravi Deepa; Bhuvanesh Kumar Shanmugam; B. V. N. Phani Kumar; Asit Baran Mandal (pp. 471-472).

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.170, #2)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.170, #2)