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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #5)

Plant Growth Promotion by an Extracellular HAP-Phytase of a Thermophilic Mold Sporotrichum thermophile by Bijender Singh; T. Satyanarayana (pp. 1267-1276).

Phytase of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorganic phosphate from Ca⁺², Mg⁺², and Co⁺² phytates more efficiently than those of Al³⁺, Fe²⁺, Fe³⁺, and Zn²⁺. The hydrolysis rate was higher at 60 °C as compared to 26 °C. Among all the organic acids tested, citrate was more effective in enhancing solubilization of insoluble phytate salts by phytase than others. The dry weight and inorganic phosphate contents of the wheat plants were high when supplemented with phytase or fungal spores. The plants provided with 5 mg phytate per plant exhibited enhanced growth and inorganic phosphate. With increase in the dosage of phytase, there was increase in growth and inorganic phosphate of plants, the highest being at 20 U per plant. The compost made employing the combined native microflora of the wheat straw and S. thermophile promoted growth of the plants. The plant-growth-promoting effect was also higher with the compost made using S. thermophile than that from only the native microflora.

Keywords: Phytase; Insoluble phytates; Sporotrichum thermophile ; Plant growth promotion; Compost

Cloning of a New Xylanase Gene from Streptomyces sp. TN119 Using a Modified Thermal Asymmetric Interlaced-PCR Specific for GC-Rich Genes and Biochemical Characterization by Junpei Zhou; Huoqing Huang; Kun Meng; Pengjun Shi; Yaru Wang; Huiying Luo; Peilong Yang; Yingguo Bai; Bin Yao (pp. 1277-1292).

A bacterial strain, Streptomyces sp. TN119, was isolated from the gut of Batocera horsfieldi larvae and showed xylanolytic activity. A degenerate primer set was designed based on the base usage of G and C in Actinobacteria xylanase-coding sequences belonging to the glycosyl hydrolases family 10 (GH 10), and used to clone the partial xylanase gene from Streptomyces sp. TN119. A modified thermal asymmetric interlaced (TAIL)-PCR specific for high-GC genes, named GC TAIL-PCR, was developed to obtain the full-length xylanase gene (xynA119; 1089 bp). Rich in GC content (67.8%), xynA119 encodes a new GH 10 xylanase (XynA119), which shares highest identity (48.8%) with an endo-1,4-β-xylanase from Cellulosimicrobium sp. HY-12. Recombinant XynA119 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 6.5 and 60 °C, was stable at pH 4.0 to 10.0 and 50 °C, was resistant to most chemicals (except for Cu²⁺, Mn²⁺, Ag⁺, Hg²⁺ and SDS) and trypsin, and produced simple products. The specific activity, K _m, V _max, and k _cat using oat-spelt xylan as substrate were 57.9 U mg⁻¹, 1.0 mg ml⁻¹, 74.8 μmol min⁻¹ mg⁻¹, and 49.2 s^–1, respectively.

Keywords: Xylanase; Batocera horsfieldi ; Gut; Streptomyces sp.; TN119; Thermal asymmetric interlaced (TAIL)-PCR; High-GC genes

Production of Lipid and Fatty Acids during Growth of Aspergillus terreus on Hydrocarbon Substrates by Adepu Kiran Kumar; Preety Vatsyayan; Pranab Goswami (pp. 1293-1300).

An Aspergillus terreus, isolated from oil contaminated soil, could degrade a wide range of petroleum hydrocarbons including the immediate oxidation products of hydrocarbons, like alkanols and alkanals. Among all the linear chain carbon substrates, highest growth of 39.1 ± 3.8 g l⁻¹ (wet weight) was observed when n-hexadecane was used as the sole source of carbon. The growth of the fungus on this highly hydrophobic substrate was associated with the morphological change of the hyphae and increase production of lipid in the cells. The lipid production in the hydrocarbon (n-hexadecane) grown cells was sevenfold higher than the corresponding glucose grown cells. The fatty acid profile of the lipid content formed in the hydrocarbon grown cells was significantly different from the glucose grown cells and was composed of fatty acids with chain length C₁₄ to C₃₃ as revealed from the liquid chromatography electrospray ionization mass spectrometry analyses. Among the ranges, the fatty acids with chain lengths C₁₄ to C₁₈ were predominant in the profile. Considering the fatty acid profile and the high level of lipid production, this A. terreus mediated production of lipid is envisaged to have potential application in the oleochemical industries including the production of biodiesel.

Keywords: Filamentous fungi; Aspergillus terreus ; Lipid; Fatty acid; Hydrocarbon

Biotransformation of Paeonol and Emodin by Transgenic Crown Galls of Panax quinquefolium by W. L. Ma; C. Y. Yan; J. H. Zhu; G. Y. Duan; R. M. Yu (pp. 1301-1308).

Two aromatic substrates, paeonol (1) and emodin (2), were biotransformed by using transgenic crown galls of Panax quinquefolium. Four biotransformed products (3–6) were isolated and identified by physicochemical and spectral methods. A β-glucoside (3, 73.2% of biotransformation yield) and a 1-(2,4-dimethoxyphenyl)- ethanone (4, 8.03%) were isolated from the suspension cultures after 7-day incubation of substrate 1. Upon administration of substrate 2, another β-glucoside [emodin-6-O-β-d-glucopyranoside (5), 19.2%] and a hydroxylated derivative, citreorosein (6, 54.6%), were also obtained. The results demonstrate that transgenic crown galls of P. quinquefolium have the capacities to catalyze glycosylation, hydroxylation, and methylation reactions in the plant cells on those aromatic compounds.

Keywords: Transgenic crown galls; Panax quinquefolium ; Biotransformation; Paeonol; Emodin

Kinetics of Zn²⁺-induced Brain Type Creatine Kinase Unfolding and Aggregation by Hang Mu; Zhi-Rong Lü; Daeui Park; Byoung-Chul Kim; Jong Bhak; Fei Zou; Jun-Mo Yang; Sen Li; Yong-Doo Park; He-Chang Zou; Hai-Meng Zhou (pp. 1309-1320).

We studied the effect of Zn²⁺ on the folding and aggregation of brain creatine kinase (CK-BB). We developed a method to purify CK-BB from rabbit brain and conducted inhibition kinetics and unfolding studies of CK-BB. Zn²⁺ conspicuously aggregated and osmolytes, such as glycine and proline, were able to suppress the formation of aggregates and protect the enzymatic activity against Zn²⁺. These results suggest that Zn²⁺ might act as a risk factor for CK-BB in the brain under certain conditions, and some osmolytes may help CK-BB to sustain the active state when Zn²⁺ is present. Our study provides useful information regarding the effect of Zn²⁺ on brain-derived metabolic enzymes, especially those that are putatively related to brain disease. Furthermore, our study suggests that although Zn²⁺ may induce CK-BB inactivation and misfolding, the ability of some abundant proteins and osmolytes to chelate Zn²⁺ nonspecifically may protect CK-BB and allow it to exist in the active form.

Keywords: Creatine kinase; Brain-type; Zn²⁺ ; Folding; Aggregation

Improvement of Aspergillus sulphureus Endo-β-1,4-Xylanase Expression in Pichia pastoris by Codon Optimization and Analysis of the Enzymic Characterization by Yihang Li; Bo Zhang; Xiang Chen; Yiqun Chen; Yunhe Cao (pp. 1321-1331).

The gene xynB from Aspergillus sulphureus encoding the endo-β-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzαA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml⁻¹, which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 °C for 3 days. The enzyme showed optimal activity at 50 °C and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na₂HPO₄–citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn²⁺. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.

Keywords: Endo-β-1,4-xylanase; Aspergillus sulphureus ; De novo synthesis; Codon optimization; Pichia pastoris

Isolation of Halotolerant Bacillus licheniformis WX-02 and Regulatory Effects of Sodium Chloride on Yield and Molecular Sizes of Poly-γ-Glutamic Acid by Xuetuan Wei; Zhixia Ji; Shouwen Chen (pp. 1332-1340).

A poly-γ-glutamic acid (γ-PGA) productive strain, halotolerant bacterium WX-02 was isolated from the saline soil of China (Yingcheng). By physiological, biochemical, and 16S rDNA sequence analysis methods, the strain was identified as Bacillus licheniformis. The effect of NaCl concentration on γ-PGA production by WX-02 was investigated in modified E (ME) medium. It was found that the γ-PGA production was salt-inducible, and the highest volumetric yield of γ-PGA (13.86 g/l) was attained with 8% of NaCl. It was also observed that the molecular size of γ-PGA decreased when the NaCl concentration increased. This was the first report of isolation and identification of a γ-PGA productive strain, halotolerant B. licheniformis. This study provided a simple strategy for controlling the yield and molecular size of γ-PGA by WX-02.

Keywords: Bacillus licheniformis ; Halotolerant; Poly-γ-glutamic acid; Molecular size; Yield

Alpha-Glucosidase Folding During Urea Denaturation: Enzyme Kinetics and Computational Prediction by Xue-Qiang Wu; Jun Wang; Zhi-Rong Lü; Hong-Min Tang; Daeui Park; Sang-Ho Oh; Jong Bhak; Long Shi; Yong-Doo Park; Fei Zou (pp. 1341-1355).

In this study, we investigated structural changes in alpha-glucosidase during urea denaturation. Alpha-glucosidase was inactivated by urea in a dose-dependent manner. The inactivation was a first-order reaction with a monophase process. Urea inhibited alpha-glucosidase in a mixed-type reaction. We found that an increase in the hydrophobic surface of this enzyme induced by urea resulted in aggregation caused by unstable folding intermediates. We also simulated the docking between alpha-glucosidase and urea. The docking simulation suggested that several residues, namely THR9, TRP14, LYS15, THR287, ALA289, ASP338, SER339, and TRP340, interact with urea. Our study provides insights into the alpha-glucosidase unfolding pathway and 3D structure of alpha-glucosidase.

Keywords: Alpha-glucosidase; Urea unfolding; Docking simulation

Antibacterial Activities of Crude Extract of Aloe barbadensis to Clinically Isolated Bacterial Pathogens by Ruchi Pandey; Avinash Mishra (pp. 1356-1361).

The antibacterial activity of Aloe barbadensis was tested on clinically isolated bacterial pathogens i.e. Enterococcus bovis, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Morganella morganii, and Klebsiella pneumoniae causing infection in human being. Ethanolic and aqueous extracts were used for the antibacterial effect, which was measured by the appearance of zone of inhibition. Relatively higher MIC concentrations were obtained for gram negative bacteria E. coli and K. pneumoniae, with ethanol extract; however, no inhibitory effect was noted for aqueous extract. Ethanolic extract possesses great inhibitory activity for gram positive bacteria, E. bovis followed by S. aureus. Among gram negative bacteria, highest inhibitory effect was observed with P. aeruginosa, followed by M. morganii, P. mirabilis, and P. vulgaris, which was significant (p < 0.01) than E. coli and K. pneumoniae. Antimicrobial activity tests of crude extract of A. barbadensis were carried out to validate the use of traditional medicinal herbal and results of this study tend to give credence to the common use of A. barbadensis gel and leaf.

Keywords: Aloe vera ; Antibacterial; Ethanolic extract; Herbal; Medicinal plant

Expression of Bacillus subtilis MA139 β-mannanase in Pichia pastoris and the Enzyme Characterization by Jiayun Qiao; Zhenghua Rao; Bing Dong; Yunhe Cao (pp. 1362-1370).

The 1014 nucleotides long gene-encoding β-mannanase from Bacillus subtilis strain MA139 was cloned using PCR. To obtain high expression levels in Pichia pastoris, the β-mannanase gene was optimized according to the codon usage bias of P. pastoris and fused downstream of GAP promoter. The reconstituted plasmid pGAP-mann was transformed into P. pastoris X-33 strain to constitutively express β-mannanase. When cultured at 28 °C for 3 days protein yields up to 2.7 mg/mL was obtained with the enzyme activity of up to 230 U/mL. In comparison, wild-type gene product yielded 1.9 mg/mL and 170 U/mL, respectively indicating that the protein yield and enzyme activity were significantly improved by codon modification. After purification, the enzyme properties were characterized. The optimal activity was at pH 6.0 and 50 °C. In the pH range of 3.0 to 9.0, β-mannanase showed above 60% of its peak activity. Among the numerous ions tested copper significantly inhibited the enzyme activity. These results suggested that codon-optimized β-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

Keywords: β-mannanase; Bacillus subtilis ; Codon optimization; Pichia pastoris ; Expression

An Enhanced Process for the Production of a Highly Purified Extracellular Lipase in the Non-conventional Yeast Yarrowia lipolytica by Saoussen Turki; Atef Ayed; Néjib Chalghoumi; Frederic Weekers; Philippe Thonart; Héla Kallel (pp. 1371-1385).

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with 10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale in Y. lipolytica LgX64.81.

Keywords: Yarrowia lipolytica ; Lipase; Fed-batch culture; Bioreactor; Purification

Immobilization of β-Galactosidase onto Magnetic Beads by Shengtang Zhang; Sufang Gao; Guoqiang Gao (pp. 1386-1393).

A study of the cross-linking of β-galactosidase on magnetic beads is reported here. The magnetic beads were prepared from artemisia seed gum, chitosan, and magnetic fluid in the presence of a cross-linking regent (i.e., glutaraldehyde). The reactive aldehyde groups of the magnetic beads allowed the reaction of the amino groups of the enzymes. The animated magnetic beads were used for the covalent immobilization of β-galactosidase. The effect of various preparation conditions on the activity of the immobilized β-galactosidase, such as immobilizing time, amount of enzyme, and the concentration of glutaraldehyde, were investigated. The influence of pH and temperature on the activity and the stability of the enzyme, both free and immobilized, have been studied. And o-nitrophenyl-β-d-galactopyranoside (ONPG) was chosen as a substrate. The β-galactosidase immobilized on the magnetic beads resulted in an increase in enzyme stability. Optimum operational temperature for immobilized enzyme was 10 °C higher than that of free enzyme and was significantly broader.

Keywords: Magnetic beads; β-galactosidase; Enzyme immobilization; Properties

Poly(Amic Acid)-Modified Biomass of Baker’s Yeast for Enhancement Adsorption of Methylene Blue and Basic Magenta by Jun-xia Yu; Bu-hai Li; Xiao-mei Sun; Jun Yuan; Ru-an Chi (pp. 1394-1406).

In this study, poly(amic acid)-modified biomass was prepared to improve the adsorption capacities for two cationic dyes, methylene blue and basic magenta. X-ray photoelectron spectroscopy and potentiometric titration demonstrated that a large number of imide, amine, and carboxyl groups were introduced on the biomass surface, and the concentrations of these functional groups were calculated to be 0.27, 1.08, and 1.08 mmol g⁻¹ by using the first derivative method. According to the Langmuir equation, the maximum uptake capacities (q _m) for methylene blue and basic magenta were 680.3 and 353.4 mg g⁻¹, respectively, which were 13- and sevenfold than that obtained on the unmodified biomass. Adsorption kinetics study showed that the completion of the adsorption process needed only 40 min, which is faster than the common sorbent such as activated carbon and resin. Experimental results showed that pH and ionic strength had little effect on the capacity of the modified biomass, indicating that the modified biomass had good potential for practical use.

Keywords: Modified biomass; Methylene blue; Basic magenta; Adsorption; First derivative method

Enhanced Enzymatic Hydrolysis of Lignocellulose by Optimizing Enzyme Complexes by Mingjia Zhang; Rongxin Su; Wei Qi; Zhimin He (pp. 1407-1414).

To enhance the conversion of the cellulose and hemicellulose, the corncob pretreated by aqueous ammonia soaking was hydrolyzed by enzyme complexes. The saturation limit for cellulase (Spezyme CP) was determined as 15 mg protein/g glucan (50 filter paper unit (FPU)/g glucan). The accessory enzymes (β-glucosidase, xylanase, and pectinase) were supplemented to hydrolyze cellobiose (cellulase-inhibiting product), hemicellulose, and pectin (the component covering the fiber surfaces), respectively. It was found that β-glucosidase (Novozyme 188) loading of 1.45 mg protein/g glucan [30 cellobiase units (CBU)/g glucan] was enough to eliminate the cellobiose inhibitor, and 2.9 mg protein/g glucan (60 CBU/g glucan) was the saturation limit. The supplementation of xylanase and pectinase can increase the conversion of cellulose and hemicellulose significantly. The yields of glucose and xylose enhanced with the increasing enzyme loading, but the increasing trend became low at high loading. Compared with xylanase, pectinase was more effective to promote the hydrolysis of cellulose and hemicellulose. The supplementation of pectinase with 0.12 mg protein/g glucan could increase the yields of glucose and xylose by 7.5% and 29.3%, respectively.

Keywords: Lignocellulose; Cellulase; β-Glucosidase; Xylanase; Pectinase

Expression, Purification, and Characterization of a Novel Soluble Form of Human Delta-like-1 by Mei Zhao; Mingyuan Wu; Lingchen Guo; Junfen Jiang; Weiwei Huang; Xiaojuan Lin; Zhonghui Zhang; Di Xiang; Huili Lu; Shunying Zhu; Yan Yu; Anja Moldenhauer; Wei Han (pp. 1415-1427).

The notch signaling pathway plays an important role in inhibiting cell differentiation and enhancing the repopulation capability of hematopoietic stem/progenitor cells. In this study, we developed rhDSL, a novel soluble form of Notch ligand Delta-like-1, which contains the DSL domain and the N-terminal sequence of the ligand, and investigated its function in ex vivo expansion of human umbilical cord blood (UCB)-primitive hematopoietic cells. The coding sequence for rhDSL was cloned into a pQE30 vector, and the recombinant rhDSL, fused with a 6× His tag, was expressed in Escherichia coli as inclusion bodies after isopropyl β-d-thiogalactoside induction. After renaturing by dilutions, the protein was purified through anion exchange followed by affinity chromatography. The purity of rhDSL protein was more than 99% with very low endotoxin. In combination with human c-kit ligand, the effect of rhDSL on ex vivo expansion of UCB CD34⁺ cells was found to be optimal at 1.5 μg/ml of rhDSL. The rhDSL protein might therefore be a potential supplement for the expansion of UCB-primitive hematopoietic cells.

Keywords: Notch signaling; rhDSL; Expression; Purification; HS/PCs

Bioproduction of Glycolic Acid from Glycolonitrile with a New Bacterial Isolate of Alcaligenes sp. ECU0401 by Yu-Cai He; Jian-He Xu; Jin-Huan Su; Li Zhou (pp. 1428-1440).

Alcaligenes sp. ECU0401 has been isolated from soil samples with high nitrilase activity against glycolonitrile using the enrichment culture technique. The preferred carbon/nitrogen sources and metal ions were sodium acetate, a composite of peptone and yeast extract, and Cu²⁺, respectively. Glycolic acid was obtained in a yield of 96.5% after 14 h of biotransformation from a total of 200 mM glycolonitrile in the mode of sequential addition during the cultivation of Alcaligenes sp. ECU0401 in a 5-L jar fermenter. Fifty micromolars of glycolonitrile could be hydrolyzed in a yield of 94.1% by resting cells after 36 h. The microbial nitrilase system could hydrolyze various nitriles with high activity, and no amidase activity and glycolic acid were observed in hydrolyzing glycolamide. It significantly exhibited high enantioselectivity in the hydrolysis of mandelonitrile and 2-chloromandelonitrile (>99.9% e.e. p). Efficient biocatalyst recycling was achieved as a result of immobilization in glutaraldehyde/polyethylenimine cross-linked carrageenan with immobilized cells exhibiting a biocatalyst productivity of 1,042.2 g glycolic acid per gram dry cell weight after 29 batch recycles.

Keywords: Nitrilase; Alcaligenes sp. ECU0401; Glycolic acid; Glycolonitrile; Immobilization

Cultivation of Rhodobacter sphaeroides in the Stirred Bioreactor with Different Feeding Strategies for CoQ₁₀ Production by Hong-Wei Yen; Chang-Yu Feng; Jia-Lin Kang (pp. 1441-1449).

The logistic growth model combined with the Luedeking-Piret equation was adopted in this study to model the batch production of CoQ₁₀ in the cultivation of Rhodobacter sphaeroides. The simulation results indicated that CoQ₁₀ production was a primary metabolite. As being a primary metabolite, a longer cell growing stage would tend to accumulate more biomass and lead to a higher CoQ₁₀ concentration being produced. In this context, a fed-batch operation by molasses feeding was performed to increase the biomass and subsequent CoQ₁₀ production. Three different molasses feeding strategies were operated in this study. Results suggested that the fed-batch operation with molasses controlled at 10 ± 1 g/l could increase the cell mass and CoQ₁₀ concentration to reach their maximum values of 18.6 g/l and 83.8 mg/l, respectively, nearly 2.2 times and 1.9 times their respective values obtained in the batch cultivation.

Keywords: Kinetic model; CoQ₁₀ ; Luedeking-Piret; Simulation; Fed-batch

Identification of Chaulmoogric Acid as a Small Molecule Activator of Protein Phosphatase 5 by Charmian Cher; Marie-Helene Tremblay; Jack R. Barber; Shi Chung Ng; Bin Zhang (pp. 1450-1459).

Protein phosphatase 5 (PP5) is an important protein phosphatase that is abundantly expressed in the central nervous system. Recent studies showed that PP5 activity in the neocortex from patients with Alzheimer’s disease (AD) is decreased significantly, suggesting that small molecule PP5 activator may have therapeutic potential for AD. We performed a biochemical screening for PP5 activators with the microsource compound library. Chaulmoogric acid was identified to be an effective activator with EC₅₀ value of 134.5 μM. Importantly, results from circular dichroism (CD) and limited proteolysis study showed that chaulmoogric acid binds to a region of tetratricopeptide repeat (TPR) domain of PP5 resulting in complete loss of helical contents. These results demonstrate a different mechanism of action from that of arachidonic acid, a known activator for PP5 dephosphorylation activity. Synergistic activation of PP5 enzymatic activity was also observed with combined application of both compounds at relatively low concentrations. Therefore, further structure activity relationship study of chaulmoogric acid may facilitate the discovery of small molecules that can synergize with endogenous arachidonic acid for PP5 activation.

Keywords: PP5; TPR; Arachidonic acid; Chaulmoogric acid; High-throughput screening

Telomere and Telomerase as Targets for Cancer Therapy by Xiaoping Tian; Bo Chen; Xiaochuan Liu (pp. 1460-1472).

Telomere maintenance and telomerase reactivation is essential for the transformation of most human cancer cells. Telomere shortening to the threshold length, mutations of the telomere-associated proteins, and/or telomerase RNA lead to telomeric dysfunction and therefore genomic instability. Telomerase up-regulation in 85% of human cancer cells has become a hallmark of cancers, hence a promising target for anticancer therapy. In this review, we discuss the mechanism of cancer due to telomere dysfunction and the resulting biological effects, the control of telomerase activity, and the new developments in cancer therapies targeting telomere and telomerase.

Keywords: Telomere; Telomerase; Telomere dysfunction; Telomerase RNA; Cancer therapy

Exploring the Role of C–H….π Interactions on the Structural Stability of Single Chain “All-Alpha” Proteins by V. Shanthi; K. Ramanathan; Rao Sethumadhavan (pp. 1473-1483).

C–H….π interactions are known to be important contributors to protein stability. In this study, we have analyzed the influence of C–H….π interactions in single chain “all-alpha” proteins. In the data set, a total of 181 C–H….π interactions were observed. The most prominent representatives are the interactions between aromatic C–H donor groups and aromatic π acceptors. Eighty-one percent of the C–H….π interactions between side chain to side chain and remaining19% of the C–H….π interactions were observed between side-chain to side-chain five-member aromatic ring. The donor atom contribution to C–H….π interactions was mainly from Phe, Tyr, and Trp residues. The acceptor atom contribution to C–H….π interactions was mainly from Phe, Tyr, Trp, and His. The highest percentage of C–H….π interactions were observed form Phe residue. The secondary structure preference analysis of all C–H….π interacting residues showed that Phe, Tyr, Trp, and His preferred to be in helix. Long-range C–H….π interactions are the predominant type of interactions in single chain all-alpha proteins data set. All the C–H….π interactions forming residues in the data set preferred to be in the buried region. Seventy-three percent of the donor residues and 65% of the acceptor residues are highly conserved.

Keywords: C–H….π interactions; Secondary structure; Interactions range; Solvent accessibility; Conservation

Expression and Characterization of the Dictyoglomus thermophilum Rt46B.1 Xylanase Gene (xynB) in Bacillus subtilis by Wei Zhang; Kai Lou; Guan Li (pp. 1484-1495).

To obtain extracellular and high-level expression of the Dictyoglomus thermophilum Rt46B.1 xylanase B gene, this gene was integrated into the α-amylase gene site of a host strain of Bacillus subtilis WB800. The extreme thermophile xylanase gene was successfully integrated and expressed in the host, measured at 24 ± 0.4 XUs/mL in the Luria broth medium supernatant. The recombinant enzyme was purified by ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. The molecular mass and pI value of xylanase were estimated to be 24 kDa and 4.3, respectively. The optimal pH level and temperature of the purified enzyme were 6.5 and 85 °C, respectively. Xylanase showed reasonable activity at temperatures up to 95 °C and remained stable at 4 °C for 1 week. The purified enzyme retained most of its activity in 1 mM ethylenediaminetetraacetic acid or dithiothreitol and 0.1% Tween-20 or Triton X-100. However, strong inhibition was observed in the presence of 5 mM Mn²⁺, 0.5% sodium dodecyl sulfate, Tween-20, or Triton X-100; a strong stimulating effect was also observed in the presence of Fe²⁺. The K _m and V _max values of the recombinant xylanase for birchwood xylan were calculated to be 2.417 ± 0.36 mg/mL and 325 ± 41 µmol/min mg, respectively. Xylanase was found to be useful in the prebleaching process of paper pulps.

Keywords: Thermostable xylanase; Purification; Characterization; Bacillus subtilis

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures by Andre Ricardo de Lima Damásio; Tony Márcio da Silva; Alexandre Maller; João Atílio Jorge; Hector Francisco Terenzi; Maria de Lourdes Teixeira de Moraes Polizeli (pp. 1496-1507).

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K _m and V _max values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 µmol/min/mg, respectively. PG was found to have temperature optimum at 65 °C and was totally stable at 45 °C for 90 min. Half-life at 55 °C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na⁺¹, K⁺¹, and Co⁺² (1 mM) and Cu⁺² (1 and 10 mM).

Keywords: Paecilomyces variotii ; Exoenzymes; Pectinases; Polygalacturonase; Pectin

Predicting Drug Interaction of Clopidogrel on Microbial Metabolism of Diclofenac by K. Srisailam; V. Raj Kumar; C. Veeresham (pp. 1508-1516).

Seven fungal cultures were studied for the metabolism of diclofenac in order to elucidate the nature of enzymes involved in biotransformation, as diclofenac is a specific substrate to cytochrome P450 (CYP) 2C9 isozyme in mammals. The metabolites were identified by high-performance liquid chromatography–diode array detection and liquid chromatography–tandem mass spectroscopy analysis. The study included clopidogrel, a selective inhibitor of CYP2C9 isozyme, to inhibit the metabolism of diclofenac. Two-stage fermentation protocol was used to study the diclofenac metabolism and its inhibition by clopidogrel. Among the cultures studied, four have shown positive indication for drug interaction, since clopidogrel inhibited the metabolism of diclofenac in a dose-dependent manner. The results indicate that microbial cultures possess enzyme systems similar to mammals and they can be used to predict drug interactions in mammalian systems.

Keywords: Biotransformation; HPLC; LC–MS–MS; Metabolites; Microorganisms; Diclofenac; Clopidogrel; Drug interaction

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321 by Gi-Wook Choi; Hyun-Woo Kang; Se-Kwon Moon; Bong-Woo Chung (pp. 1517-1527).

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.

Keywords: Bioethanol; Continuous production; Simultaneous saccharification and fermentation; Cassava; Self-flocculating yeast

Maximization of β-Galactosidase Production: A Simultaneous Investigation of Agitation and Aeration Effects by Fernanda Germano Alves; Francisco Maugeri Filho; Janaína Fernandes de Medeiros Burkert; Susana Juliano Kalil (pp. 1528-1539).

In this work, the agitation and aeration effects in the maximization of the β-galactosidase production from Kluyveromyces marxianus CCT 7082 were investigated simultaneously, in relation to the volumetric enzyme activity and the productivity, as well as the analysis of the lactose consumption and production of glucose, and galactose of this process. Agitation and aeration effects were studied in a 2 L batch stirred reactor. A central composite design (2² trials plus three central points) was carried out. Agitation speed varied from 200 to 500 rpm and aeration rate from 0.5 to 1.5 vvm. It has been shown in this study that the volumetric enzyme production was strongly influenced by mixing conditions, while aeration was shown to be less significant. Linear models for activity and productivity due to agitation and aeration were obtained. The favorable condition was 500 rpm and 1.5 vvm, which lead to the best production of 17 U mL⁻¹ for enzymatic activity, 1.2 U mL⁻¹ h⁻¹ for productivity in 14 h of process, a cellular concentration of 11 mg mL⁻¹, and a 167.2 h⁻¹ volumetric oxygen transfer coefficient.

Keywords: Agitation; Aeration; Enzymatic activity; Productivity; Stirred reactor

The New Application of Biosorption Properties of Enteromorpha prolifera by Izabela Michalak; Katarzyna Chojnacka (pp. 1540-1556).

The main goal of this paper was to elaborate the possibility of industrial application of biosorption properties of Enteromorpha prolifera (production of mineral feed additives for livestock). In this study, biosorption process was used in the binding of chromium(III) ions from aqueous solution by the green macroalga. The kinetics of biosorption process was studied in a batch system with respect to the initial pH, temperature, initial metal ion concentration, and initial biomass concentration. E. prolifera demonstrated good biosorption properties. The equilibrium biosorption capacity increased with pH and with initial concentration of metal ions. The uptake of chromium(III) ions by the dried alga was affected by the temperature, but in small extent. With increase of the biomass concentration, the decrease of biosorption capacity at equilibrium was observed. The best biosorption conditions were determined as the initial pH 5, temperature 25 °C, the initial chromium(III) ions concentration 400 mg/L, and biosorbent concentration 1.0 g/L. Biosorption capacity at equilibrium reached at these conditions was 100 mg/g. The mechanism of the biosorption of chromium(III) ions by E. prolifera was analyzed in equilibrium experiments. Equilibrium data were fitted to Langmuir, Dubinin–Radushkevich, and Freundlich adsorption isotherms. The most suitable model for describing the obtained data was Langmuir model. The experimental results and the analysis of the solution before and after biosorption process suggested ion-exchange mechanism.

Keywords: Enteromorpha prolifera ; Biosorption; Chromium(III) ions; Kinetics; Isotherm; Mineral feed additives

Microwave Pretreatment of Substrates for Cellulase Production by Solid-State Fermentation by Xuebing Zhao; Yujie Zhou; Guangjian Zheng; Dehua Liu (pp. 1557-1571).

The agricultural residues, wheat bran and rice hulls, were used as substrates for cellulase production with Trichoderma sp 3.2942 by solid-state fermentation. Microwave irradiation was employed to pretreat the substrates in order to increase the susceptibility. Although the highest cellulase yield was obtained by the substrates pretreated by 450 W microwave for 3 min, pretreatment time and microwave power had no significant effect on cellulase production. The initial reducing sugar content (RSC) of substrates was decreased by microwave irradiation, but more reducing sugars were produced in later fermentation. Alkali pretreatment combined with microwave pretreatment (APCMP) of rice hulls could significantly increase cellulase yields and reducing sugar. The maximum filter paper activity, carboximethylcellulase (CMC)ase, and RSC were increased by 35.2%, 21.4%, and 13%, respectively, compared with those of untreated rice hulls. The fermented residues could produce more cellulase and reducing sugars than fresh rice hulls after they were treated by APCMP. The increased accessibility of the substrates by microwave pretreatment was mainly achieved by rupture of the rigid structure of rice hulls. However, for alkali pretreatment and APCMP, delignification and removal of ash played very important roles for increasing the acceptability of substrates.

Keywords: Solid state fermentation; Microwave pretreatment; Alkali pretreatment; Cellulase; Agricultural residue

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #5)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.160, #5)