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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #8)

Effects of Ionic Liquids on the Growth of Arthrobacter simplex and Improved Biodehydrogenation in an Ionic Liquid-Containing System with Immobilized Cells by Jin Huang; Songlin Xie; Junyao He; Pu Wang (pp. 2131-2143).

Dehydrogenated derivatives of corticosteroids are usually more effective than their precursors in treating diseases. In this study, the toxicity of seven water-miscible ionic liquid and three organic solvents to the biocatalyst Arthrobacter simplex UR016 was tested to evaluate the possibility of biodehydrogenation from 17α-hydroxy-16β-methyl-pregna-4,9(11)-diene-3,20-dione (HMPDD) to 17α-hydroxy-16β-methyl-pregna-1,4,9(11)-triene-3,20-dione (HMPTD) in an ionic liquid-containing system. Although most tested room temperature ionic liquids (RTILs) showed higher toxicities to A. simplex UR016 than organic solvents, bacterial growth was promoted in the presence of [EMIM](l)-Lac or [BMIM](l)-Lac at concentrations below 2.5 mmol/l, especially [EMIM](l)-Lac, presented the lowest toxicity to A. simplex. Following immobilization investigations, a conversion ratio of 89.9 % was achieved with a cell biomass of 10 g/l (dry cell weight/reaction mixture volume) in the polyethylene glycol (PEG)-modified calcium alginate gel bead, a suitable matrix for cell immobilization. Further studies indicated that the conversion ratio can be improved by increasing cell loading to 60 beads per flask (containing 30 ml reaction mixture). Under optimal conditions with a [EMIM](l)-Lac content of 0.3 %, the conversion ratio reached 93.4 %, the highest value ever reported.

Keywords: Arthrobacter simplex ; Ionic liquids; Biodehydrogenation; Immobilized cells; Growth

l-Asparaginase as Potent Anti-leukemic Agent and Its Significance of Having Reduced Glutaminase Side Activity for Better treatment of Acute Lymphoblastic Leukaemia by L. N. Ramya; Mukesh Doble; V. P. B. Rekha; K. K. Pulicherla (pp. 2144-2159).

Acute lymphoblastic leukaemia (ALL) is one of the leading types of malignant disorder seen in children. Viral infections, genetic factors and exposure to chemical carcinogens are some of the factors responsible for causing ALL. Treatment strategies followed for curing ALL include chemotherapy or radiation therapy, wherein, chemotherapy involves the use of the enzymatic drug l-Asparaginase. The enzyme can be produced from various plants, animals, bacterial and fungal sources but, among them, bacterial sources are widely used for production of this enzyme. The enzyme is non-human in origin having certain bottle necks with l-Asparaginase therapy in the form of side effects such as pancreatitis, thrombosis which are mainly due to glutaminase side activity. Hence, present-day research is mainly focussed on minimizing or completely eliminating the glutaminase activity of the enzyme l-Asparaginase. This review is focussed on the complications associated with glutaminase side activity and use of glutaminase free enzymatic drug l-Asparaginase in treating ALL and the other developments related to the modification of the drug for quality treatment.

Keywords: Acute lymphoblastic leukaemia; l-Asparaginase; Dual substrate specificity; Pectobacterium carotovorum ; Glutaminase side activity

Molecular Identification Using ITS Sequences and Genome Shuffling to Improve 2-Deoxyglucose Tolerance and Xylanase Activity of Marine-Derived Fungus, Aspergillus Sp. NRCF5 by Ahmed M. A. El-Bondkly (pp. 2160-2173).

During the screening of xylanolytic enzyme from marine-derived fungi isolated from the inner tissue of Egyptian soft coral Rhytisma sp., one strain, NRCF5, exhibited high enzyme activity with 0.1 % (w/v) antimetabolite 2-deoxyglucose (2DG) tolerance. This fungal strain was identified as Aspergillus sp. NRCF5 based on its morphological characteristics and internal transcribed spacer (ITS) sequences. The ITS region of hyperactive xylanolytic strain (NRCF5) was amplified, sequenced, and submitted to GenBank (accession no. JQ277356). To apply the fundamental principles of genome shuffling in breeding of xylanase-producing fungi, marine-derived fungus Aspergillus sp. NRCF5 was used as starting strain in this work and applied for induction of genetic variability using different combinations and doses of mutagens. Five mutants with high xylanase activity and 0.25 % (w/v) antimetabolite 2DG tolerance were obtained from the populations generated by the mutation of combination between ultraviolet irradiation (UV, 5 min) and N-methyl-N-nitro-N-nitrosoguanidine (NTG, 100 μg/ml) for 30 (UNA) and 60 (UNB) min as well as NTG (100 μg/ml) and ethidium bromide (250 μg/ml) for 30 (NEA) and 60 (NEB) min. Then, they were subjected for recursive protoplast fusion. Seven hereditarily stable recombinants with high xylanase activity and 1.0 % (w/v) 2DG tolerance were obtained by four rounds of genome shuffling. Among them, a high xylanase-producing recombinant, R4/31, was obtained, which produced 427.5 U/ml xylanase. This value is 6.13-fold higher than that of the starting strain NRCF5 and 2.48-fold higher than that of the parent strain (mutant NEA51). The subculture experiments indicated that the high producer of marine Aspergillus sp. R4/31 fusant was stable.

Keywords: Marine fungi; Xylanase; ITS sequences; Mutation; Genome shuffling

A Molecular Description of Acid Phosphatase by Asha Anand; Pramod Kumar Srivastava (pp. 2174-2197).

Acid phosphatase is ubiquitous in distribution in various organisms. Although it catalyzes simple hydrolytic reactions, it is considered as an interesting enzyme in biological systems due to its involvement in different physiological activities. However, earlier reviews on acid phosphatase reveal some fragmentary information and do not give a holistic view on this enzyme. So, the present review summarizes studies on biochemical properties, structure, catalytic mechanism, and applications of acid phosphatase. Recent advancement of acid phosphatase in agricultural and clinical fields is emphasized where it is presented as potent agent for sustainable agricultural practices and diagnostic marker in bone metabolic disorders. Also, its significance in prostate cancer therapies as a therapeutic target has been discussed. At the end, current studies and prospects of immobilized acid phosphatase are included.

Keywords: Acid phosphatase; Phosphate scavenger; Bone remodelling; Biomarker; Prostatic acid phosphatase

Cloning and Expression of a Novel Xylanase Gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris by Huimin Zhang; Minchen Wu; Jianfang Li; Shujuan Gao; Yanjun Yang (pp. 2198-2211).

A full-length complementary DNA (cDNA) of Auxyn11D, a gene that encodes a novel endo-β-1,4-d-xylanase of Aspergillus usamii E001 (abbreviated to AuXyn11D), was obtained using 3′- and 5′-rapid amplification of cDNA ends (RACE) methods. The cDNA sequence is 855 bp in length, containing 5′- and 3′-untranslated regions and a 696-bp open reading frame (ORF) that encodes a 32-aa signal peptide and a 199-aa mature peptide (namely AuXyn11D). Multiple homology alignment of amino acid sequences verified that AuXyn11D belongs to glycoside hydrolase family 11. Moreover, a mature peptide-encoding cDNA fragment of Auxyn11D was cloned and expressed in Pichia pastoris GS115. One P. pastoris transformant expressing the highest recombinant AuXyn11D (reAuXyn11D) activity of 15.0 U/mL, labeled as P. pastoris GSAuXyn4-16, was chosen by shake flask test. SDS–PAGE assay demonstrated that the reAuXyn11D, a glycosylated protein with an apparent molecular mass of 32.0 kDa, was secreted into the medium. The purified reAuXyn11D displayed the highest activity at pH 4.5 and 55 °C. It was stable at a pH range of 3.5–6.5 and at a temperature of 50 °C or below. Its activity was not significantly affected by most of metal ions tested and EDTA, but increased by Ca²⁺ and inhibited by Mn²⁺. The K _m and V _max of the reAuXyn11D towards birchwood xylan were 6.32 mg/mL and 391.6 U/mg, respectively.

Keywords: Aspergillus usamii ; Cloning; Expression; Xylanase; Pichia pastoris

Laboratory Production of Human Prolactin from CHO Cells Adapted to Serum-Free Suspension Culture by Fernanda Santos Arthuso; Paolo Bartolini; Carlos Roberto Jorge Soares (pp. 2212-2224).

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23 kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40 days, to grow in suspension in serum-free medium. High cell densities of up to 4.0 × 10⁶ cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30 days. Two harvesting strategies were set up, 50 or 100 % of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5–7 μg hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.

Keywords: Human prolactin; Suspension culture; CHO cells

Silver Nanoparticle-Mediated Enhancement in Growth and Antioxidant Status of Brassica juncea by Priyadarshini Sharma; Deepesh Bhatt; M. G. H. Zaidi; P. Pardha Saradhi; P. K. Khanna; Sandeep Arora (pp. 2225-2233).

Metal nanoparticles can potentially be used as tools for engineering biological redox reactions. Present study underlines the effect of silver metal nanoparticles (at 0, 25, 50, 100, 200 and 400 ppm) on the growth and antioxidant status of 7-day-old Brassica juncea seedlings. Fresh weight, root and shoot length, and vigor index of seedlings is positively affected by silver nanoparticle treatment. It induced a 326 % increase in root length and 133 % increase in vigor index of the treated seedlings. Improved photosynthetic quantum efficiency and higher chlorophyll contents were recorded in leaves of treated seedlings, as compared to the control seedlings. Levels of malondialdehyde and hydrogen peroxide decreased in the treated seedlings. Nanoparticle treatment induced the activities of specific antioxidant enzymes, resulting in reduced reactive oxygen species levels. Decrease in proline content confirmed the improvement in antioxidant status of the treated seedlings. The observed stimulatory affects of silver nanoparticles are found to be dose dependent, with 50 ppm treatment being optimum for eliciting growth response. Present findings, for the first time indicate that silver nanoparticles promote the growth of B. juncea seedlings by modulating their antioxidant status.

Keywords: Silver nanoparticles; Growth; Antioxidant status; Redox potential; Quantum efficiency; Reactive oxygen species

Antioxidant, Anti-inflammatory, and Anti-senescence Activities of a Phlorotannin-Rich Natural Extract from Brown Seaweed Ascophyllum nodosum by Mélody Dutot; Roxane Fagon; Marc Hemon; Patrice Rat (pp. 2234-2240).

Aging at the cellular level is characterized by oxidative stress, inflammation, and cell senescence. An extract of the brown seaweed Ascophyllum nodosum rich in phlorotannins has been studied for its inhibitory activity against oxidative stress, inflammation, and senescence. A. nodosum extract at 0.2 % prevented tBHP-induced reactive oxygen species production (evaluated using the H2DCF-DA test in cytofluorometry) in epithelial cells and LPS-induced TNF-α and IL-6 release (evaluated using ELISA technique) in macrophages. A. nodosum extract also increased nuclear SIRT1 activity in epithelial cells. Altogether, these beneficial cellular effects of phlorotannin-rich A. nodosum extract could be used in topical therapeutic formulations against aging.

Keywords: Oxidative stress; Inflammation; Sirtuin; Aging; Seaweed; Phlorotannins

Purification and Characterization of Alkaline Pectin Lyase from a Newly Isolated Bacillus clausii and Its Application in Elicitation of Plant Disease Resistance by Zuming Li; Zhihui Bai; Baoguo Zhang; Baojv Li; Bo Jin; Michael Zhang; Francis Lin; Hongxun Zhang (pp. 2241-2256).

Alkaline pectin lyase (PNL) shows potential as a biological control agent against several plant diseases. We isolated and characterized a new Bacillus clausii strain that can produce 4,180 U/g of PNL using sugar beet pulp as a carbon source and inducer. The PNL was purified to apparent homogeneity using ultrafiltration, ammonium sulfate fractionation, DEAE Sepharose Fast Flow, and Sephadex G-75 gel filtration. The purified PNL was found to be a monomeric protein with a molecular weight of 35 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It demonstrated optimal activity with K _m of 0.87 mg/ml at pH 10.0 and 60 °C. The enzyme is stable in the pH range of 8.0–10.0 and temperature ≤40 °C. Ca²⁺ was found to stimulate the enzymatic activity of the PNL by up to 410 %. Mass spectrometric results gave 38 % match coverage with pectate lyase from B. clausii KSM-K16 (gi|56961845). The PNL was found to elicit disease resistance in cucumber seedlings, suggesting that it may have applications in biocontrol and sustainable agriculture.

Keywords: Bacillus clausii ; Alkaline pectin lyase; Sugar beet pulp; Mass spectrometric sequencing; Plant disease resistance

Detection and Clinical Significance of CD44v6 and Integrin-β1 in Pancreatic Cancer Patients using a Triplex Real-Time RT-PCR Assay by Gang Zhou; David Chiu; Dajiang Qin; Lizhi Niu; Jinlei Cai; Lihua He; Wenhao Huang; Kecheng Xu (pp. 2257-2268).

The cell adhesion molecules CD44v6 and integrin-β1 are associated with the progression and metastasis of cancer. A novel triplex real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify CD44v6 and integrin-β1 gene expression in peripheral blood mononuclear cells from 30 pancreatic cancer (PC) patients and 12 healthy individuals. The standard curve of the triplex qRT-PCR was constructed by optimizing the reaction condition and the amplification efficiency was 102.5, 101.1, and 100.6 % for CD44v6, integrin-β1 and endogenous gene (β-actin) amplification. Nonspecific bands were not observed from the triplex qRT-PCR amplification and the detection limit of this assay was 100 copies. Expression levels of CD44v6 and integrin-β1 gene were significantly lower in healthy individuals than PC patients (P < 0.05). CD44v6 and integrin-β1 gene expression were not associated with the sex, age, and tumor position in PC (P > 0.05). CD44v6 gene expression was significantly associated with clinical stage, liver metastasis, and tumor size (P < 0.05). Integrin-β1 gene expression was significantly associated with clinical stage and liver metastasis (P < 0.05). This triplex qRT-PCR assay may provide a useful tool for diagnosis, prognosis, and therapeutic evaluation in PC.

Keywords: CD44v6; Integrin-β1; Quantitative; Real-time RT-PCR; Pancreatic cancer

l-myo-Inositol-1-Phosphate Synthase Expressed in Developing Organ: Isolation and Characterisation of the Enzyme from Human Fetal Liver by Dhani Raj Chhetri; Seema Gupta; Asok Kumar Mukherjee; Jukta Adhikari (pp. 2269-2282).

l-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0–60 % ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46 % of MIPS could be recovered. The human fetal liver MIPS specifically utilised d-glucose-6-phosphte and NAD⁺ as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40 °C. The enzyme activity was remarkably stimulated by NH ₄ ⁺ , slightly stimulated by K⁺ and Ca²⁺ and highly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺. The K _m values of MIPS for d-glucose-6-phosphate and NAD⁺ were found to be as 1.15 and 0.12 mM respectively while the V _max values were 280 nM and 252 nM for d-glucose-6-phosphate and NAD⁺ correspondingly. The apparent molecular weight of the native enzyme was determined to be 170 kDa.

Keywords: Human fetal liver; Enzyme purification; l-myo-inositol-1-phosphate synthase; Myo-inositol; d-glucose-6-phosphate; l-myo-inositol-1-phosphate

Mathematical Tool from Corn Stover TGA to Determine Its Composition by Cesare Freda; Francesco Zimbardi; Francesco Nanna; Egidio Viola (pp. 2283-2294).

Corn stover was treated by steam explosion process at four different temperatures. A fraction of the four exploded matters was extracted by water. The eight samples (four from steam explosion and four from water extraction of exploded matters) were analysed by wet chemical way to quantify the amount of cellulose, hemicellulose and lignin. Thermogravimetric analysis in air atmosphere was executed on the eight samples. A mathematical tool was developed, using TGA data, to determine the composition of corn stover in terms of cellulose, hemicellulose and lignin. It uses the biomass degradation temperature as multiple linear function of the cellulose, hemicellulose and lignin content of the biomass with interactive terms. The mathematical tool predicted cellulose, hemicellulose and lignin contents with average absolute errors of 1.69, 5.59 and 0.74 %, respectively, compared to the wet chemical method.

Keywords: Corn stover; Thermogravimetric analysis; Cellulose; Hemicellulose; Lignin; Steam explosion

Mobility Study of Individual Residue Sites in the Carbohydrate Recognition Domain of LSECtin Using SDSL–EPR Technique by Changzhen Wang; Juntao Yang; Yu Zhou; Jianbo Cong; Guofu Dong; Xiangjun Hu; Li Tang; Ke Wu (pp. 2295-2304).

Conformational changes in proteins profoundly influence their functional profiles. With site-directed spin labeling (SDSL)–electron paramagnetic resonance (EPR) spectroscopy, we investigated the mobility features of individual residue sites in the carbohydrate recognition domain (CRD) of LSECtin, a type II integral membrane protein. The mobility of six different residue sites scatting around the Ca²⁺-1-binding site were investigated by comparing their EPR spectra rotational correlation time τ _c in order to obtain the information of conformational changes of relevant region. The results showed that the overall mobility of LSECtin-CRD increased after addition of Ca²⁺ and N-acetylglucosamine, but different sites in the CRD exhibited different mobility features, suggesting that these sites may have different functional profiles. The preliminary observations thus demonstrated that SDSL–EPR spectroscopy is not only an effective technique to reveal the mobility of single residue sites in LSECtin-CRD but also that the functions of single residue sites may be indicated by their conformational dynamics.

Keywords: SDSL–EPR spectroscopy; LSECtin-CRD; Cysteine; Mobility

Characterization of Bioimprinted Tannase and Its Kinetic and Thermodynamics Properties in Synthesis of Propyl Gallate by Transesterification in Anhydrous Medium by Guangjun Nie; Zhiming Zheng; Guohong Gong; Genhai Zhao; Yan Liu; Junying Song; Jun Dai (pp. 2305-2317).

Tannase has been extensively applied to synthesize gallic acid esters. Bioimprinting technique can evidently enhance transesterification-catalyzing performance of tannase. In order to promote the practical utilization of the modified tannase, a few enzymatic characteristics of the enzyme and its kinetic and thermodynamics properties in synthesis of propyl gallate by transesterification in anhydrous medium have been studied. The investigations of pH and temperature found that the imprinted tannase holds an optimum activity at pH 5.0 and 40 °C. On the other hand, the bioimprinting technique has a profound enhancing effect on the adapted tannase in substrate affinity and thermostability. The kinetic and thermodynamic analyses showed that the modified tannase has a longer half-time of 1,710 h at 40 °C; the kinetic constants, the activation energy of reversible thermal inactivation, and the activation energy of irreversible thermal inactivation, respectively, are 0.054 mM, 17.35 kJ mol⁻¹, and 85.54 kJ mol⁻¹ with tannic acid as a substrate at 40 °C; the free energy of Gibbs (ΔG) and enthalpy (ΔH) were found to be 97.1 and 82.9 kJ mol^-1 separately under the same conditions.

Keywords: Tannase; Bioimprinting; Kinetic; Thermodynamics; Transesterification

Expression, Purification of Recombinant Human Mitochondrial Transcription Termination Factor 3 (hMTERF3) and Preparation of Polyclonal Antibody Against hMTERF3 by Wei Xiong; Yonghui Luo; Cuixiang Zhang; Deyong Tan; Shaoyuan Zuo (pp. 2318-2329).

In mammalian cells, a family of mitochondrial transcription termination factors (MTERFs) regulates mitochondrial gene expression. Mitochondrial transcription termination factor 3 (MTERF3) is the most conserved member of the MTERF family and a negative regulator of mammalian mitochondrial DNA transcription. To create a specific polyclonal antibody against human MTERF3 (hMTERF3), we first cloned hMTERF3 into prokaryotic expression vector pGEX-4T-1, and GST-hMTERF3 was efficiently expressed in Escherichia coli after induction by IPTG. The expressed GST-tagged hMTERF3 fusion protein was purified by passive electro-elution process and then used to immunize BALB/c mice, we obtained anti-GST-hMTERF3 polyclonal antibody purified by protein A column and determined the sensitivity and specificity of the antibody against human MTERF3 by enzyme-linked immunosorbent assay and Western blot assay. Furthermore, the full-length hMTERF3 protein expressed in human embryonic kidney 293T cells was detected by anti-GST-hMTERF3 in western blot analysis and immunofluorescence staining. Taken together, our results demonstrate the functionality of the mouse anti-GST-hMTERF3 polyclonal antibody which will provide a useful tool for further characterization of hMTERF3.

Keywords: Mitochondrial transcription termination factor 3 (MTERF3); Prokaryotic expression; Recombinant protein; Purification; Polyclonal antibody

An Improved Process of Ethanol Production from Hemicellulose: Bioconversion of Undetoxified Hemicellulosic Hydrolyzate from Steam-Exploded Corn Stover with a Domesticated Pichia stipitis by Qiang Yong; Xin Li; Yun Yuan; Chenhuan Lai; Nannan Zhang; Qiulu Chu; Yong Xu; Shiyuan Yu (pp. 2330-2340).

Bioconversion of undetoxified hemicellulosic hydrolyzate from steam-exploded corn stover was investigated with a domesticated Pichia stipitis CBS 5776. The countercurrent washing was applied to recover sugars from the steam-exploded corn stover, which could enrich sugars in washing liquor and give an efficient saving of water. Acid concentration, reaction temperature, and time were optimized for the acid post-hydrolysis of oligosaccharides in steam-exploded prehydrolyzate by a central composite design and response surface methodology. The domestication of P. stipitis to the hydrolyzate resulted in improving sugar consumption and ethanol yield by gradually increasing the ratio of hydrolyzate in the medium. Recycling utilization of the domesticated yeast demonstrated that the yeast kept a stable ability of fermenting both hexose and pentose in the undetoxified hydrolyzate. The sugar consumption and ethanol yield were over 90 and 80 %, respectively.

Keywords: Corn stover prehydrolyzate; Acid post-hydrolysis; Countercurrent washing; Domestication; Pichia stipitis

Expression and Characterization of Codon-Optimized Carbonic Anhydrase from Dunaliella Species for CO₂ Sequestration Application by Bashistha Kumar Kanth; Kiha Min; Shipra Kumari; Hancheol Jeon; Eon Seon Jin; Jinwon Lee; Seung Pil Pack (pp. 2341-2356).

Carbonic anhydrases (CAs) have been given much attention as biocatalysts for CO₂ sequestration process because of their ability to convert CO₂ to bicarbonate. Here, we expressed codon-optimized sequence of α-type CA cloned from Dunaliella species (Dsp-aCAopt) and characterized its catalyzing properties to apply for CO₂ to calcite formation. The expressed amount of Dsp-aCAopt in Escherichia coli is about 50 mg/L via induction of 1.0 mM isopropyl-β-d-thiogalactopyranoside at 20 °C (for the case of intact Dsp-aCA, negligible). Dsp-aCAopt enzyme shows 47 °C of half-denaturation temperature and show wide pH stability (optimum pH 7.6/10.0). Apparent values of K _m and V _max for p-nitrophenylacetate substrate are 0.91 mM and 3.303 × 10⁻⁵ μM min⁻¹. The effects of metal ions and anions were investigated to find out which factors enhance or inhibit Dsp-aCAopt activity. Finally, we demonstrated that Dsp-aCAopt enzyme can catalyze well the conversion of CO₂ to CaCO₃, as the calcite form, in the Ca²⁺ solution [8.9 mg/100 μg (172 U/mg enzyme) with 10 mM of Ca²⁺]. The obtained expression and characterization results of Dsp-aCAopt would be usefully employed for the development of efficient CA-based system for CO₂-converting/capturing processes.

Keywords: Alpha-carbonic anhydrase; Codon optimization; Dunaliella species; Inhibitory effects; CO₂ sequestration

Identification of Newly Zeaxanthin-Producing Bacteria Isolated from Sponges in the Gulf of Thailand and their Zeaxanthin Production by Patcharee Thawornwiriyanun; Somboon Tanasupawat; Chutiwan Dechsakulwatana; Somkiet Techkarnjanaruk; Worapot Suntornsuk (pp. 2357-2368).

Sponge-associated bacteria have been found to produce a variety of bioactive compounds including natural pigments. Here, we report the molecular identification of zeaxanthin-producing sponge-associated bacteria isolated from sponges in the Gulf of Thailand and the effect of environmental factors on zeaxanthin production from a bacterium. Three colorful sponge-associated bacteria (CHOB06-6, KODA19-6, and MAKB08-4) were identified based on the 16S rDNA profile. The 16S rDNA sequence-based analyses revealed that CHOB 06-6 and MAKB 08-4 were the closest relatives to Sphingomonas phyllosphaerae FA2^T, and KODA19-6 was a relative of Shingomonas (Blastomonas) natatoria DSM 3183^T. After all bacteria were cultivated in a modified Zobell medium, S. natatoria KODA19-6 was found to produce the highest zeaxanthin at 0.62 mg/l. pH and temperature considerably affected its zeaxanthin production. Its optimal condition for zeaxanthin production was found at a pH of 7 and 30 °C. The bacterium had a maximum specific growth rate (μ _max) of 0.06 1/h with zeaxanthin productivity (Q _p) of 6.27 μg/l·h. Therefore, this newly zeaxanthin-producing bacterium has a potential to produce natural zeaxanthin for the food, feed, pharmaceutical, and cosmetic industries.

Keywords: Identification; Zeaxanthin; Sponge-associated bacteria; Production; Sphingomonas phyllosphaerae ; Sphingomonas natatoria

Potential of Thermo and Alkali Stable Xylanases from Thielaviopsis basicola (MTCC-1467) in Biobleaching of Wood Kraft Pulp by Baby Rani Goluguri; Chiranjeevi Thulluri; Madhu Cherupally; Nagaraju Nidadavolu; Das Achuthananda; Lakshmi Narasu Mangamuri; Uma Addepally (pp. 2369-2380).

Thermo- and alkali-stable xylanases produced from Thielaviopsis basicola (MTCC-1467) on low-cost carbon source like rice straw were evaluated for their potential application in biobleaching of wood kraft pulp. Enzyme treatment at retention time of 240 min with 20 IU/gm of dried pulp resulted in ~85.2 % of reduction in kappa number. When compared to control, 110.8, 93, and 72.2 % of enhancement in brightness (percent International Organization of Standardization), whiteness, and fluorescence, respectively, were observed for enzyme-treated pulp. Spectroscopic analysis showed significant release of chromophoric compounds from enzyme-treated pulp. Furthermore, scanning electron microscope studies of unbleached and enzyme bleached pulp revealed the effectiveness of enzymatic treatment. The enzyme-treated pulp subjected to later stages of chemical bleaching resulted in 16 % decrease in chlorine consumption along with considerable reduction in chemical oxygen demand percentage (14.5 %) level of effluent. Various pulp properties like fiber length, fiber width, burst strength, burst index, tear strength, tear index, tensile strength, and breaking length were also significantly improved after enzyme treatment when compared to control.

Keywords: Xylanase; Thermostable; Alkalistable; Kraft pulp; Biobleaching

Investigation of Coculture of Human Adipose-Derived Stem Cells and Mature Adipocytes by Kedong Song; Wenfang Li; Hong Wang; Hai Wang; Tianqing Liu; Ruiming Ning; Ling Wang (pp. 2381-2387).

The purpose of this study was to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into adipocytes by coculturing them with human mature adipocytes. The transwell culture system was utilized for indirect coculture of hADSCs and human mature adipocytes at four different hADSCs-to-mature adipocytes ratios, i.e., 1:5, 1:1, 2:1, and 5:1. After 8 days of coculture, the Oil Red O and Trypan Blue stainings were performed for the evaluation of adipogenic differentiation of hADSCs. In addition, flow cytometric analysis and Hoechst 33342/PI double staining were performed after 20 days of coculture. The Oil Red O and Trypan Blue stainings showed that hADSCs with high viability could not differentiate into mature adipocytes after 8 or 20 days of coculture. However, flow cytometric analysis indicated that CD105 expression of hADSCs decreased after 20 days of coculture. These results indicated that hADSCs cocultured with human adult adipocytes could not successfully differentiate into adipocytes.

Keywords: Adipocytes; Adipose-derived stem cells; Coculture; Adipogenic differentiation; Oil Red O staining

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #8)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.167, #8)