Applied Biochemistry and Biotechnology (v.180, #8)

Chiral lactic acid and its ester derivatives are crucial building blocks and platforms in the generation of high value-added drugs, fine chemicals and functional materials. Optically pure D-lactic acid and its ester derivatives cannot be directly generated from fermentation and are quite expensive. Herein, we identified, heterologously expressed and functionally characterized one Bacillus esterase BSE01701 from the deep sea of the Indian Ocean. Esterase BSE01701 could enzymatically resolve inexpensive racemic methyl lactate and generate chiral D-methyl lactate. The enantiomeric excess of desired chiral D-methyl lactate and the substrate conversion could reach over 99 % and 60 %, respectively, after process optimization. Notably, the addition of 60 % (v/v) organic co-solvent heptane could greatly improve both the enantiomeric excess of D-methyl lactate and the conversion. BSE01701 was a very promising marine microbial esterase in the generation of chiral chemicals in industry.
Keywords: Bacillus esterase; Deep-sea microorganism; Kinetic resolution; D-methyl lactate; High stereo-selectivity

Protective Effect of Zingiber officinale Against Dalton’s Lymphoma Ascites Tumour by Regulating Inflammatory Mediator and Cytokines by Sundararaj Rubila; Thottiam Vasudevan Ranganathan; Kunnathur Murugesan Sakthivel (1482-1496).
The aim of the present investigation was to evaluate Zingiber officinale paste against Dalton’s lymphoma ascites (DLA)-induced tumours in Swiss albino mice. Experimental animals received Z. officinale paste (low dose 100 mg/kg bw and high dose 500 mg/kg bw) orally for eight alternative days. Treatment with Z. officinale paste showed significant increase in haemoglobin level and decrease in aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (γ-GT) level. Z. officinale paste reduced the inflammatory mediators and cytokine levels, such as inducible nitric oxide (iNOS), tumour necrosis factor level (TNF-α) and interleukin-1β (IL-1β). Treatment with Z. officinale paste also significantly increased the antioxidant enzyme level, such as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione transferase (GST), and decreased the lipid peroxidation. Treatment also increased the vitamin C and E levels in treated animals compared with the DLA-bearing host. Histopathological studies also confirmed the protective influence of Z. officinale paste against DLA. The present study suggested that Z. officinale paste could be used as natural spice and a potent antitumour agent.
Keywords: Zingiber officinale paste; Antitumour; GC-MS; Antioxidant enzymes; Cytokines; 6-gingerol

Overexpression of Δ12-Fatty Acid Desaturase in the Oleaginous Yeast Rhodosporidium toruloides for Production of Linoleic Acid-Rich Lipids by Yanan Wang; Sufang Zhang; Markus Pötter; Wenyi Sun; Li Li; Xiaobing Yang; Xiang Jiao; Zongbao K. Zhao (1497-1507).
Under nutrient-limited conditions, the red yeast Rhodosporidium toruloides can accumulate neutral lipids, of which the compositional fatty acids are mainly saturated and mono-unsaturated ones with 16 or 18 carbon atoms. To improve the linoleic acid content in the lipids, we enabled galactose-inducible expression of the gene encoding Δ12-fatty acid desaturase (FADS) from Mortierella alpina or Fusarium verticillioides by integration of the corresponding expression cassettes into the genome of R. toruloides haploid and diploid strains. The relative linoleic acid content increased up to fivefold and the final linoleic acid titer reached 1.3 g/L under flask culture conditions. Our results suggested that R. toruloides may be further explored as cell factory for production of high-valued lipids and other fatty acid derivatives as bio-based chemicals and fuels.
Keywords: Rhodosporidium toruloides ; Δ12-fatty acid desaturase; Linoleic acid; Microbial lipids; Genetic engineering

In this work, to elucidate why the acid-pretreated bamboo shows disappointingly low enzymatic digestibility comparing to the alkali-pretreated bamboo, residual lignins in acid-pretreated and kraft pulped bamboo were isolated and analyzed by adsorption isotherm to evaluate their extents of nonproductive enzyme adsorption. Meanwhile, physicochemical properties of the isolated lignins were analyzed and a relationship was established with non-productive adsorption. Results showed that the adsorption affinity and binding strength of cellulase on acid-pretreated bamboo lignin (MWLa) was significantly higher than that on residual lignin in pulped bamboo (MWLp). The maximum adsorption capacity of cellulase on MWLp was 129.49 mg/g lignin, which was lower than that on MWLa (160.25 mg/g lignin). When isolated lignins were added into the Avicel hydrolysis solution, the inhibitory effect on enzymatic hydrolysis efficiency of MWLa was found to be considerably stronger than that with MWLp. The cellulase adsorption on isolated lignins was correlated positively with hydrophobicity, phenolic hydroxyl group, and degree of condensation but negatively with surface charges and aliphatic hydroxyl group. These results suggest that the higher nonproductive cellulase adsorption and physicochemical properties of residual lignin in acid-pretreated bamboo may be responsible for its disappointingly low enzymatic digestibility.
Keywords: Moso bamboo; Pretreatment; Nonproductive adsorption; Adsorption isotherm; Physicochemical properties

Food Waste Fermentation to Fumaric Acid by Rhizopus arrhizus RH7-13 by Huan Liu; Jingyuan Ma; Meng Wang; Weinan Wang; Li Deng; Kaili Nie; Xuemin Yue; Fang Wang; Tianwei Tan (1524-1533).
Fumaric acid as a four-carbon unsaturated dicarboxylic acid is widely used in the food and chemical industries. Food waste (FW), rich in carbohydrates and protein, is a promising potential feedstock for renewable bio-based chemicals. In this research, we investigated the capability of Rhizopus arrhizus RH7-13 in producing fumaric acid from FW. The liquid fraction of the FW (L-FW) was proven to be the best seed culture medium in our research. When it was however used to be fermentation medium, the yield of fumaric acid reached 32.68 g/L, at a volumetric production of 0.34 g/L h. The solid fraction of FW mixed with water (S-FW) could also be used as fermentation medium when a certain amount of glucose was added, and the yield of fumaric acid reached 31.26 g/L. The results indicated that both fractions of FW could be well utilized in fermentation process and it could replace a part of common carbon, nitrogen, and nutrient. The process has an application potential since reducing the costs of raw materials.
Keywords: Food waste; Fumaric acid; Glucose; Rhizopus arrhizus ; Fermentation

The investigation for the first time assesses the efficacy of recycled de-oiled algal biomass extract (DABE) as a cultivation media to boost lipid productivity in Chlorella minutissima and its comparison with Bold’s basal media (BBM) used as control. Presence of organic carbon (3.8 ± 0.8 g/l) in recycled DABE resulted in rapid growth with twofold increase in biomass productivity as compared to BBM. These cells expressed four folds higher lipid productivity (126 ± 5.54 mg/l/d) as compared to BBM. Cells cultivated in recycled DABE showed large sized lipid droplets accumulating 54.12 % of lipid content. Decrement in carbohydrate (17.76 %) and protein content (28.12 %) with loss of photosynthetic pigments compared to BBM grown cells were also recorded. The fatty acid profiles of cells cultivated in recycled DABE revealed the dominance of C16:0 (39.66 %), C18:1 (29.41 %) and C18:0 (15.82 %), respectively. This model is self-sustained and aims at neutralizing excessive feedstock consumption by exploiting recycled de-oiled algal biomass for cultivation of microalgae, making the process cost effective.
Keywords: Biodiesel; Chlorella minutissima ; De-oiled algal extract; Lipid productivity

Overexpression of a Chitinase Gene from Trichoderma asperellum Increases Disease Resistance in Transgenic Soybean by Fuli Zhang; Xianle Ruan; Xian Wang; Zhihua Liu; Lizong Hu; Chengwei Li (1542-1558).
In the present study, a chi gene from Trichoderma asperellum, designated Tachi, was cloned and functionally characterized in soybean. Firstly, the effects of sodium thiosulfate on soybean Agrobacterium-mediated genetic transformation with embryonic tip regeneration system were investigated. The transformation frequency was improved by adding sodium thiosulfate in co-culture medium for three soybean genotypes. Transgenic soybean plants with constitutive expression of Tachi showed increased resistance to Sclerotinia sclerotiorum compared to WT plants. Meanwhile, overexpression of Tachi in soybean exhibited increased reactive oxygen species (ROS) level as well as peroxidase (POD) and catalase (SOD) activities, decreased malondialdehyde (MDA) content, along with diminished electrolytic leakage rate after S. sclerotiorum inoculation. These results suggest that Tachi can improve disease resistance in plants by enhancing ROS accumulation and activities of ROS scavenging enzymes and then diminishing cell death. Therefore, Tachi represents a candidate gene with potential application for increasing disease resistance in plants.
Keywords: Overexpression; Tachi ; Soybean transformation; Sclerotinia sclerotiorum resistance; Physiology

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting by Junko Masuda; Hiroshi Kawamoto; Warren Strober; Eiji Takayama; Akifumi Mizutani; Hiroshi Murakami; Tomokatsu Ikawa; Atsushi Kitani; Narumi Maeno; Tsukasa Shigehiro; Ayano Satoh; Akimasa Seno; Vaidyanath Arun; Tomonari Kasai; Ivan J. Fuss; Yoshimoto Katsura; Masaharu Seno (1559-1573).
Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.
Keywords: TCF3 (E2A); Artificial transcription factor; TALE technology

Lactic acid is widely used in chemical, pharmaceutical, cosmetic, and food industries, besides it is the building block to produce polylactic acid, which is a sustainable alternative biopolymer to synthetic plastic due to its biodegradability. Aiming at producing an optically pure isomer, the present work evaluated the potential of pulp mill residue as feedstock to produce d(−)-lactic acid by a strain of the bacterium Lactobacillus coryniformis subsp. torquens using separate hydrolysis and fermentation process. Enzymatic hydrolysis, optimized through response surface methodology for 1 g:4 mL solid/liquid ratio and 24.8 FPU/gcellulose enzyme loading, resulted in 140 g L−1 total reducing sugar and 110 g L−1 glucose after 48 h, leading to 61 % of efficiency. In instrumented bioreactor, 57 g L−1 of d(−)-lactic acid was achieved in 20 h of fermentation, while only 0.5 g L−1 of l(+)-lactic acid was generated. Furthermore, product yield of 0.97 g/g and volumetric productivity of 2.8 g L−1 h−1 were obtained.
Keywords: Pulp mill residue; Separate hydrolysis and fermentation; Enzymatic hydrolysis; d(−)-lactic acid; Lactobacillus coryniformis subsp. torquens

Elucidating the Effect of Glycerol Concentration and C/N Ratio on Lipid Production Using Yarrowia lipolytica SKY7 by Mathiazhakan Kuttiraja; Ayed Douha; Jose R. Valéro; Rajeswar Dayal Tyagi (1586-1600).
The high demand for renewable energy and increased biodiesel production lead to the surplus availability of crude glycerol. Due to the above reason, the bio-based value addition of crude glycerol into various bioproducts is investigated; among them, microbial lipids are attractive. The present study was dedicated to find the optimal glycerol concentration and carbon/nitrogen (C/N) ratio to produce maximum lipid using Yarrowia lipolytica SKY7. The glycerol concentration (34.4 to168.2 g/L) and C/N ratio (25 to 150) were selected to investigate to maximize the lipid production. Initial glycerol concentration 112.5 g/L, C/N molar ratio of 100, and with 5 % v/v inoculum supplementation were found to be optimum for biomass and lipid production. Based on the above optimal parameters, lipid concentration of 43.8 % w/w with a biomass concentration of 14.8 g/L was achieved. In the case of glycerol concentration, the maximum Yp/s (0.192 g/g); Yx/s (0.43 g/g) was noted when the initial glycerol concentration was 112.5 g/L with C/N molar ratio 100 and inoculum volume 5 % v/v. The glycerol uptake was also noted to increase with the increase in glycerol concentration. At low C/N ratio, the glycerol consumption was found to be high (79.43 g/L on C/N 25) whereas the glycerol consumption was observed to decrease when the C/N ratio was raised to 150 (40.8 g/L).
Keywords: Y. lipolytica ; Crude glycerol; Biodiesel; Microbial lipid; C/N ratio

Genome Shuffling and Gentamicin-Resistance to Improve ε-Poly-l-Lysine Productivity of Streptomyces albulus W-156 by Liang Wang; Xusheng Chen; Guangyao Wu; Xin Zeng; Xidong Ren; Shu Li; Lei Tang; Zhonggui Mao (1601-1617).
Genome shuffling has been a recently effective method for screening the desirable phenotypes of industrial strains. Here, we combined genome shuffling and gentamicin resistance to improve the production of ε-poly-l-lysine in Streptomyces albulus W-156. Five starting mutants with higher ε-poly-l-lysine (ε-PL) productivities were firstly obtained by atmospheric and room temperature plasma (ARTP) mutagenesis. After three rounds of genome shuffling with increasing concentration of gentamicin for selection, S. albulus AG3-28, was finally got with a production of 3.43 g/L in shaking flask. In a 5-L fermenter, AG3-28 exhibited a higher ε-PL productivity (56.5 g/L) than the initial strain W-156 (37.5 g/L). Key enzyme activities in primary and secondary metabolic pathways were analyzed, and the transcription levels of hrdD and pls were determined by quantitative real time-polymerase chain reaction (qRT-PCR). Increase of key enzyme activities and the upregulation of the gene transcriptional levels demonstrated that ε-PL synthetic pathway in AG3-28 was obviously strengthened, which might be responsible for the high productivity. Moreover, hyper-yield strain AG3-28 was found to produce a slightly lower ε-PL polymerization degree than the parent strain. Amplified fragment length polymorphism (AFLP) analysis reflects the genetic diversity among the derivates after genome shuffling.
Keywords: Genome shuffling; Gentamicin resistance mutation; ε-Poly-l-lysine; Key enzyme activities; qRT-PCR; AFLP; Polymerization

The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.
Keywords: Fed-batch fermentation; High-temperature process; Ogataea thermomethanolica ; Phytase; Table sugar; Thermotolerant methylotrophic yeast

Overproduction, Purification and Characterization of Adenylate Deaminase from Aspergillus oryzae by Shubo Li; Yi Qian; Yunlong Liang; Xinkuan Chen; Mouming Zhao; Yuan Guo; Zongwen Pang (1635-1643).
Adenylate deaminase (AMPD, EC 3.5.4.6) is an aminohydrolase that widely used in the food and medicine industries. In this study, the gene encoding Aspergillus oryzae AMPD was cloned and expressed in Escherichia coli. Induction with 0.75 mM isopropyl β-d-l-thiogalactopyranoside resulted in an enzyme activity of 1773.9 U/mL. Recombinant AMPD was purified to electrophoretic homogeneity using nickel affinity chromatography, and its molecular weight was calculated as 78.6 kDa. Purified AMPD exhibited maximal activity at 35 °C, pH 6.0 and 30 mM K+, with apparent K m and V max values of 2.7 × 10−4 M and 77.5 μmol/mg/min under these conditions. HPLC revealed that recombinant AMPD could effectively catalyse the synthesis of inosine-5′-monophosphate (IMP) with minimal by-products, indicating high specificity and suggesting that it could prove useful for IMP production.
Keywords: Adenylate deaminase; Aspergillus oryzae ; Purification; Catalytic properties; Inosine-5ʹ-monophosphate

Selection and Identification of Chloramphenicol-Specific DNA Aptamers by Mag-SELEX by Ye Duan; Zhiqiang Gao; Lihui Wang; Huishan Wang; Hexiao Zhang; Hao Li (1644-1656).
Chloramphenicol (CAP) has been widely used to treat bacterial infections in livestock and aquatic animals. To reduce the risk of CAP residues, an efficient technology to rapidly detect CAP residues in animal-sourced food is expressly needed. In this study, magnetic bead-based systematic evolution of ligands by exponential enrichment (Mag-SELEX) strategy was performed to select and identify CAP-specific single-stranded DNA (ssDNA) aptamers from a random oligonucleotide library. After nine rounds of selection, five potential ssDNA aptamers were selected. Low homology indicated that they might belong to different families. To identify an aptamer with the highest affinity for CAP, the dissociation constant (K d) values of these selected aptamers were determined. The lowest K d values of two potential aptamers (i.e., No. 4 and No. 5) were, respectively, 0.10162 ± 0.0111 and 0.03224 ± 0.00819 μM, which were much lower than previously reported lowest K d value (i.e., 0.766 μM) of CAP aptamer. Moreover, compared with No. 4, aptamer No. 5 had higher binding rate, which is quite different among those with CAP and with CAP’s structural analogs (i.e., thiamphenicol (TAP) and florfenicol (FF)). These results indicated that the potential aptamer No. 5 with highest specificity and affinity for CAP would be an ideal aptamer for future detection of residual CAP in animal-sourced food.
Keywords: ssDNA aptamer; Chloramphenicol; Drug residue detection; Mag-SELEX; Specificity and affinity

Sudden death syndrome (SDS) is a complex of two diseases of soybean (Glycine max), caused by the soil borne pathogenic fungus Fusarium virguliforme. The root rot and leaf scorch diseases both result in significant yield losses worldwide. Partial SDS resistance has been demonstrated in multiple soybean cultivars. This study aimed to highlight proteomic changes in soybean roots by identifying proteins which are differentially expressed in near isogenic lines (NILs) contrasting at the Rhg1/Rfs2 locus for partial resistance or susceptibility to SDS. Two-dimensional gel electrophoresis resolved approximately 1000 spots on each gel; 12 spots with a significant (P < 0.05) difference in abundance of 1.5-fold or more were picked, trypsin-digested, and analyzed using quadruple time-of-flight tandem mass spectrometry. Several spots contained more than one protein, so that 18 distinct proteins were identified overall. A functional analysis performed to categorize the proteins depicted that the major pathways altered by fungal infection include disease resistance, stress tolerance, and metabolism. This is the first report which identifies proteins whose abundances are altered in response to fungal infection leading to SDS. The results provide valuable information about SDS resistance in soybean plants, and plant partial resistance responses in general. More importantly, several of the identified proteins could be good candidates for the development of SDS-resistant soybean plants.
Keywords: Soybean; Proteomics; 2D gel electrophoresis; Sudden death syndrome; Rhg1/Rfs2 locus