Applied Biochemistry and Biotechnology (v.180, #6)

The textile-dyeing industry is rated as one of the foremost industrial sectors that explodes large amount of pollutants to the environment. Reactive azo dye degradation, being a major constituent of these pollutants and perilous material, has been constantly receiving scientific attention. In textile industry, use of Remazole Brilliant Violet 5R (RBV5R) as reactive azo dyes is more frequent. Highly competent, RBV5R-degrading bacterial consortium VIE6 was developed from the soil of the Vatva Industrial Estate, Gujarat, India. Consortium VIE6 comprised of five bacterial strains Bacillus sp. DMB1, Staphylococcus sp. DMB2, Escherichia sp. DMB3, Enterococcus sp. DMB4, and Pseudomonas sp. DMB5. These strains convened a better decolorization efficiency between 200 and 1000 mg/L of dye concentration and were much stable at pH 6.5, 37 °C. Azoreductase, laccase, and lignin peroxidase activities of consortium showed significant variation throughout the degradation process indicating the different metabolic capabilities of the existing microflora. The community interactions and synergism were shown to facilitate the biotransformation of RBV5R by combination of various electron donors. Voltammograms revealed the variations in electron discharge properties which coincide with the dynamics of community derived using qPCR assays. The variation in catabolic capabilities of the individual strains was observed during active metabolism of RBV5R degradation pertaining to the aerobic and facultative functions.
Keywords: Azo dye; Metabolism; Dynamics; Microaerophilic; Consortium; Phytotoxicity; Biotransformation

Enhancement of Lipid Production of Chlorella Pyrenoidosa Cultivated in Municipal Wastewater by Magnetic Treatment by Songfang Han; Wenbiao Jin; Yangguang Chen; Renjie Tu; Abd El-Fatah Abomohra (1043-1055).
Despite the significant breakthroughs in research on microalgae as a feedstock for biodiesel, its production cost is still much higher than that of fossil diesel. One possible solution to overcome this problem is to optimize algal growth and lipid production in wastewater. The present study examines the feasibility of using magnetic treatment for enhancement of algal lipid production and wastewater treatment in outdoor-cultivated Chlorella pyrenoidosa. Results confirmed that magnetic treatment significantly enhances biomass and lipid productivity of C. pyrenoidosa by 12 and 10 %, respectively. Application of magnetic field in a semi-continuous culture resulted in highly treated wastewater with total nitrogen maintained under 15 mg L−1, ammonia nitrogen below 5 mg L−1, total phosphorus less than 0.5 mg L−1, and CODCr less than 50 mg L−1. In addition, magnetic treatment resulted in a decrease of wastewater turbidity, an increase of bacterial numbers, and an increase of active oxygen in wastewater which might be attributed to the enhancement of growth and lipid production of C. pyrenoidosa.
Keywords: Magnetic treatment; Chlorella pyrenoidosa ; Lipid enhancement; Wastewater treatment; Biodiesel

Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity by Edgar Omar Mendoza-Llerenas; David Javier Pérez; Zeferino Gómez-Sandoval; Pilar Escalante-Minakata; Vrani Ibarra-Junquera; Rodrigo Said Razo-Hernández; Vittorio Capozzi; Pasquale Russo; Giuseppe Spano; Daniela Fiocco; Juan Alberto Osuna-Castro; Abel Moreno (1056-1075).
β-Fructosidase, a glycoside hydrolase of a biotechnologically important strain, was studied for its biochemical, physicochemical, and three-dimensional structure characteristics. This enzyme was heterologously expressed in Escherichia coli as a C-terminal His-tagged protein (SacB). β-Fructosidase catalyzes the cleavage of glycoside bonds toward certain carbohydrates with β-fructofuranosyl linkages; however, SacB exhibited selectivity toward sucrose and an optimum activity at pH 6.0–6.5 and 37 °C. In such optimum enzymatic activity conditions, the SacB was commonly observed as a monodisperse protein by dynamic light scattering (DLS). As β-fructosidase belongs to glycoside hydrolase family 32 (GH32), a β-sandwich and a five-bladed β-propeller domain are typical predicted folds in its structure. Docking and molecular dynamic simulations revealed for the first time a funnel-like channel perfectly exposed in the β-propeller domain of the Lactobacillus plantarum β-fructosidase (this allows the interaction between its entire catalytic triad and substrates that are larger than sucrose). In contrast, SacB showed a closed central tunnel collaterally induced by its His-tag.
Keywords: Lactobacillus plantarum ; β-Fructosidase; Invertase; β-Propeller domain; Selectivity; Computer simulations

Prunella vulgaris L. (P. vulgaris) is an important medicinal plant with a wide range of antiviral properties. Traditionally, it is known as self-heal because of its faster effects on wound healing. It is commonly known as a natural antiseptic due to the presence of various polyphenols. There is lack of research efforts on its propagation and production of bioactive compounds under field and in vitro conditions. In this study, the effects of different ratios (1:2, 1:3, 2:1, and 3:1) of silver (Ag) and gold (Au) nanoparticles (NPs) alone or in combination with naphthalene acetic acid (NAA) were investigated for callus culture development and production of secondary metabolites. The Ag (30 μg l−1), AgAu (1:2), and AgAu (2:1) NPs in combination with NAA (2.0 mg l−1) enhanced callus proliferation (100 %) as compared to the control (95 %). Among the different NPs tested, AuNPs with or without NAA produced higher biomass in log phases (35–42 days) of growth kinetics. Furthermore, AgAu (1:3) and AuNPs alone enhanced total protein content (855 μg-BSAE/mg-fresh weight (FW)), superoxide dismutase (0.54 nM/min/mg-FW), and peroxidase (0.39 nM/min/mg-FW) enzymes in callus cultures. The AgAuNPs (1:3) in combination with NAA induced maximum accumulation of phenolics (TPC 9.57 mg/g-dry weight (DW)) and flavonoid (6.71 mg/g-DW) content. Moreover, AgAuNPs (3:1) without NAA enhanced antioxidant activity (87.85 %). This study provides the first evidence of NP effect on callus culture development and production of natural antioxidants in P. vulgaris.
Keywords: Prunella vulgaris ; Nanoparticles; In vitro cultures; Secondary metabolism; Naphthalene acetic acid

Endophytes from medicinal plants represent a potential source of bioactive compounds. During the present investigation, fungal endophytes were isolated from turmeric (Curcuma longa), an important medicinal plant. A total of 207 endophytic fungal isolates were obtained from the rhizome of C. longa L. They were grouped into seven genera based on morphological and molecular data. The fungal endophytes of C. longa were evaluated for antifungal activity against Colletotrichum gloeosporioides, the causal organism of leaf spot of turmeric. The disease is a major cause for economic loss in turmeric cultivation. Endophytic Phoma herbarum showed significant activity against C. gloeosporioides and was therefore selected for further studies. A compound gentisyl alcohol was isolated from P. herbarum which showed effective antagonism against C. gloeosporioides. The organism could therefore be used as a biocontrol agent against C. gloeosporioides.
Keywords: Turmeric; Endophytic fungi; Phoma ; Leaf spot; Colletotrichum gloeosporioides ; Gentisyl alcohol

Simultaneous Biosynthesis of Polyhydroxyalkanoates and Extracellular Polymeric Substance (EPS) from Crude Glycerol from Biodiesel Production by Different Bacterial Strains by Denilson de Jesus Assis; Gleice Valéria Pacheco Gomes; Diego Roberto da Cunha Pascoal; Lorena Silva Pinho; Lilian Brandão Oliveira Chaves; Janice Izabel Druzian (1110-1127).
Simultaneous synthesis of polyhydroxyalkanoates (PHAs) and polyglutamic acid (PGA) was investigated in cultures of Cupriavidus necator IPT 026, C. necator IPT 027, C. necator IPT 029, and Bacillus megaterium INCQS 425 strains in a medium containing 2.0 % sucrose or crude glycerol from biodiesel (CGB), in an orbital shaker (35 °C, 180 rpm, 72 h). All the strains tested simultaneously produced PHA and PGA in a medium containing CGB. The C. necator IPT026 culture provided higher molecular mass PHA and PGA (1128.55 and 835.56 kDa, respectively). B. megaterium INCQS 425 promoted PGA production (1.90 g L−1) with higher crystalline melting temperature (84.04 °C) and higher initial decomposition temperature (247.10 °C). Furthermore, the latter culture promoted the production of medium- and long-chain PHA (0.78 g L−1) with high crystalline melting temperatures (∼170 °C) and high initial decomposition temperature (307.53 °C) and low degree of crystallinity (20.2 %). These characteristics render these PHAs more appropriate and suitable for processes that require high temperatures, such as extrusion, increasing the possibility of industrial applications, especially in the packaging sector.
Keywords: Polyhydroxyalkanoates; Polyglutamic acid; Crude glycerol; Simultaneous synthesis; Properties

The visceral mass of the gastropod, Harpa ventricosa was hydrolysed using trypsin, alcalase and pepsin for 12 h to produce protein hydrolysates. Subsequently, the active hydrolysate was observed in the 3rd hour of tryptic hydrolysate (29.17 ± 0.62 and 34.85 ± 0.55 %) using human red blood cell (HRBC) membrane stabilization and albumin denaturation (AD) assays. The active hydrolysate was fractionated by membrane filtration unit, where <10-kDa fraction revealed better anti-inflammatory activity with IC50 value 6.27 ± 0.05 and 5.38 ± 0.02 mg/ml for HRBC and AD assays, respectively. Additionally, the active fraction contains essential and non-essential (aspartic acid, arginine, glutamic acid and leucine) amino acids and, sequentially, the active fraction was further purified using consecutive chromatography, in which fraction C-II exhibited strong anti-inflammatory activity (HRBC 56.02 ± 0.52 and AD 50.71 ± 1.10 % assays). The non-toxic, low molecular weight (690.2 Da) hexapeptide (Ala-Lys-Gly-Thr-Trp-Lys) suppressed the nitric oxide (NO) and pro-cytokine production in a dose-dependent manner on THP-1 cell lines.
Keywords: Hydrolysate; Peptide; ESI-MS/MS; Gastropod; Anti-inflammatory

In the present study, the effect of the type of sugar cane bagasse (non-depithed or depithed) and its particle size on the production of xylose and its subsequent fermentation to xylitol by Debaryomyces hansenii CBS767 was investigated using a full factorial experimental design. It was found that the particle size range and whether bagasse was depithed or not had a significant effect on the concentration and yield of xylose in the resulting hemicellulose hydrolysate. Depithed bagasse resulted in higher xylose concentrations compared to non-depithed bagasse. The corresponding detoxified hemicellulose hydrolysates were used as fermentation media for the production of xylitol. The hemicellulose hydrolysate prepared from depithed bagasse also yielded meaningfully higher xylitol fermentation rates compared to non-depithed bagasse. However, in the case of non-depithed bagasse, the hemicellulose hydrolysate prepared from larger particle size range resulted in higher xylitol fermentation rates, whereas the effect in the case of non-depithed bagasse was not pronounced. Therefore, depithing of bagasse is an advantageous pretreatment when it is to be employed in bioconversion processes.
Keywords: Sugar cane bagasse; Depithing; Particle size; Xylose fermentation; Xylitol

Carbon Catabolite Regulation of Secondary Metabolite Formation and Morphological Differentiation in Streptomyces coelicolor by A. Romero-Rodríguez; B. Ruiz-Villafán; V. H. Tierrafría; R. Rodríguez-Sanoja; S. Sánchez (1152-1166).
In the genus Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. However, there is little information about the mechanism involved in these effects. In the present work, it was found that glucose exerted a suppressive effect on the Streptomyces coelicolor actinorhodin (Act) and undecylprodigiosin (Red) production, as well as in its morphological differentiation. Accordingly, using a high-density microarray approach in S. coelicolor grown under glucose repression, at early growth stages, a negative effect was exerted on the transcription of genes involved in Act and Red production, when compared with non-repressive conditions. Seven genes of Act and at least ten genes of Red production were down-regulated by glucose. Stronger repression was observed on the initial steps of antibiotics formation. On the contrary, the coelimycin P1 cluster was up-regulated by glucose. Regarding differentiation, no sporulation was observed in the presence of glucose and expression of a set of genes of the bld cascade was repressed as well as chaplins and rodlins genes. Finally, a series of transcriptional regulators involved in both processes were up- or down-regulated by glucose. This is the first global transcriptomic approach performed to understand the molecular basis of the glucose effect on the synthesis of secondary metabolism and differentiation in the genus Streptomyces. The results of this study are opening new avenues for further exploration.
Keywords: Transcriptomics; Streptomycetes; Microarray; Gene expression; Antibiotics

A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding by Zhanping Zhou; Shuangzhi Zhao; Yang Liu; Zhengying Chang; Yanhe Ma; Jian Li; Jiangning Song (1167-1179).
The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.
Keywords: Chitosanase; Glycoside hydrolase family 8; Aspartic acid; Enzyme-substrate interaction

The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine by Jixue Lu; Yonghui Zhang; Dongfang Sun; Wei Jiang; Shizhen Wang; Baishan Fang (1180-1195).
Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99 % e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.
Keywords: Enzyme complex; Leucine dehydrogenase; Formate dehydrogenase; L-tert-leucine; Scaffoldin; Acceleration factor

One novel microbial esterase PHE21 was cloned from the genome of Pseudomonas oryzihabitans HUP022 identified from the deep sea of the Western Pacific. PHE21 was heterologously expressed and functionally characterized to be a robust esterase which behaved high resistance to various metal ions, organic solvents, surfactants, and NaCl. Despite the fact that the two enantiomers of ethyl 3-hydroxybutyrate were hard to be enzymatically resolved before, we successfully resolved racemic ethyl 3-hydroxybutyrate through direct hydrolysis reactions and generated chiral ethyl (S)-3-hydroxybutyrate using esterase PHE21. After process optimization, the enantiomeric excess, the conversion rate, and the yield of desired product ethyl (S)-3-hydroxybutyrate could reach 99, 65, and 87 %, respectively. PHE21 is a novel marine microbial esterase with great potential in asymmetric synthesis as well as in other industries.
Keywords: Deep-sea microorganism; Robust esterase; Kinetic resolution; Ethyl (S)-3-hydroxybutyrate; High stereo-selectivity

Construction and Analysis of an Adipose Tissue-Specific and Methylation-Sensitive Promoter of Leptin Gene by Qinkai Zhang; Denggao Xu; Min Zhang; Xiao Dong; Huansheng Dong; Qingjie Pan (1213-1226).
DNA methylation plays a very important role in the regulation of gene expression. Under general situations, methylation in a gene promoter region is frequently accompanied by transcriptional suppression, and those genes that are highly methylated display the phenomenon of low expression. In contrast, those genes whose methylation level is low display the phenomenon of active expression. In this study, we conducted DNA methylation analysis on the CpG sites within the promoter regions of five adipose tissue-specific transcriptional factors—Adiponectin, Chemerin, Leptin, Smaf-1, and Vaspin—and examined their messenger RNA (mRNA) expression levels in different mouse tissues. We also performed analyses on the correlation between the DNA methylation levels of these genes and their mRNA expression levels in these tissues. The correlation coefficient for Leptin was the highest, and it displayed a high expression in an adipose tissue-specific manner. Thus, we cloned the regulatory region of Leptin gene and incorporated its promoter into the eukaryotic expression vector pEGFP-N1 and constructed a recombinant plasmid named pEGFP-N1-(p-Lep). This recombinant plasmid was first verified by DNA sequencing and then transfected into mouse pre-adipocytes via electroporation. Measurement of the activity of luciferase (reporter) indicated that p-Lep was capable of driving the expression of the reporter gene. This study has paved a solid basis for subsequent studies on generating transgenic animals.
Keywords: Adipose tissue; Gene expression; DNA methylation; Leptin ; Promoter

Enhancement of Nutritional and Antioxidant Properties of Peanut Meal by Bio-modification with Bacillus licheniformis by Xinjian Yang; Da Teng; Xiumin Wang; Qingfeng Guan; Ruoyu Mao; Ya Hao; Jianhua Wang (1227-1242).
Peanut meal (PM) is limited in practical use (feed or food) from imbalance of amino acid profile and denaturation of protein. Fermentation was used to promote its nutritional and functional properties by single-factor experiments and orthogonal experiments. Results showed that the nutritional properties of fermented peanut meal (crude protein content, dry matter content, ash content, acid soluble oligopeptides content, in vitro digestibility, and content of organic acids) had a significant increase (P < 0.05 or P < 0.01) and more importantly, the content of amino acids was balanced by fermentation. In addition, fermented peanut meal possessed better antioxidant activities in the areas of reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radical scavenging activity, hydroxyl radical scavenging activity, and metal chelating activity (P < 0.05 or P < 0.01). These results implied that the nutritional and antioxidant properties of peanut meal were improved effectively by biological modification, which could be valuable in terms of nutrition and protein resources. It is great of importance to meet requirement of raw materials for husbandry in China when facing a huge lacking of feedstuff, especially for protein feed with an over 80 % import amount depending from other countries yearly.
Keywords: Bio-modification; Solid-state fermentation optimization; Peanut meal; Bacillus licheniformis ; Nutritional property; Antioxidant activity

Cloning and Expression Analysis of Eight Upland Cotton Pentatricopeptide Repeat Family Genes by Zongfu Han; Yuxiang Qin; Fanjin Kong; Yongsheng Deng; Zongwen Wang; Guifang Shen; Jinghui Wang; Bing Duan; Ruzhong Li (1243-1255).
The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants. Most PPR genes are localized in mitochondria and chloroplasts functioning in regulation of plant growth and development, fertility restoration for cytoplasmic male sterility (CMS), and stress defense. In this study, using in silico cloning and PCR amplification with degenerate primers based on Arabidopsis PPR genes, we cloned eight new full-length PPR genes encoding protein sequences ranging from 458 to 875 amino acids, with 8 to 16 repetitive PPR elements in upland cotton and all of them lack introns. Expression analysis revealed that eight PPR genes were differently expressed in roots, stems, leaves, and floral buds. As for GhI12, its expression in floral buds at days 3–5 was significantly higher in line 777R (restorer line) than in line 777A (CMS line). Further tests with real-time PCR showed that GhI12 expression peaked at day 3 in 777R, followed by a gradual decline, while its expression fluctuated in 777A, peaking at day 5 and day 13. In addition, Gh155c17 and GhI12 were upregulated under salt stress. This is the first report of upland cotton PPR genes involved in salt stress response.
Keywords: Cotton; PPR protein; Cloning; Expression; Salt tolerance