Applied Biochemistry and Biotechnology (v.179, #3)

Production of Butyrate from Lactate by a Newly Isolated Clostridium sp. BPY5 by Yong Tao; Xiaohong Hu; Xiaoyu Zhu; Hong Jin; Zhancheng Xu; Qinglan Tang; Xiangzhen Li (361-374).
Lactate-utilizing bacteria play important roles in the production of Chinese strong-flavored liquor (CSFL). However, the identity of these bacteria and their lactate-utilizing properties are largely unknown. Here, a lactate-utilizing, butyrate-producing bacterium BPY5 was isolated from an old fermentation pit for CSFL production. The isolate represented a novel species belonging to Clostridium cluster XIVa of family Lachnospiraceae based on phylogenetic analysis using 16S rRNA gene sequences. Strain BPY5 could ferment lactate into butyrate as the major metabolic product. Butyrate was significantly formed at initial lactate concentration from 66 to 104 mM, but substantially declined when initial lactate exceeded 133 mM. At initial lactate concentration of 66 mM, lactate conversion was independent on initial pH from 5.5 to 7.0, but the conversion was completely inhibited when pH dropped below 4.8. Nevertheless, the inhibition on lactate conversion was largely relieved by the addition of acetate, suggesting that exogenous acetate could enhance lactate conversion at low pH condition. Additionally, lactate in CSFL-brewing wastewater was dramatically removed when inoculated with strain BPY5. These results implicate that the isolate may be applied for the industrial production of butyrate or the recovery of butyrate from lactate-containing wastewater.
Keywords: Clostridium cluster XIVa; Lactate conversion; Butyrate production; CSFL brewing fermentation; Wastewater

Development of an in-House TaqMan Real-Time PCR-Based Method to Detect Residual Host Cell DNA in HBV Vaccine by Mahdi Paryan; Mana Khodayar; Vahid Kia; Samira Mohammadi–Yeganeh; Hooman Kaghazian (375-382).
Biological therapeutic products such as recombinant hepatitis B virus (HBV) vaccine, produced by microbial fermentation in complex media, should be evaluated for host cell DNA contamination in purification steps. Eliminating these contaminations increases the efficacy of the vaccine and decreases its side effects. The objective of the present study is to trace the residual host cell DNA (HCD) in recombinant HBV vaccine by developing a TaqMan Real-Time PCR method which is more sensitive, specific, and reproducible than traditional methods such as Picogreen analysis and Threshold DNA assay. Primers and a probe were designed for the most highly conserved regions of Pichia pastoris genome. To determine the specificity of the assay, in addition to performing a BLAST for the primers and the probe in NCBI nucleotide database, 20 different human genomes and 8 bacterial and viral genomes were used. Moreover, serial dilutions of plasmids, from 102 to 107 copies/μL (from 0.00064 to 6.4 pg/μL), were prepared to find the sensitivity and the limit of detection (LOD) of the assay. Using 28 different genome samples, the specificity of the assay was determined to be 100 %. In addition, the sensitivity and LOD of the method was 0.39 × 10−5 pg/μL. Moreover, the reproducibility of the assay based on intra- and inter-assay was 1.03 and 1.06 %, respectively. Considering the suitable specificity and sensitivity, ease of use, relatively low cost, and rapidity of the assay, it can be a reproducible and sensitive method to examine recombinant vaccines for P. pastoris residual DNA.
Keywords: HBV vaccine; Pichia pastoris ; TaqMan real-time PCR; Residual host DNA; Contamination

Polyacrylamide Gel-Entrapped Maltase: An Excellent Design of Using Maltase in Continuous Industrial Processes by Muhammad Asif Nawaz; Afsheen Aman; Haneef Ur Rehman; Zainab Bibi; Asma Ansari; Ziaul Islam; Ishtiaq Ahmad Khan; Shah Ali Ul Qader (383-397).
Bacterial maltase catalyzes the hydrolysis of maltose and is known as one of the most significant hydrolases. It has several applications in different industrial processes but widely used in food fermentation technology and alcohol production. In the current study, entrapment technique was comprehensively examined using polyacrylamide gel as a matrix support to improve the stability and catalytic efficiency of maltase for continuous use. Maximum entrapment yield of maltase was achieved at 10 % polyacrylamide concentration with 3.0-mm bead size. Optimized conditions indicated an increase in the reaction temperature from 45 to 55 °C after maltase entrapment while no change was observed in the reaction time and pH. An increase in the K m value of entrapped maltase was attained whereas V max value decreased from 8411.0 to 6813.0 U ml−1 min−1 with reference to its free counterpart. Entrapped maltase showed remarkable thermal stability and retained 16 % activity at 70 °C even after 120.0 min. Entrapped maltase also exhibited excellent recycling efficiency up to eight consecutive reaction cycles. With respect to economic feasibility, entrapped maltase indicates its high potential to be used in various biotechnological applications.
Keywords: Maltase; Polyacrylamide; Entrapment; Thermal stability; Recycling efficiency

Challenges and Opportunities in the Development of Aptamers for TNFα by Claudia Nübel; Bettina Appel; Ingeborg Hospach; Michaela Mai; Nadejda Krasteva; Gabriele Nelles; Lothar Petruschka; Sabine Müller (398-414).
RNA aptamers for tumor necrosis factor-alpha (TNFα), for which functionality was demonstrated in L929 cells, show only little affinity for the protein in vitro. Detailed investigation of the aptamer-protein interaction by surface plasmon resonance and quartz crystal microbalance analysis revealed that affinity is not the only crucial parameter for efficacy and functionality of those aptamers. Instead, the sensitive equilibrium of the monomeric and homotrimeric form of soluble TNFα decides on aptamer binding. Our results show that the field of application and the source of TNFα have to be carefully defined before selection of aptamer sequences.
Keywords: Aptamer; Biotin linker; Cytokine; In vitro selection; QCM; Rational design; SPR; ELISA; TNFα

GH53 Endo-Beta-1,4-Galactanase from a Newly Isolated Bacillus licheniformis CBMAI 1609 as an Enzymatic Cocktail Supplement for Biomass Saccharification by Evandro Antonio de Lima; Carla Botelho Machado; Letícia Maria Zanphorlin; Richard John Ward; Hélia Harumi Sato; Roberto Ruller (415-426).
Galactanases (endo-β-1,4-galactanases—EC 3.2.1.89) catalyze the hydrolysis of β-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-β-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5–8 and 40 °C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75 % activity even after 24 h of incubation at pH 4–10. The enzyme was thermostable, retaining nearly 100 % activity after 1-h incubation at pH 7.0 at 25–45 °C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2 s−1 mg−1 mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE®1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25 %.
Keywords: Bacillus licheniformis ; Bioenergy; Biomass saccharification; Cocktail supplementation; Heterologous expression; Galactanases; Pectinases

The methanolic extract of E. scaber Linn was evaluated for anti-inflammatory activity by determining its effects on production of pro-inflammatory cytokines like Tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) in Lipopolysaccharide (LPS) stimulated monocytes. The cytotoxicity of the extract was analyzed prior to the cytokine quantification assays. The extract was further subjected to UPLC MS Q-TOF, for the identification of bioactive components present in the crude extract. The extract was found not to be cytotoxic against monocytes, and exhibited significant inhibition in the production of pro-inflammatory cytokines. The presence of 34 components in the methanolic extract was detected through mass spectrum analysis.
Keywords: Elephantopus scaber ; Inflammation; Monocytes; Pro-inflammatory cytokines; Tumor necrosis factor-α; Interleukin-1β; Mass spectrometry

Microbial 5-aminolevulinic acid (ALA) produced from wastewater is considered as potential renewable energy. However, many hurdles are needed to be overcome such as the regulation of key influencing factors on ALA yield. Biomass and ALA production by Rhodobacter sphaeroides was optimized using response surface methodology. The culturing medium was artificial volatile fatty acids wastewater. Three additives were optimized, namely succinate and glycine that are precursors of ALA biosynthesis, and D-glucose that is an inhibitor of ALA dehydratase. The optimal conditions were achieved by analyzing the response surface plots. Statistical analysis showed that succinate at 8.56 mmol/L, glycine at 5.06 mmol/L, and D-glucose at 7.82 mmol/L were the best conditions. Under these optimal conditions, the highest biomass production and ALA yield of 3.55 g/L and 5.49 mg/g-biomass were achieved. Subsequent verification experiments at optimal values had the maximum biomass production of 3.41 ± 0.002 g/L and ALA yield of 5.78 ± 0.08 mg/g-biomass.
Keywords: Rhodobacter sphaeroides ; Biomass; 5-Aminolevulinic acid yield; Influencing factor; Response surface methodology; Wastewater

Control of Grifola frondosa Morphology by Agitation and Aeration for Improving Mycelia and Exo-Polymer Production by Feng-Jie Cui; Xiao-Xiao Chen; Wei-Min Liu; Wen-Jing Sun; Shuhao Huo; Yan Yang (459-473).
The present study describes the improved mycelia and exo-polymer production under control of Grifola frondosa morphology by changing the aeration rate and agitation intensity in a 25-L stirred fermentor. The aeration rate of 1.0 vvm yielded a highest mycelia biomass of 24.754 g/L with the lowest pellet percentage of 20.5 %. The maximum exo-polymer (2.324 g/L) was achieved at 0.75 vvm with mycelia polysaccharide production (0.321 g/g), whereas clumps and filaments dominated the ratios of 45.6 and 33.9 %, respectively. The change of aeration rate and agitation intensity had slight influence on the monosaccharide compositions in exo-polymers and significantly affected glucose and mannose contents in the mycelia polysaccharides. These findings will provide a clue for exploring the relationship between fermentation parameters, morphologies, and polysaccharide synthesis pathway of G. frondosa.
Keywords: Grifola frondosa ; Agitation intensity; Aeration rate; Morphology; Exo-polymer; Polysaccharide compositions

Semi-Rational Design of Geobacillus stearothermophilus L-Lactate Dehydrogenase to Access Various Chiral α-Hydroxy Acids by Aşkın Sevinç Aslan; William R. Birmingham; Nevin Gül Karagüler; Nicholas J. Turner; Barış Binay (474-484).
Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids.
Keywords: Chiral α-hydroxy acids (AHAs); Geobacillus stearothermophilus L-lactate dehydrogenase (bsLDH); Protein engineering; Modeling

Investigation of the Reuse of Immobilized Lipases in Biodiesel Synthesis: Influence of Different Solvents in Lipase Activity by Erika C. G. Aguieiras; Douglas S. Ribeiro; Pedro P. Couteiro; Caenam M. B. Bastos; Danielle S. de Queiroz; Juliana M. Parreira; Marta A. P. Langone (485-496).
Biodiesel production catalyzed by immobilized lipases offers the possibility of easy reuse of the catalyst, which is very important to minimize costs and to make this process economically feasible. In this study, the reuse of three commercial immobilized lipases (Novozym 435, Lipozyme RM IM, and Lipozyme TL IM) was investigated in ethanolysis of soybean oil. The effect of the use of solvents (ethanol, butanol, and hexane) to wash the immobilized lipases before the enzyme reuse was evaluated, as well as the lipase reuse without solvent washing. The washing with butanol and ethanol led to the lowest decrease in ester yield after the first batch and allowed the highest glycerol removal (>85 %) from biocatalysts. The biocatalysts were incubated at 50 °C for 2 h in these three solvents. Esterification activities of the enzyme preparations, scanning electron microscopy (SEM) analyses of the beads, and protein content in organic phase were evaluated before and after incubation in the solvent. SEM analysis showed a significant change in beads morphology of Novozym 435 after contact with hexane. For Lipozyme TL IM lipase, this effect was visualized with ethanol.
Keywords: Biodiesel; Immobilized lipase; Soybean oil; Reuse; Ethanol; Butanol; Hexane; Transesterification

Identification and Functional Characterization of a Fructooligosaccharides-Forming Enzyme from Aspergillus aculeatus by José Juan Virgen-Ortíz; Vrani Ibarra-Junquera; Pilar Escalante-Minakata; Sara Centeno-Leija; Hugo Serrano-Posada; José de Jesús Ornelas-Paz; Jaime David Pérez-Martínez; Juan Alberto Osuna-Castro (497-513).
Although fructosyltransferases from Aspergillus aculeatus have received a considerable interest for the prebiotics industry, their amino acid sequences and structural features remain unknown. This study sequenced and characterized a fructosyltransferase from A. aculeatus (AcFT) isolated by heat treatment of Pectinex Ultra SP-L. The AcFT enzyme showed two isoforms, low-glycosylated AcFT1 and high-glycosylated AcFT2 forms, with similar optimum activity at 60 °C. The purified heat-resistant AcFT1 and AcFT2 isoforms produced identical patterns of fructooligosaccharides (FOS; kestose, nystose and fructosylnystose) with a notable transfructosylation capability (~90 % transferase/hydrolase ratio). In contrast, the pI and optimum pH values exhibited discrete differences, attributable to their glycosylation pattern. Partial protein sequencing showed that AcFT enzyme corresponds to Aspac1_37092, a putative 654-residue fructosyltransferase encoded in the genome of A. aculeatus ATCC16872. A homology model of AcFT also revealed the typical fold common to members of the glycoside hydrolase family 32 (GH32), with an N-terminal five-blade β-propeller domain enclosing catalytic residues D60, D191, and E292, linked to a C-terminal β-sandwich domain. To our knowledge, this is the first report describing the amino acid sequence and structural features of a heat-resistant FOS-forming enzyme from A. aculeatus, providing insights into its potential applications in the prebiotics industry.
Keywords: Amino acid sequence; Fructosyltransferase; Heat treatment; Transfructosylation

Treatment of Common Effluent Treatment Plant Wastewater in a Sequential Anoxic–Oxic Batch Reactor by Developed Bacterial Consortium VN11 by Sananda Chattaraj; Hemant J. Purohit; Abhinav Sharma; Niti B. Jadeja; Datta Madamwar (514-529).
A laboratory-scale anoxic–oxic sequential reactor system was seeded with acclimatized mixed microbial consortium for the treatment of common effluent treatment plant (CETP) wastewater having 7000–7400 mg L−1 of COD and 3000–3400 mg L−1 of BOD. Initially, CETP wastewater was treated under anoxic reactor at 5000 mg L−1 of MLSS concentrations, 5.26 ± 0.27 kg COD m−3 day−1 of organic loading rate (OLR) and 36 h of hydraulic retention time (HRT). Further, the effluent of anoxic reactor was treated in oxic reactor with an OLR of 6.6 ± 0.31 kg COD m−3 day−1 and 18 h HRT. Maximum color and COD removal were found to be 72 and 85 % at total HRT of 2.25 days under anoxic–oxic sequential reactor at 37 °C and pH 7.0. The UV–VIS, FTIR, NMR and GCMS studies showed that majority of peaks observed in untreated wastewater were either shifted or disappeared after sequential treatment. Phytotoxicity study with the seeds of Vigna radiata and Triticum aestivum showed more sensitivity toward the CETP wastewater, while the products obtained after sequential treatment does not have any inhibitory effects. The results demonstrated that the anoxic–oxic reactor fed with bacterial consortium VN11 could bring about efficient bioremediation of industrial wastewaters.
Keywords: Common effluent treatment plant; Wastewater treatment; Organic loading rate; Hydraulic retention time; Sequential activated sludge process