Characterization of a novel manganese dependent endoglucanase belongs in GH family 5 from Phanerochaete chrysosporium

Huy, N.D.,Nguyen, C.L.,Park, H.S.,Loc, N.H.,Choi, M.S.,Kim, D.H.,Seo, J.W.,Park, S.M. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.2

<P>The cDNA encoding a putative glycoside hydrolase family 5, which has been predicted to be an endoglucanase (PcEg5A), was cloned from Phanerochaete chrysosporium and expressed in Pichia pastoris. PcEg5A contains a carbohydrate-binding domain and two important amino acids, E209 and E319, playing as proton donor and nucleophile in substrate catalytic domain. SDS-PAGE analysis indicated that the recombinant endoglucanase 5A (rPcEg5A) has a molecular size of 43 kDa which corresponds with the theoretical calculation. Optimum pH and temperature were found to be 4.5-6.0, and 50 degrees C-60 degrees C, respectively. Moreover, rPcEg5A exhibited maximal activity in the pH range of 3.0-8.0, whereas over 50% of activity still remained at 20 degrees C and 80 degrees C. rPcEg5A was stable at 60 degrees C for 12 h incubation, indicating that rPcEg5A is a thermostable enzyme. Manganese ion enhanced the enzyme activity by 77%, indicating that rPcEg5A is a metal dependent enzyme. The addition of rPcEg5A to cellobiase (cellobiohydrolase and beta-glucosidase) resulted in a 53% increasing saccharification of NaOH-pretreated barley straw, whereas the glucose release was 47% higher than that cellobiase treatment alone. Our study suggested that rPcEg5A is an enzyme with great potential for biomass saccharification. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Mixed-culture H₂ fermentation performance and the relation between microbial community composition and hydraulic retention times for a fixed bed reactor fed with galactose/glucose mixtures

Anburajan, P.,Park, J.H.,Sivagurunathan, P.,Pugazhendhi, A.,Kumar, G.,Choi, C.S.,Kim, S.H. Society for Bioscience and Bioengineering, Japan ; 2017 Journal of bioscience and bioengineering Vol.124 No.3

<P>This study examined the mesophilic continuous biohydrogen fermentation from galactose and glucose mixture with an initial substrate concentration of 15 g/L (galactose 12 g/L and glucose 3 g/L) as a resembling carbon source of pre-treated red algal hydrolyzate. A fixed bed reactor was fed with the sugar mixture at various hydraulic retention times (HRTs) ranging 12 to 1.5 h. The maximum hydrogen production rate of 52.6 L/L-d was found at 2 h HRT, while the maximum hydrogen yield of 2.3 +/- 0.1 mol/mol hexose(added), was achieved at 3 h HRT. Microbial communities and species distribution were analyzed via quantitative polymerase chain reaction (qPCR) and the dominant bacterial population was found as Clostridia followed by Lactobacillus sp. Packing material retained higher 16S rRNA gene copy numbers of total bacteria and Clostridium butyricum fraction compared to fermentation liquor. The finding of the study has demonstrated that H-2 production from galactose and glucose mixture could be a viable approach for hydrogen production. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.</P>

Reduction of d-lactate content in sauerkraut using starter cultures of recombinant Leuconostoc mesenteroides expressing the ldhL gene

Jin, Q.,Li, L.,Moon, J.S.,Cho, S.K.,Kim, Y.J.,Lee, S.J.,Han, N.S. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.5

<P>The n-form of lactate, which causes metabolic stress upon excessive dietary intake, is mainly produced by Leuconostoc sp., the predominant species in sauerkraut. To shift the metabolic flux of D-lactate from pyruvate to L-lactate, we expressed the L-lactate dehydrogenase (ldhL) gene in Leuconostoc mesenteroides ATCC 8293. The IdhL gene from Lactobacillus plantarum was introduced into L. mesenteroides using the shuttle vectors pLeuCM and pLeuCM42. To elevate the expression level of IdhL in L. mesenteroides, the nucleotides for pyruvate kinase promoter were fused to IdhL and cloned into above vectors to construct pLC18pkL and pLC42pkL. As results, introduction of pLC42pkL in L. mesenteroides significantly improved both L-LDH activity and L-lactate productivity during fermentation, decreasing the D-/L-lactate ratio. When used as a starter culture for sauerkraut fermentation, recombinant L. mesenteroides harboring pLC42pkL increased L-lactate concentration and decreased D-lactate concentration compared to the wild type strain. We newly developed a recombinant L. mesenteroides which has high L-lactate dehydrogenase activity and applied this strain to minimize the harmful effect of D-lactate during the sauerkraut fermentation. To the best of our knowledge, we demonstrate for the first time the effective use of recombinant Leuconostoc sp. for quality improvement of fermented foods. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Formation of chitosan-fucoidan nanoparticles and their electrostatic interactions: Quantitative analysis

Lee, E.J.,Lim, K.H. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.1

<P>The stoichiometric distributions of both positive amino groups and negative sulfate ions loaded in chitosan-fucoidan nanoparticles (CFNs) were predicted quantitatively by correlating the separate yields of loaded chitosan and fucoidan, and a proposed relative charge density model (case 1). In addition, those distributions of both positive amino groups and negative sulfate ions loaded in CFNs were obtained by deriving the expression of their loaded concentrations directly from the experimental data (case 2). Both the model-prediction and experimental derivations were remarkably consistent with each other except at pH 2. The discrepancy between cases 1 and 2 at pH 2 was explained by an increase in the sulfate group loading because of the most intensive electrostatic (specific ion) interactions at pH 2. The ratio of the CFN-free net charge density shielded by counter-ions in the solution entrapped in CFNs to their counter-ion-crosslinking charge density was suggested to be a quantitative criterion for determining the size distribution of CFNs. The formation of CFNs ranked according to size was predicted well and explained reasonably by the suggested criterion, considering both the ionic strength of the entrapped solution in CFNs and the nonspecific binding (interaction) of the positive amino groups among the chitosan molecules. Furthermore, the fraction of nonspecifically-bound positive amino groups causing hysteresis was quantified from the positive net charged amino groups per unit-mass CFN. Thus, its magnitude was predicted to have a strong correlation with the CFN-preparation conditions, such as pH and fucoidan to chitosan mass ratio. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD

Eom, G.T.,Oh, J.Y.,Park, J.H.,Lim, H.J.,Lee, S.J.,Kim, E.Y.,Choi, J.E.,Jegal, J.,Song, B.K.,Yu, J.H.,Song, J.K. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.122 No.3

<P>An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33 degrees C. To isolate a mutant TliDEF capable of secreting TliA at 35 degrees C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type WE, tliF and tliA in Escherichia con. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35 degrees C. At the growth temperature of 35 degrees C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20 degrees C, 25 degrees C and 30 degrees C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.</P>

Modified harvest system for enhancing Factor VIII yield in alternating tangential flow perfusion culture

Kim, S.C.,An, S.,Kim, H.K.,Park, B.S.,Na, K.H.,Kim, B.G. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.5

<P>This study describes the development and experimental verification of a modified harvest system to enhance Factor VIII (FVIII) yield in an alternating tangential flow (ATF) perfusion culture. The main innovation of the modified harvest system is the use of check and pinch valves, eliminating the need of a peristaltic pump for harvest. The system was applied to perfusion cultures of Chinese hamster ovary cells, which co-express both recombinant human FVIII (rhFVIII) and von Willebrand factor (vWF). The modified harvest system showed comparable cell growth with the conventional harvest system using a peristaltic pump. The perfusion rate was successfully controlled using the system. In addition, the modified harvest system achieved an approximately 13.6-fold increase in the final concentration yield of FVIII activity and a 1.47-fold increase in the production yield of FVIII activity compared with a peristaltic pump. Enhancement of the yield of FVIII activity resulted from the reduction of FVIII antigen (FVIII:Ag) retention. As a result of transmembrane pressure (TMP) measurement, the reduction of the retained FVIII:Ag was due to the increased TMP, which was caused by the characteristic function of a check valve, compared with a peristaltic harvest system. The modified harvest system developed in this study could be useful to enhance the production yield of other recombinant proteins in ATF perfusion culture. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Characterization of a thermostable glycoside hydrolase family 36 α-galactosidase from Caldicellulosiruptor bescii

Lee, A.,Choi, K.H.,Yoon, D.,Kim, S.,Cha, J. Society for Bioscience and Bioengineering, Japan ; 2017 Journal of bioscience and bioengineering Vol.124 No.3

<P>The putative gene cluster involved in the degradation of the raffinose family oligosaccharides (RFO) was identified in Caldicellulosiruptor bescii. Within the cluster, the gene encoding a putative alpha-galactosidase (CbAga36) was cloned and expressed in Escherichia coli. Size exclusion chromatography of the purified rCbAga36 indicated that the native form was a tetramer. Its primary sequence was similar to the family of glycoside hydrolase 36. The purified recombinant CbAga36 (rCbAga36) was optimally active at pH 5.0 and 70 degrees C and had a half-life of 15 h and 10 h at 70 degrees C and 80 degrees C, respectively. rCbAga36 showed high activity with the artificial substrate (p-nitrophenyl cc-D-galactopyranoside, pNPaGal) exhibiting lower Km and higher kat than natural substrates such as melibiose and raffinose. Although rCbAga36 demonstrated preferential activity toward the hydrolysis of RFO such as raffinose and stachyose, it did not degrade the polymeric galactomannans. Our results imply that CbAga36 may play a role in the degradation of RFO, transported into the cytoplasm via a transporter into galactose, which is further utilized as an energy source in C. bescii. Furthermore, its ability to synthesize novel oligosaccharides by transglycosylation renders this enzyme potentially useful for the production of dietary oligosaccharides with novel function. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.</P>

Production of d-psicose from d-fructose by whole recombinant cells with high-level expression of d-psicose 3-epimerase from Agrobacterium tumefaciens

Park, C.S.,Park, C.S.,Shin, K.C.,Oh, D.K. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.2

<P>The specific activity of recombinant Escherichia coli cells expressing the double-site variant (133L-S213C) D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was highest at 24 h of cultivation time in Terrific Broth (TB) medium among the media tested. The contents of crude protein and DPEase in recombinant cells at 24 h were 37.0 and 8.6% (w/w), respectively, indicating that the enzyme was highly expressed. The reaction conditions for the production of D-psicose from D-fructose by whole recombinant cells with the highest specific activity were optimal at 60 degrees C, pH 8.5, 4 g/l cells, and 700 g/l D-fructose. Under these conditions, whole recombinant cells produced 230 g/I D-psicose after 40 min, with a conversion yield of 33% (w/w), a volumetric productivity of 345 g/l/h, and a specific productivity of 86.2 g/g/h. These are the highest conversion yield and volumetric and specific productivities of D-psicose from D-fructose by cells reported thus far. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase

Park, E.S.,Park, S.,Shin, J.S. Society for Bioscience and Bioengineering, Japan ; 2017 Journal of bioscience and bioengineering Vol.123 No.4

<P>Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to K-M = 0.73 +/- 0.09 mM and k(cat) = 400 +/- 20 s(-1) which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 10(4)-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.</P>

Nitrification resilience and community dynamics of ammonia-oxidizing bacteria with respect to ammonia loading shock in a nitrification reactor treating steel wastewater

Cho, K.,Shin, S.G.,Lee, J.,Koo, T.,Kim, W.,Hwang, S. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.122 No.2

<P>The aim of this study was to investigate the nitrification resilience pattern and examine the key ammonia-oxidizing bacteria (AOB) with respect to ammonia loading shocks (ALSs) in a nitrification bioreactor treating steel wastewater. The perturbation experiments were conducted in a 4-L bioreactor operated in continuous mode with a hydraulic retention time of 10 d. Three sequential ALSs were given to the bioreactor (120, 180 and 180 mg total ammonia nitrogen (TAN)/L. When the first shock was given, the nitrification process completely recovered after 14 d of further operation. However, the resilience duration was significantly reduced to 1 d after the second and third ALSs. In the bioreactor, Nitrosomonas aestuarii dominated the other AOB species, Nitrosomonas europaea and N. nitrosa, throughout the process. In addition, the population of N. aestuarii increased with ammonia utilization following each ALS; i.e., this species responded to acute ammonia overloadings by contributing to ammonia oxidation. This finding suggests that N. aestuarii could be exploited to achieve stable nitrification in industrial wastewaters that contain high concentrations of ammonia. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.</P>