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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #6)

Breeding an Amylolytic Yeast Strain for Alcoholic Beverage Production by Ming-Chung Cheng; Rei-Chu Chang; Der-Feng Dent; Pao-Chuan Hsieh (pp. 693-706).

A starch-utilizing, yeast-like fusant was successfully created from fused protoplasts of Schizosaccharomyces pombe and Monascus anka, and the feasibility of using this fusant as a new strain for alcoholic beverage development was reported. The new fusant utilized various carbon sources more efficiently than its parent cells did. Rice koji prepared separately by cultivating the fusant and its parental strains on rice was compared to explore the effect of yeast strain on the production of α-amylase, glucoamylase, and acid protease that are crucial in wine making using cereal grains. It was found that the fusant produced greater levels of the above-mentioned enzymes than its parental strain does. Consequently, the usage of this fusant in the alcoholic fermentation of polished rice was found to reduce approximately 50% consumption of added glucoamylase than when its parental strain was used. Besides, at the end of fermentation, the fusant yeast resulted in a mash with distribution of flavor components very different from that produced by its parental strains. Thus, the fusant can be used as a new yeast strain for creating novel alcoholic beverages.

Keywords: Protoplast fusion; Schizosaccharomyces pombe ; Monascus anka ; Glucoamylase consumption reduction; Flavor components; Novel alcoholic beverages

Enhanced Production of l-Arginine by Expression of Vitreoscilla Hemoglobin Using a Novel Expression System in Corynebacterium crenatum by Meijuan Xu; Zhiming Rao; Hong Xu; Chunyan Lan; Wenfang Dou; Xiaomei Zhang; Hongyu Xu; Jian Jin; Zhenghong Xu (pp. 707-719).

Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g⁻¹, and the oxygen uptake rates reached 0.25 mg A ₅₆₂ ⁻¹ h⁻¹ which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L⁻¹, and the biomass was largely increased to 6.45 g L⁻¹, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.

Keywords: Amino acids; Corynebacterium crenatum ; vgb ; Fermentation; Dissolved oxygen

Pretreatment of Synthetic Dairy Wastewater Using the Sophorolipid-Producing Yeast Candida bombicola by Achlesh Daverey; Kannan Pakshirajan (pp. 720-728).

The presence of high strength fats and oils in dairy industry wastewaters poses serious challenges for biological treatment systems, and, therefore, its pretreatment is necessary in order to remove them. In the present study, synthetic dairy wastewater prepared in the laboratory was pretreated using the sophorolipid-producing yeast Candida bombicola in a laboratory-scale bioreactor under batch, fed-batch, and continuous modes of operation. To support the yeast growth, the wastewater was supplemented with sugarcane molasses (1% w/v) and yeast extract (0.1% w/v). Results from the batch operated fermentor revealed complete utilization of fats present in the wastewater within 96 h with more than 93% COD removal efficiency. The yeast was, however, able to pretreat the wastewater more quickly and efficiently under fed-batch mode of operation than under batch operated condition in the same fermentor. Continuous experiments were carried out with a wastewater retention time of 28 h in the reactor; results showed very good performance of the system in complete utilization of fats and COD removal efficiency of more than 90%. The study proved the excellent potential of the biosurfactant-producing yeast in pretreating high-fat- and oil-containing dairy industry wastewater.

Keywords: Dairy wastewater; Pretreatment; Candida bombicola ; Fats and oils removal; Sophorolipid

Strain Improvement of Streptomyces roseosporus for Daptomycin Production by Rational Screening of He–Ne Laser and NTG Induced Mutants and Kinetic Modeling by Guanghai Yu; Xiaoqiang Jia; Jianping Wen; Wenyu Lu; Guoying Wang; Qinggele Caiyin; Yunlin Chen (pp. 729-743).

To improve the yield of daptomycin by Streptomyces roseosporus, a method of rational screening of He–Ne Laser and N-methyl-N-nitro-N-nitrosoguanidine (NTG)-induced mutants was employed in this work. Under the optimal mutagenesis conditions (NTG of 0.3 mg/L, 40 min; irradiation at 15 mW, 15 min), two steps of combined mutations were conducted. Screening of mutants was done according to the individual resistance to n-decanoic acid and daptomycin. A mutant strain LC-54-16, with the highest daptomycin production ability of 616 mg/L was obtained, which was over 5 times higher than the wild strain LC-52-6. The transcription levels of the main dpt genes in the mutant were approximately five times higher than those in the wild. The superiority of the mutant to the wild strain was further proved by the comparative studies on the kinetics of the mutant and the wild. The decrease of the inhibition of substrate and product was further confirmed, as well. It was concluded that the method of rational screening of He–Ne Laser and NTG-induced mutants could efficiently improve the daptomycin production ability of S. roseosporus.

Keywords: He–Ne laser irradiation; NTG; Rational screening; Daptomycin; Streptomyces roseosporus ; RT-PCR; Kinetics

Extraction of Bovine Serum Albumin Using Reverse Micelles Formed by Hexadecyl Trimethyl Ammonium Chloride by Qingchi Sun; Yanzhao Yang; Yanmin Lu; Wenjuan Lu (pp. 744-755).

The extraction of bovine serum albumin (BSA) has been investigated using reverse micelles of hexadecyl trimethyl ammonium chloride/n-octanol/isooctane. Forward extraction process parameters such as the surfactant concentration, co-solvent concentration, pH, ionic strength, and species of the initial aqueous phase were important factors affecting the extraction performance. These parameters were varied to optimize the extraction efficiency. Under the optimized conditions, forward extraction efficiencies of BSA can reach practically 99.55%. The thermodynamic study revealed that the extraction of BSA is controlled by entropy changes. Maximum back-extraction efficiency of 85.16% can be obtained at low pH values and high salt concentrations. The structures of BSA during reverse micelle extraction did not change by comparing the circular dichroism spectra of BSA back-extracted to the aqueous phase with that of feed BSA.

Keywords: BSA; Reverse micelles; Forward extraction; Back extraction; CD spectra

Bioprocess Optimization of Furanocoumarin Elicitation by Medium Renewal and Re-elicitation: A Perfusion-Based Approach by Renuka Diwan; Nutan Malpathak (pp. 756-764).

Effect of various abiotic (methyl jasmonate, salicylic acid) and biotic (yeast extract, Aspergillus niger) elicitors on furanocoumarin production and in situ product removal was studied using shoot cultures of Ruta graveolens L. Elicitation by yeast extract (1% w/v) on day 15 was most effective. It led to 7.8-fold higher furanocoumarin production that was attained 24 h after elicitation and 43% of the product was released into the medium. Changes in the relative concentration of furanocoumarins produced depend on the elicitor used. Molar ratio of bergapten increased to 93% in response to yeast extract. With the perspective of developing a commercially feasible process, an approach for preserving viability of biomass and its reuse needs to be developed. For this, medium renewal strategy was investigated. Removal of the spent medium 48 h after elicitation allowed in situ product removal and proved effective in revival of cultures, allowing reuse of biomass. A week after medium renewal, the revived biomass was re-elicited and a second furanocoumarin production peak was obtained. A perfusion-based bioprocess optimization approach, employing elicitation coupled with medium renewal with subsequent re-elicitation, as a new strategy for improved furanocoumarin production, has been suggested.

Keywords: Bioprocess optimization; Medium renewal; Re-elicitation; Furanocoumarins; Elicitors; Ruta graveolens L

Recovery and Characterization of a Serine Collagenolytic Extract from Snow Crab (Chionoecetes opilio) By-products by Nathalie Souchet; Serge Laplante (pp. 765-779).

Sequential acidic precipitation followed by a single chromatographic step (gel filtration) allowed the recovery of a collagenolytic fraction containing several proteases from by-products of snow crab (Chionoecetes opilio). The partial purification was particularly efficient to recover tryptic (purification fold = 1,352.5; yield = 110%) but also chymotryptic, elastolytic, and collagenolytic activities. A temperature of 40 °C and pH 8.0–8.5 were optimal for enzyme activity, which was stable for 2 h under these conditions. Calcium was not required for stability and thus activity. The isoelectric points of the protein components ranged from 3.7 to 4.6. Zymography revealed 29 and 48 kDa major components and others from 22 to 56 kDa. Enzymes were inhibited by PMSF and TLCK but were insensitive to TPCK. In view of these properties, the proteases likely belong to the serine collagenase group. Inhibition by EDTA could be due to a mechanism other than Ca²⁺ chelation. Using a food system (ground fish), the fraction was more proteolytic than a commercial bacterial protease, suggesting potential applications in enzymatic hydrolysis processes.

Keywords: Chionoecetes opilio ; By-products; Serine collagenases; Acidic precipitation; Purification; Activity

Studies on Amendment of Different Biopolymers in Sandy Loam and Their Effect on Germination, Seedling Growth of Gossypium herbaceum L. by Satish Vitthalrao Patil; B. K. Salunke; C. D. Patil; R. B. Salunkhe (pp. 780-791).

Different biopolymers, agar, cellulose, alginate, psyllium gaur gum, and bacterial exopolysaccharide (EPS) powders were amended to check their efficacy in enhancing maximum water holding capacity (MWHC), permanent wilting point (PWP), and germination and seedling growth of the Gossypium herbaceum in a laboratory scale. The efficacy of all biopolymers for enhancement of MWHC, PWP, and growth was also analyzed by measuring organic carbon, organic matter, total nitrogen, respiration rate, and microflora in amended and control sandy loams. The range of concentrations (0.2–2%) of all biopolymers was incorporated in sandy loam containing pots. The soil without polymer was considered as control. The psyllium (0.6%) and bacterial EPS (1%) amended soil has 242 and 233% increase in MWHC and thus delaying in the permanent wilting point by 108 and 84 h at 37 °C, respectively, as compared to control. All biopolymers found to increase more or less MWHC, organic matter, total nitrogen, microflora, and PWP as compared to control. The psyllium and bacterial EPS show the highest increase organic matter, biomass, and microflora. The highest reduction in MWHC after 12 weeks were observed in cellulose, gaur gum, and alginate; besides, psyllium, bacterial EPS, and agar showed comparatively less reduction MWHC, i.e., 24% and 14.5%, respectively. The toxicity studies of biopolymer were carried out on earthworm (Eisenia foetida). It revealed their nontoxic nature. The biopolymer amendment in sandy loam can be an effective strategy to improved soil texture, fertility, and thereby crop yield.

Keywords: Biopolymer; Psyllium; MWHC; PWP; Gossypium herbaceum L.; Eisenia foetida

Plant Oil Bodies: Novel Carriers to Deliver Lipophilic Molecules by Stefania Bonsegna; Simona Bettini; Rosanna Pagano; Antonella Zacheo; Viviana Vergaro; Giovanna Giovinazzo; Gabriella Caminati; Stefano Leporatti; Ludovico Valli; Angelo Santino (pp. 792-802).

Oil bodies (OBs) are specialised organelles ubiquitously detected in plant oil seeds, which serve as lipid storage compartments. OBs consist of a hydrophobic core of triacylglycerol (TAGs), surrounded by a monolayer of phospholipids (PLs) embedded with some specific proteins with a size ranging from 0.5 to 2 μm. In this work, we report an easy method to reconstitute OBs starting from their constituents and to encapsulate lipophilic molecules, i.e. the fluorescent fluorescein isothiocyanate (FITC) and carboxyfluorescein (CF), into reconstituted OBs. This methods allowed us to produce OBs 4- to 10-fold smaller (50–200 nm) than the native one and to obtain a good recovery (about 40%) of both the fluorescent compounds used in the present work. The properties of reconstituted OBs were investigated by a combination of Brewster angle microscopy, scanning force microscopy, ζ-potential techniques. OBs were stable and formed ordered monolayers when patterned on hydrophobic substrates whereas they showed a higher tendency to aggregate into larger, coalescing OBs when were deposited onto hydrophilic substrates or at the air/water interface. Furthermore, we verified the uptake of FITC-loaded OBs by the MCF-7 breast cancer cell line. Our results indicated that OBs could be envisaged as novel carriers to deliver hydrophobic bioactive compounds.

Keywords: AFM analysis; Air/water interface characterization; Cellular uptake; Langmuir–Schaefer films; MCF-7 cells; Oil bodies

Development of a Protein Chip to Measure PKCβ Activity by Dong-Woo Lee; Hae Jong Kim; Chi-Ho Choi; Jeong-Hyun Shin; Eun-Ki Kim (pp. 803-812).

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.

Keywords: PKCβ; Tyrosinase; Protein chip; HTS; Depigmenting agents

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #6)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #6)