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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.119, #1)

RNA interference silencing the transcriptional message by Shantanu Karkare; Saurabha Daniel; Deepak Bhatnagar (pp. 1-12).

RNA interference (RNAi) is silencing of gene expression by double-stranded RNA (dsRNA) having complementary sequence to the target gene to be silenced. This phenomonon has transformed into a complete technology for functional genomic studies. Small interfering RNAs (siRNAs) are 21- to 23-nucleotide dsRNAs, in which the sense strand is the same as the target mRNA and the antisense strand is the complement of the target mRNA sequence. These are the effector molecules for inducing RNAi, leading to posttranscriptional gene silencing with RNA-induced silencing complex. Besides siRNA, which can be chemically synthesized, various other systems in the form of potential effector molecules for posttranscriptional gene silencing are available, such as short hairpin RNAs (shRNAs), long dsRNAs, short temporal RNAs, and micro RNAs (miRNAs). These effector molecules either are processed into siRNA such as in the case of shRNA or directly aid gene silencing as in the case of miRNA. RNAi for various unknown genes may facilitate to elucidate inherited genetic diseases and provide drug candidates for viral and oncogenic diseases. This can be achieved by targeting mRNA from oncogenic genes or mRNA for viral cellular receptor and viral structural proteins for RNAi. In this article, we evaluate various aspects and applications of RNAi technology and provide comprehensive information for the system currently available for inducing RNAi.

Keywords: Small interfering RNA; short hairpin RNA; micro RNA; RNA-induced silencing complex; siRNA expression cassettes; siRNA expression vectors

Effect of detoxification of dilute-acid corn fiber hydrolysate on xylitol production by J. Buhner; F. A. Agblevor (pp. 13-30).

Four different detoxification methods were evaluated for the production of xylitol from corn fiber dilute-acid hydrolysate using Candida tropicalis. Although C. tropicalis could ferment the dilute partially neutralized hydrolysate to xylitol in low yields (0.1 g/g), it could not ferment the concentrated hydrolysate. Overliming, calcium hydroxide neutralization, neutralization combined with activated charcoal, and overliming combined with activated charcoal methods were used to improve the fermentation of the concentrated hydrolysates. The partial neutralization combined with activated charcoal treatment was the most effective method with respect to xylitol yield and productivity. The highest xylitol yield (0.4 g of xylitol/g of xylose) was obtained for the highest concentration of hydrolysate (three times the original) that had been treated with calcium hydroxide and activated charcoal. The corresponding productivity was 0.23 g/(L.h). Overliming caused reduction in xylitol yield.

Keywords: Detoxification; overliming; Candida tropicalis ; corn fiber; hydrolysate

Decolorization and partial degradation of selected azo dyes by methanogenic sludge by Natalia Yemashova; Anna Telegina; Irina Kotova; Alexander Netrusov; Sergey Kalyuzhnyi (pp. 31-40).

The toxicity potential and decolorization of three acid azo dyes (Acid Orange 6, Acid Orange 7, and Acid Orange 52) by methanogenic granular sludge from an anaerobic expanded granular sludge bed reactor was assayed. Complete bioreduction was found for all three azo dyes. Sulfanilic acid and 4-aminoresorcinol were detected from the decolorization of Acid Orange 6, sulfanilic acid and 1-amino-2-naphtol were detected from the reduction of Acid Orange 7, and sulfanilic acid and N,N-dimethyl-1,4-phenylenediamine (DMP) were found to be intermediates of Acid Orange 52 degradation. Sulfanilic acid and 1-amino-2-naphtol were persistent in the anaerobic conditions, whereas 4-aminoresorcinol was completely mineralized by anaerobic sludge and DMP was transformed into 1,4-phenylenediamine. Enrichment cultures obtained via consecutive passages on basal medium with only azo dye as a carbon and an energy source seemed to be morphologically heterogeneous. Baculiform and coccus cells were found when viewed under a light microscope. Cocci were joined in chains. Because anaerobic sludge contains sulfate-reducing bacteria and therefore may generate sulfide, azo dyes were tested for chemical decolorization by sulfide to compare rates of chemical and biologic reduction.

Keywords: Azo dyes; methanogetic sludge; toxicity; bioreduction; chemical reduction

Purification, characterization, and structural investigation of a new moderately thermophilic and partially calcium-independent extracellular α-amylase from Bacillus sp. TM1 by Reza Hassan Sajedi; Hossein Naderi-Manesh; Khosro Khajeh; Bijan Ranjbar; Nasser Ghaemi; Mehdi Naderi-Manesh (pp. 41-50).

A new α-amylase was extracted from a recently found strain of Bacillus sp. and purified by ion-exchange chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band for the purified enzyme with an apparent molecular weight of 59 kDa. The optimum temperature and pH range of the enzyme were 40–60°C and 4.5–7.5, respectively, and its activation energy was 1.974 kcal/mol. The K m value for the enzyme activity on solubie starch was 4 mg/mL, and the T m values obtained from the circular dichroism (CD) results of thermal unfolding were 78.7 and 80.2°C in the absence and presence of the calcium, respectively. The enzyme was almost completely inhibited by the addition of Fe³⁺, Mn²⁺, and Zn²⁺ and was activated by EDTA, Cr³⁺, and Al³⁺. Moreover, it was partially inhibited by Ca²⁺, Ba²⁺, Ni²⁺, and Co²⁺. Proteolytic digestion of the enzyme using trypsin combined with results from T m using CD and irreversible thermoinactivation suggests that this enzyme can be considered a moderate thermophile with both mild flexibility and rigidity.

Keywords: α-Amylase; Bacillus sp.; characterization; proteolytic digestion; thermal stability

High-Cell-Density cultivation of pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates by Simone Cardoso Diniz; Marilda Keico Taciro; José Gregório Cabrera Gomez; José Geraldo da Cruz Pradella (pp. 51-69).

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHA_MCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65h⁻¹. Limitation and inhibition owing to NH₄ ⁺ ions were observed, respectively, at 400 and 3500 mg of NH₄ ⁺/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHA_MCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHA_MCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHA_MCL content. Using the last strategy, 21% PHA_MCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH₄ ⁺ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHA_MCL and a productivity of 0.8 g/(L·h) were obtained in 42 h of fed-batch operation. The PHA_MCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.

Keywords: Polyhydroxyalkanoate; Pseudomonas putida ; high cell-density; sugarcane; kinetics studies

Production of optically pure d-lactic acid by Nannochlorum sp. 26A4 by Shin Hirayama; Ryohei Ueda (pp. 71-77).

Microalgae were screened from seawater for greenhouse gas CO₂ fixation and d-lactic acid production by self-fermentation and tested for their growth rate, starch content, and conversion rate from starch into d-lactic acid. More than 300 strains were isolated, and some of them were found to have suitable properties for this purpose. One of the best strains, Nannochlorum, sp. 26A4, which was isolated from Sakito Island, had a starch content of 40% (dry weight), and a conversion rate from consumed starch into d-lactic acid of 70% in the dark under anaerobic conditions. The produced d-lactic acid showed a high optical purity compared with the conventional one. The proposed new d-lactic acid production system using Nannochlorum sp. 26A4 should also be an effective technology for greenhouse gas CO₂ fixation and/or conversion into industrial raw materials.

Keywords: d-lactic acid; optical purity; microalgae; self-fermentation; Nannochlorum sp.; Sakito Island

Effect of ion and surfactant choice on the recovery of a histidine-tagged protein from tobacco extract using foam fractionation by Czarena Crofcheck; Indu Maiti; Sitakanta Pattanaik; Michael Jay (pp. 79-91).

Tobacco plants can be used for the production of proteins for pharmaceutical applications. One of the most difficult and expensive tasks associated with this technology is isolating the product of interest from the hundreds of other chemicals found in tobacco. We describe a new recovery strategy in which the protein of interest is “tagged” with a histidine structure, which forms a complex with metal ions and a surfactant that will accumulate in the foamate of a foam fractionation step. His-gus, a histidine-tagged enzyme, was selectively recovered in the presence of two different surfactants and two different metal ions. The foam fractionation with N-∈-dodecylamido-N-α, N-α,-bis(carboxymethyl)-l-lysine surfactant and Ni²⁺ ions resulted in an average His-gus activity recovery value of 88% and an activity enrichment of 2.27. The performance of the recovery strategy without tobacco extract resulted in an average activity recovery value of 63.32% and an average activity enrichment value of 5.16, utilizing lauroylethylenediaminetriacetate surfactant and Ni²⁺ ions. It was shown that even though a majority of the native tobacco proteins are removed during the prefoaming step, the presence of tobacco extract does affect the recovery of His-gus.

Keywords: Downstream processing; protein recovery; transgenic tobacco; tobacco extract; yeast extract; histidine-tagged protein

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.119, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.119, #1)