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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.151, #1)

Bacillus Phytases: Present Scenario and Future Perspectives by Shijun Fu; Jianyi Sun; Lichun Qian; Zhiyu Li (pp. 1-8).

Phytases are a special class of phosphatases that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Bacillus phytases, which exhibit their desirable activity profile under neutral pH, higher thermal stability, and strict substrate specificity for the calcium–phytate complex, have considerable potential in commercial and environmental applications. This review describes recent findings concerning the production, biochemical properties, molecular characteristics, and expression of Bacillus phytases. Several potential applications of the Bacillus phytases in animal nutrition, human health, and synthesis of lower myo-inositol phosphates are also summarized.

Keywords: Phytate; Phytase; Bacillus ; Feed; Food; Lower inositol phosphates

Strategies for Enhancing Laccase Yield from Streptomyces psammoticus and Its Role in Mediator-based Decolorization of Azo Dyes by K. N. Niladevi; P. S. Sheejadevi; P. Prema (pp. 9-19).

Enhanced production of laccases from Streptomyces psammoticus in solid-state fermentation was carried out using two different strategies: laccase inducers and scale-up process. Laccase yield was enhanced by a wide range of aromatic inducers. The best inducer was pyrogallol, which yielded 116 U/g as compared to the control (55.4 U/g). Scale-up studies in packed bed bioreactor was performed at different aeration rates. Aeration at 1.5 vvm was identified as the optimum condition for laccase production (75.4 U/g) in the column bioreactor. The enzyme yield was enhanced further by combining the best conditions from the first two experiments. Fermentation was carried out in bioreactors in the presence of 1 mM pyrogallol, which resulted in 3.9-fold increase in laccase yield (215.6 U/g). The role of laccase in azo dye decolorization was evaluated in the presence of four different laccase mediators, at different concentrations. 1-Hydroxybenzotriazole (HOBT) proved to be the best mediator for S. psammoticus laccase and decolorized the azo dyes efficiently. Acid orange, Methyl orange, and Bismarck brown were decolorized at the rates of 86%, 71%, and 75% respectively, by HOBT.

Keywords: Laccase; Aromatic inducers; Packed bed reactor; Laccase-mediators; Dye decolorization; Streptomyces psammoticus

Highly Efficient Regioselective Synthesis of 5′-O-lauroyl-5-azacytidine Catalyzed by Candida antarctica Lipase B by Xi-Yu Chen; Min-Hua Zong; Wen-Yong Lou; Hong Wu (pp. 21-28).

Enzymatic regioselective acylation of 5-azacytidine with vinyl laurate was successfully conducted with an immobilized lipase from Candida antarctica type B (i.e., Novozym 435) for the first time. The acylation of 5-azacytidine took place at its primary hydroxyl group and the desired product 5′-O -lauroyl-5-azacytidine could be prepared with high reaction rate, high conversion, and excellent regioselectivity. The influences of several key variables on the enzymatic acylation were also systematically examined. Pyridine was found to be the best reaction medium. The optimum initial water activity, the molar ratio of vinyl laurate to 5-azacytidine and reaction temperature were 0.07, 30:1, and 50 °C, respectively. Under the optimized conditions described above, the initial reaction rate, the substrate conversion, and the regioselectivity were as high as 0.58 mM/min, 95.5%, and >99%, respectively, after a reaction time of around 5 h.

Keywords: 5-Azacytidine; Novozym 435; Organic solvent; Regioselective acylation; Vinyl laurate

Refolding of a Staphylokinase Variant Y1-Sak by Reverse Dilution by Jintian He; Gaizhen Wang; Ruiguang Xu; Jinlin Feng; Jinlong Wang; Huabo Su; Houyan Song (pp. 29-41).

To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive l-arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of l-arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.

Keywords: Staphylokinase; Refolding; Gel filtration refolding; Reverse dilution refolding; RGD motif; Antiplatelet aggregation

Sugarcane Bagasse Mild Alkaline/Oxidative Pretreatment for Ethanol Production by Alkaline Recycle Process by Ke-Ke Cheng; Jian-An Zhang; Wen-Xiang Ping; Jing-Ping Ge; Yu-Jie Zhou; Hong-Zhi Ling; Jing-Ming Xu (pp. 43-50).

In order to decrease the alkali and water consumptions in the sugarcane bagasse alkaline/oxidative pretreatment for ethanol production, an alkaline recycle process was carried out. Two recycles of NaOH/H₂O₂ pretreatment did not decrease the pretreatment and enzymatic hydrolysis efficiencies and the consumptions of NaOH and water would be saved by 26% and 40%, respectively. A simultaneous saccharification and fermentation (SSF) culture with pretreated bagasse as substrate was developed giving 25 g ethanol l⁻¹ with a yield of 0.2 g g⁻¹ bagasse and productivity of 0.52 g l⁻¹ h⁻¹.

Keywords: Bagasse; Cellulase; Enzymatic hydrolysis; Ethanol; Fermentation; Pretreatment

Enhancing the Thermostability of a Novel β-agarase AgaB through Directed Evolution by Chao Shi; Xinzhi Lu; Cuiping Ma; Yiming Ma; Xiaoyan Fu; Wengong Yu (pp. 51-59).

To increase the thermostability of β-agarase AgaB by directed evolution, the mutant gene libraries were generated by error-prone polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) shuffling. Mutants with high thermostability were screened by a simple method based on agarase-degrading agar to generate a clear zone on the agar plate. A mutant S2 was obtained through two rounds of error-prone PCR and a single round of DNA shuffling and selection. It has higher thermostability and slightly increased catalytic activity than wild-type AgaB. Melting temperature (T _m) of S2, as determined by circular dichroism, is 4.6 °C higher than that of wild-type AgaB, and the half-life of S2 is 350 min at 40 °C, which is 18.4-fold longer than that of the wild-type enzyme. Saturation mutagenesis and hydrophobic cluster analysis indicated that hydrophobic interaction might be the key factor that enhances the enzyme stability.

Keywords: Agarase; Thermostability; Directed evolution; Clear zone screening; DNA shuffling

Practical Aspects of Hydrophobic Polycationic Bactericidal “Paints” by Koushik Mukherjee; Jaime J. Rivera; Alexander M. Klibanov (pp. 61-70).

We previously discovered that coating solid surfaces with long-chained linear N-dodecyl,N-methyl-polyethylenimine makes them bactericidal and virucidal. In the present study, focusing on the use of this microbicidal paint to kill airborne Escherichia coli and Staphylococcus aureus, we have systematically investigated the dependence of this effect on the concentration and mode of application of the hydrophobic polycation, the number of coats, the nature of the solvent, and the presence of a dye in such paint. In addition, the latter’s ability to be regenerated after use, stability upon repeated washings, and mammalian toxicity has been evaluated.

Keywords: Polyethylenimine; Antibacterial; Coating; Toxicity; Escherichia coli ; Staphylococcus aureus ; Biocidal materials

Inhibitory Effects of Tunisian Marine Algal Extracts on Digestive Lipases by Faouzi Ben Rebah; Sana Smaoui; Fakher Frikha; Youssef Gargouri; Nabil Miled (pp. 71-79).

The lipase inhibitory activity of ethanol extracts obtained from some marine algae collected on the Tunisian coast was evaluated. Caulerpa prolifera extract markedly reduced both dog gastric (DGL) and human pancreatic lipase (HPL) activities. Generally, the inhibition reached 100% after 40 to 60 min of incubation depending on lipase types and on substrates used. Moreover, the inhibitory effect of C. prolifera extract on lipases appeared to be accelerated by adding bile salts, which likely modified the interface and allowed the inhibitory compound to inactivate the lipase. The separation of C. prolifera extract by thin-layer chromatography (TLC) resulted in eight fractions showing efficient inhibition rate against DGL, compared to the crude extract. In the case of HPL, TLC fractionation reduced the inhibitory rates, suggesting that the effect of algal extract on lipases may be caused by a synergetic action of several compounds within the extract. High-performance liquid chromatograph separation resulted in isolation of a major compound displaying high inhibition capacity of HPL activity. Caulerpa prolifera extract may therefore be useful in developing antiobesity drugs.

Keywords: Algae; Extraction; Inhibition; Lipase; TLC; HPLC

A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization by Yong Zhang; Chaoneng Ji; Xiaoxiao Zhang; Zhenxing Yang; Jing Peng; Rui Qiu; Yi Xie; Yumin Mao (pp. 81-92).

A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 °C, respectively. The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min, exhibiting a half-life of 8 min at 70 °C. The K _m and V _max for pNPP were determined to be 31.5 μM and 430 μM/min at optimal conditions. A divalent cation is essential, with a combination of Mg²⁺ and Co²⁺ or Zn²⁺ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.

Keywords: Alkaline phosphatase; Cloning; Characterization; Geobacillus ; Thermostable

Expression and Production of Human Interleukin-7 in Insect Cells Using Baculovirus Expression Vector System (BEVS) by Maryam Mirzaei; Barbara Jardin; Cynthia B. Elias; Satya Prakash (pp. 93-103).

Interleukin-7 (IL-7) is a glycoprotein cytokine with significant clinical and biomedical potential, such as cancer therapy and HIV infections. Earlier it has been cloned and expressed in various protein expression systems; however, they are not efficient for large-scale production. To address this inadequacy, we report in this paper the production of recombinant human interleukin-7 (hIL-7) in insect cells. A recombinant bacmid containing hIL-7 was constructed, purified, and characterized. It was used to infect Trichoplusia ni (BT1-TN-5B1/High Five™) insect cells. Result shows that T. ni cells successfully produce hIL-7 in shake flask cultures. A scale up to 2.5-L laboratory batch bioreactor showed the efficacy of this system for large-scale production. Our results offer a highly efficient, inexpensive, and convenient system for the large-scale expression and production of recombinant hIL-7.

Keywords: Interleukin-7; Insect cells; Trichoplusia ni cells; Glycoprotein cytokine production; Baculovirus vector system; Batch bioreactor

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.151, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.151, #1)