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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #5)

Enzymatic Hydrolysis of Recovered Office Printing Paper with Low Enzyme Dosages to Produce Fermentable Sugars by Hui Chen; Richard A. Venditti; Hasan Jameel; Sunkyu Park (pp. 1121-1136).

The use of recovered paper and paper manufacturing wastes are a potentially large, concentrated, and convenient raw material for ethanol production via enzyme hydrolysis and fermentation. However, many previous studies in the area have investigated impractically high enzyme charges. In this research, low dosages of enzymes on copy paper (CP) were investigated for the conditions of 5% consistency (w/v) and 50 °C for 48 h. The removal of inorganic filler (mainly calcium carbonate) by washing prior to hydrolysis led to higher sugar yields than the unwashed CP as well as CP acidified to remove the ash. Enzyme adsorption measurement showed that both acid-soluble ash and acid-insoluble ash adsorb enzymes with a greater affinity than fibers. Drying of the fibers (termed hornification) decreased the efficiency of enzyme hydrolysis, confirming previous results. The mechanical refining at 10% consistency in a laboratory refining mill of previously dried fibers improved the sugar recovery to similar or higher levels as never-dried fibers. By plotting water retention value (WRV) versus corresponding sugar recovery, it was shown that WRV is more useful at low enzyme charges reflecting the use of refining and the reversal of hornification. For de-ashed and refined copy paper, the sugar recovery was determined to be 82% and 97% with enzyme dosages of 4 and 8 Filter Paper Unit (FPU)/g oven-dried (OD) substrate.

Keywords: Office recovered paper; Low enzyme dosage; Sugar recovery; Ash removal; Mechanical refining; Cellulase adsorption; Water retention value (WRV); Hornification

Amentoflavone Inhibits UVB-Induced Matrix Metalloproteinase-1 Expression Through the Modulation of AP-1 Components in Normal Human Fibroblasts by Chan-Woo Lee; Yongjoo Na; Nok-hyun Park; Han-Sung Kim; Soo Mi Ahn; Jin Woong Kim; Han-Kon Kim; Young Pyo Jang (pp. 1137-1147).

Amentoflavone is a well-known biflavonoid that has diverse biological effects. Previously, we reported that amentoflavone suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression in normal human fibroblasts (NHF). We investigated the effects of amentoflavone on UVB-induced MMP-1 expression in order to elucidate its mode of action. NHF were treated with amentoflavone for indicated times and doses with UVB irradiation. The expressions of MMP-1 gene and protein were determined by RT-PCR and ELISA, respectively. MAP kinase phosphorylation and the expression of c-Fos protein were determined by Western blot. The treatment of amentoflavone completely blocked the upregulation of MMP-1 which is induced by UVB irradiation in HaCaT–NHF co-culture in a dose-dependent manner as well as in NHF monoculture. Also, amentoflavone inhibited UVB-induced activation of extracellular signal-regulated kinase (ERK) without changing total ERK protein level, and did not affect p38 or JNK activation. Finally, AP-1 transcription factor components, phospho-c-Jun and c-Fos protein expressions were decreased by amentoflavone treatment. The major finding of this study shows that amentoflavone inhibits intracellular cell signaling ERK pathway leading to the prevention of MMP-1 expression in human skin fibroblasts. Therefore, these results strongly suggest that amentoflavone should be investigated as a potential agent for the prevention and the treatment of skin photoaging.

Keywords: Amentoflavone; Ultraviolet-B; Matrix metalloproteinase-1; MAP kinase; AP-1; Human fibroblasts

Synthesis, Characterization, and Oil Recovery Application of Biosurfactant Produced by Indigenous Pseudomonas aeruginosa WJ-1 Using Waste Vegetable Oils by Wen-Jie Xia; Zhi-bin Luo; Han-Ping Dong; Li Yu; Qing-Feng Cui; Yong-Qiang Bi (pp. 1148-1166).

A bacterial strain was isolated and cultured from the oil excavation areas in tropical zone in northern China. The biochemical characteristics and partial sequenced 16S rRNA gene of isolate, WJ-1, was identical to those of cultured representatives of the species Pseudomonas aeruginosa. This bacterium was able to produce a type of biosurfactant. Compositional analysis revealed that the extracted biosurfactant was composed of high percentage lipid (∼74%, w/w) and carbohydrate (∼20%, w/w) in addition to a minor fraction of protein (∼6%, w/w). The best production of 50.2 g/l was obtained when the cells were grown on minimal salt medium containing 6.0% (w/v) glucose and 0.75% (w/v) sodium nitrate supplemented with 0.1% (v/v) element solution at 37 °C and 180 rpm after 96 h. The optimum biosurfactant production pH value was found to be 6.0–8.0. The biosurfactant of WJ-1, with the critical micelle concentration of 0.014 g/L, could reduce surface tension to 24.5 mN/m and emulsified kerosene up to EI₂₄ ≈95. The results obtained from time course study indicated that the surface tension reduction and emulsification potential was increased in the same way to cell growth. However, maximum biosurfactant production occurred and established in the stationary growth phase (after 90 h). Thin layer chromatography, Fourier transform infrared spectrum, and mass spectrum analysis indicate the extracted biosurfactant was affiliated with rhamnolipid. The core holder flooding experiments demonstrated that the oil recovery efficiency of strain and its biosurfactant was 23.02% residual oil.

Keywords: Pseudomonas aeruginosa ; Biosurfactant; Rhamnolipid; Surface tension; Emulsification; Oil recovery

Hydrolysis of Native and Heat-Treated Starches at Sub-Gelatinization Temperature Using Granular Starch Hydrolyzing Enzyme by U. Uthumporn; Y. N. Shariffa; A. A. Karim (pp. 1167-1182).

The effect of heat treatment below the gelatinization temperature on the susceptibility of corn, mung bean, sago, and potato starches towards granular starch hydrolysis (35°C) was investigated. Starches were hydrolyzed in granular state and after heat treatment (50°C for 30 min) by using granular starch hydrolyzing enzyme for 24 h. Hydrolyzed heat-treated starches showed a significant increase in the percentage of dextrose equivalent compared to native starches, respectively, with corn 53% to 56%, mung bean 36% to 47%, sago 15% to 26%, and potato 12% to 15%. Scanning electron microscopy micrographs showed the presence of more porous granules and surface erosion in heat-treated starch compared to native starch. X-ray analysis showed no changes but with sharper peaks for all the starches, suggested that hydrolysis occurred on the amorphous region. The amylose content and swelling power of heat-treated starches was markedly altered after hydrolysis. Evidently, this enzyme was able to hydrolyze granular starches and heat treatment before hydrolysis significantly increased the degree of hydrolysis.

Keywords: Starch; Enzymes; Hydrolysis; Heat treatment and dextrose equivalent

Biogas Production Potential and Kinetics of Microwave and Conventional Thermal Pretreatment of Grass by Lianhua Li; Xiaoying Kong; Fuyu Yang; Dong Li; Zhenhong Yuan; Yongming Sun (pp. 1183-1191).

Pretreatment methods play an important role in the improvement of biogas production from the anaerobic digestion of energy grass. In this study, conventional thermal and microwave methods were performed on raw material, namely, Pennisetum hybrid, to analyze the effect of pretreatment on anaerobic digestion by the calculation of performance parameters using Logistic function, modified Gompertz equation, and transference function. Results indicated that thermal pretreatment improved the biogas production of Pennisetum hybrid, whereas microwave method had an adverse effect on the performance. All the models fit the experimental data with R ² > 0.980, and the Reaction Curve presented the best agreement in the fitting process. Conventional thermal pretreatment showed an increasing effect on maximum production rate and total methane produced, with an improvement of around 7% and 8%, respectively. With regard to microwave pretreatment, maximum production rate and total methane produced decreased by 18% and 12%, respectively.

Keywords: Pennisetum hybrid; Anaerobic fermentation; Mathematical model; Pretreatment

Role of Tyr-435 of Vibrio harveyi Chitinase A in Chitin Utilization by Natchanok Sritho; Wipa Suginta (pp. 1192-1202).

Vibrio harveyi chitinase A or VhChiA (EC.3.2.1.14) is a member of GH-18 chitinases that catalyzes chitin degradation from marine biomaterials. Our earlier structural data of VhChiA suggested that Tyr-435 marks the ending of subsite +2 and may influence binding of the interacting substrate at the aglycone binding sites. This study reports the effects of Tyr-435 using site-directed mutagenesis technique. Mutation of Tyr-435 to Ala (mutant Y435A) enhanced both binding and catalytic efficiency of VhChiA, whereas substitution of Tyr-435 to Trp (mutant Y435W) lessened the ability of the enzyme to bind and hydrolyze chitin substrates. The increased activity of Y435A can be explained by partial removal of a steric clash around subsite (+2), thereby allowing a chitin chain to move beyond or to access the enzyme’s active site from the aglycone side more straightforwardly.

Keywords: Chitin; Chitinase; Marine bacterium; Site-directed mutagenesis; Vibrio harveyi

Identification of Highly Active Flocculant Proteins in Bovine Blood by George J. Piazza; Alberto Nuñez; Rafael A. Garcia (pp. 1203-1214).

Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits α and β) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, γ-globulin, α-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, α-globulin, and β-globulin were not flocculants. On a mass basis, hemoglobin, γ-globulin, α-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity.

Keywords: Bovine blood; Fibrinogen; Flocculation; Globulin; Hemoglobin; MALDI TOF MS

Selenium Enrichment on Cordyceps militaris Link and Analysis on Its Main Active Components by Jing Z. Dong; C. Lei; Xun R. Ai; Y. Wang (pp. 1215-1224).

To investigate the effects of selenium on the main active components of Cordyceps militaris fruit bodies, selenium-enriched cultivation of C. militaris and the main active components of the fruit bodies were studied. Superoxide dismutase (SOD) activity and contents of cordycepin, cordycepic acid, and organic selenium of fruit bodies were sodium selenite concentration dependent; contents of adenosine and cordycep polysaccharides were significantly enhanced by adding sodium selenite in the substrates, but not proportional to sodium selenite concentrations. In the cultivation of wheat substrate added with 18.0 ppm sodium selenite, SOD activity and contents of cordycepin, cordycepic acid, adenosine, cordycep polysaccharides, and total amino acids were enhanced by 121/145%, 124/74%, 325/520%, 130/284%, 121/145%, and 157/554%, respectively, compared to NS (non-selenium-cultivated) fruit bodies and wild Cordyceps sinensis; organic selenium contents of fruit bodies reached 6.49 mg/100 g. So selenium-enriched cultivation may be a potential way to produce more valuable medicinal food as a substitute for wild C. sinensis.

Keywords: Cordyceps militaris ; Selenium; Active components; Trace minerals; Medicinal value

Rapid Establishment of p-Nitrophenol Biodegradation in Acetate-Fed Aerobic Granular Sludge by Venkata Nancharaiah Yarlagadda; Renu Kadali; Naresh Sharma; Raju Sekar; Venugopalan Vayalam Purath (pp. 1225-1235).

The aim of the study was to investigate the acclimation of precultivated acetate-fed aerobic granular sludge to a toxic xenobiotic biodegradation. Establishment of p-nitrophenol (PNP) biodegradation in acetate-fed aerobic granular sludge and concomitant changes in the microstructure and bacterial community were determined. Rapid establishment of PNP utilization was observed in the granular sludge when fed with PNP as the sole carbon source. The specific PNP removal was 36-mg h^-1 g^-1 granular biomass at an initial PNP concentration of 50 mg L^-1. The presence of PNP resulted in significant membrane damage in a subpopulation of the bacterial consortium, as shown by BacLight viability staining. This was coincided with a significant decrease in the culturable bacterial diversity of the granular biomass. PCR-DGGE analysis revealed a shift and decrease in number of bands during the establishment of PNP biodegradation. Scanning electron microscopy showed the dominance of rod-shaped bacteria in the PNP-utilizing microbial granules. Our results suggest that acetate-fed granular sludge could be quickly adapted for PNP biodegradation.

Keywords: Aerobic granules; Aerobic granular sludge; Biodegradation; p-Nitrophenol

Substitution of the Catalytic Metal and Protein PEGylation Enhances Activity and Stability of Bacterial Phosphotriesterase by Lucia Perezgasga; Lorena Sánchez-Sánchez; Sergio Aguila; Rafael Vazquez-Duhalt (pp. 1236-1247).

Phosphotriesterase, a pesticide-degrading enzyme, from Flavobacterium sp. was cloned and expressed in Escherichia coli. The catalytic zinc ions were replaced by cobalt atoms increasing the catalytic activity of phosphotriesterase on different pesticides. This metal substitution increased the catalytic activity from 1.4 times to 4 times according to the pesticide. In order to explain this catalytic increase, QM/MM calculations were performed. Accordingly, the HOMO energy of the substrate is closer to the LUMO energy of the cobalt-substituted enzyme. The chemical modification of the enzyme surface with poly(ethylene glycol) increased the thermostability and stability against metal chelating agents of both metal phosphotriesterase preparations.

Keywords: Phosphotriesterase; Chemical modification; Cobalt substitution; Stability; QM/MM

Effect of Substrate Concentration on Dark Fermentation Hydrogen Production Using an Anaerobic Fluidized Bed Reactor by Eduardo Lucena Cavalcante de Amorim; Leandro Takano Sader; Edson Luiz Silva (pp. 1248-1263).

The effect of substrate (glucose) concentration on the stability and yield of a continuous fermentative process that produces hydrogen was studied. Four anaerobic fluidized bed reactors (AFBRs) were operated with a hydraulic retention time (HRT) from 1 to 8 h and an influent glucose concentration from 2 to 25 g L⁻¹. The reactors were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C with an influent pH around 5.5 and an effluent pH of about 3.5. The AFBRs with a HRT of 2 h and a feed strength of 2, 4, and 10 g L⁻¹ showed satisfactory H₂ production performance, but the reactor fed with 25 g L⁻¹ of glucose did not. The highest hydrogen yield value was obtained in the reactor with a glucose concentration of 2 g L⁻¹ when it was operated at a HRT of 2 h. The maximum hydrogen production rate value was achieved in the reactor with a HRT of 1 h and a feed strength of 10 g L⁻¹. The AFBRs operated with glucose concentrations of 2 and 4 g L⁻¹ produced greater amounts of acetic and butyric acids, while AFBRs with higher glucose concentrations produced a greater amount of solvents.

Keywords: Hydrogen production; Anaerobic fluidized bed reactor; Substrate concentration; Hydraulic retention time

High pH Solubilization and Chromatography-Based Renaturation and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor from Inclusion Bodies by Ming Li; Hua Fan; Jiahua Liu; Minhong Wang; Lili Wang; Chaozhan Wang (pp. 1264-1274).

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

Keywords: Recombinant human G-CSF; Inclusion bodies; High pH solubilization; Protein refolding; Protein purification; Liquid chromatography

ANCUT2, an Extracellular Cutinase from Aspergillus nidulans Induced by Olive Oil by Denise Castro-Ochoa; Carolina Peña-Montes; Augusto González-Canto; Ana Alva-Gasca; Ricardo Esquivel-Bautista; Arturo Navarro-Ocaña; Amelia Farrés (pp. 1275-1290).

Cutinases are versatile carboxylic ester hydrolases with great potential in many biocatalytic processes, including biodiesel production. Genome sequence analysis of the model organism Aspergillus nidulans reveals four genes encoding putative cutinases. In this work, we purified and identified for the first time a cutinase (ANCUT2) produced by A. nidulans. ANCUT2 is a 29-kDa protein which consists of 255 amino acid residues. Comparison of the amino acid sequence of ANCUT2 with other microbial cutinase sequences revealed a high degree of homology with other fungal cutinases as well as new features, which include a serine-rich region and conserved cysteines. Cutinase production with different lipidic and carbon sources was also explored. Enzyme activity was induced by olive oil and some triacylglycerides and fatty acids, whereas it was repressed by glucose (1%) and other sugars. In some conditions, a 22-kDa post-translational processing product was also detected. The cutinase nature of the enzyme was confirmed after degradation of apple cutin.

Keywords: Carboxylesterases; Cutinase; Production; Cutin; Aspergillus nidulans

Purification and Characterisation of a 31-kDa Chitinase from the Myzus Persicae Aphid: A Target for Hemiptera Biocontrol by Frédéric Francis; Julien Saguez; Anas Cherqui; Sophie Vandermoten; Charles Vincent; Marie-France Versali; Jacques Dommès; Edwin De Pauw; Philippe Giordanengo; Eric Haubruge (pp. 1291-1300).

Hydrolytic enzymes involved in chitin degradation are important to allow moulting during insect development. Chitinases are interesting targets to disturb growth and develop alternative strategies to control insect pests. In this work, a chitinase from the aphid Myzus persicae was purified with a 36-fold purification rate in a three step procedure by ammonium sulphate fractionation, anion-exchange chromatography on a DEAE column and on an affinity Concanavalin A column. The purified chitinase purity assessed by 1D and 2D SDS–PAGE revealed a single band and three spots at 31 kDa, respectively. Chitinases were found to have high homologies with Concanavalins A and B, two chitinase-related proteins, a fungal endochitinase and an aphid acetylhydrolase by peptide identification by Maldi-Tof-Tof. The efficiency of two potent chitinase inhibitors, namely allosamidin and psammaplin A, was tested and showed significant rate of enzymatic inhibition.

Keywords: Aphid; Chitinase; Purification; Inhibitors; Biocontrol

Cloning, Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from Pseudomonas sp. K in Escherichia coli by Dan-Feng Liu; Hai-Tao Ding; Yi-Qing Du; Yu-Hua Zhao; Xiao-Ming Jia (pp. 1301-1313).

A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg⁻¹ at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K _m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na⁺, NH ₄ ⁺ , Mg²⁺, Fe²⁺, Fe³⁺, Co²⁺, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.

Keywords: Phosphite dehydrogenase; Cofactor regeneration; Pseudomonas sp. K; Wide pH range stability; Low K _m value

Biomarkers in Catfish Sciades herzbergii (Teleostei: Ariidae) from Polluted and Non-polluted Areas (São Marcos’ Bay, Northeastern Brazil) by Raimunda Nonata Fortes Carvalho-Neta; Audalio Rebelo Torres Jr.; Ana Lúcia Abreu-Silva (pp. 1314-1327).

Biomarkers based on specific enzyme activities and histological alterations are useful tools for evaluating toxicological effects of xenobiotics in wild fish. In this work, an experimental system of biomarkers with enzyme glutathione S-transferase (GST) and branchial lesions in catfish (Sciades herzbergii) was mathematically modeled. The fish were collected along known pollution gradients (S1) and from areas regarded relatively free of anthropogenic input (S2) in São Marcos’ Bay, Brazil. GST was measured spectrophotometrically, and branchial lesions were examined by light microscope. The databases from this analysis were compiled, and non-linear models were used to analyze the dependence of the enzyme activity on the areas of sampling and on selected biological parameters of the fish. Fish weight, length, and somatic indices (gonadosomatic index) were significant in the model of GST activity only in A2. Brachial lesions were significant in the model of GST activity only in A1. The obtained model indicates that when the GST ceases to act, serious branchial lesions are observed in the fish of the contaminated regions.

Keywords: Glutathione S-transferase; Branchial lesion; Gonadosomatic index; Biometry; Catfish; Sciades herzbergii

Characterization of Xyn10J, a Novel Family 10 Xylanase from a Compost Metagenomic Library by Yu Seok Jeong; Han Beur Na; Sung Kyum Kim; Yong Ho Kim; Eun Ju Kwon; Jungho Kim; Han Dae Yun; Jung-Kul Lee; Hoon Kim (pp. 1328-1339).

A gene encoding an extracellular xylanase was cloned from a compost metagenomic library. The xylanase gene, xyn10J, was 1,137 bp in length and was predicted to encode a protein of 378 amino acid residues with a putative signal peptide of 27 amino acid residues. The molecular mass of the mature Xyn10J was calculated to be 39,882 Da with a pI of 6.09. Xyn10J had a motif GVKVHFTEMDI characteristic of most members of glycosyl hydrolase family 10. The amino acid sequence of Xyn10J showed 60.0% identity to that of XynH, a xylanase from an uncultured soil bacterium and 55% identity to XylC of Cellvibrio mixtus. Site-directed mutagenesis of the expected active site based on the sequence analysis indicated that an aspartic acid residue (Asp207), in addition to the identified catalytic residues Glu165 and Glu270, plays a crucial role for the catalytic activity. The purified Xyn10J had a mass of about 40 kDa and was optimally active at pH 7.0 and 40 °C. Xyn10J hydrolyzed beechwood xylan > birchwood xylan > oat spelt xylan > arabinoxylan. Xyn10J hydrolyzed xylotetraose and xylohexaose exclusively to xylobiose, xylopentaose, and xylotriose mainly to xylobiose with transglycosylation activity. The saccharification of reed (Phragmites communis) powder by commercial enzymes was significantly increased by the addition of a small amount of Xyn10J to the commercial preparation. Xyn10J is the first xylanase screened directly from a compost metagenomic library, and the enzyme has the potential to be used in the conversion of biomass to fermentable sugars for biofuel production.

Keywords: Metagenomic library; Compost; Family 10 xylanase; Transglycosylation; Biofuel production

Intracellular Detection and Evolution of Site-Specific Proteases Using a Genetic Selection System by Kathryn D. Verhoeven; Olvia C. Altstadt; Sergey N. Savinov (pp. 1340-1354).

Development of endoproteases, programmed to promote degradation of peptides or proteins responsible for pathogenic states, represents an attractive therapeutic strategy, since such biocatalytic agents could be directed against a potentially unlimited repertoire of extracellular proteinaceous targets. Difficulties associated with engineering enzymes with tailor-made substrate specificities have, however, hindered the discovery of proteases possessing both the efficiency and selectivity to act as therapeutics. Here, we disclose a genetic system, designed to report on site-specific proteolysis through the survival of a bacterial host, and the implementation of this method in the directed evolution of proteases with a non-native substrate preference. The high sensitivity potential of this system was established by monitoring the activity of the Tobacco Etch Virus protease (TEV-Pr) against co-expressed substrates of various recognition level and corroborated by both intracellular and cell-free assays. The genetic selection system was then used in an iterative mode with a library of TEV-Pr mutants to direct the emergence of proteases favoring a nominally poor substrate of the stringently selective protease. The retrieval of mutant enzymes displaying enhanced proteolytic properties against the non-native sequence combined with reduced recognition of the cognate hexapeptide substrate demonstrates the potential of this system for evolving proteases with improved or completely unprecedented properties.

Keywords: Proteases; Genetic reporter; Protein engineering; Genetic selection; Directed evolution

Constitutive Expression of Yarrowia lipolytica Lipase LIP2 in Pichia pastoris Using GAP as Promoter by Xiaofeng Wang; Yongchuan Sun; Feng Ke; Heyun Zhao; Tao Liu; Li Xu; Yun Liu; Yunjun Yan (pp. 1355-1367).

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.

Keywords: Lipase; Yarrowia lipolytica ; Pichia pastoris ; Constitutive expression; GAP promoter; Fed-batch culture

One-Step Expression and Tyrosine O-Sulfonation of Ax21 in Escherichia coli by Hou Shuguo; Zhang Wei; Zhang Chao; Wu Daoji (pp. 1368-1379).

Ax21 (activator of Xa21-mediated immunity), a pathogen-associated molecular pattern secreted by Xanthomonas oryzae pv. oryzae, can be perceived by a membrane-located pattern recognition receptor Xa21 and triggered immune responses in rice. An Ax21-derived peptide (17-amino acid) containing a sulfated tyrosine-22 (axY^S22) is sufficient for Ax21 activity. Here, we expressed Ax21 and O-sulfated its tyrosine-22 through coexpressing a putative tyrosine sulfotransferase, raxST, and two other genes involved in the synthesis of 3′-phosphoadenosine 5′-phosphosulfate in Escherichia coli BL21 (DE3). The sulfated Ax21 fused with a histidine tag in its N-terminus was extracted and bound onto a Ni-NTA agarose and then cleaved with Factor Xa and CNBr in turn. Δax21Y^S22, a 36-amino acid peptide covering axY^S22 in the lysate supernatant, was finally yielded after ultrafiltration. The purified peptide was further verified by Tricine-SDS-PAGE and isoelectrofocusing electrophoresis. Lesion length analysis, reactive oxygen species production, and mitogen-activated protein kinase (MAPK) activation of rice leaves inoculated with Δax21Y^S22 confirmed the activity of the sulfated peptide. Overall, this study successfully established an efficient system for expression and purification of a sulfated peptide. In addition, the sulfotransferase activity of RaxST was confirmed for the first time.

Keywords: Xanthomonas oryzae pv. oryzae ; PAMP; Tyrosine O-sulfation; Plant immunity; Escherichia coli

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #5)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #5)