Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Recombinant production of <Emphasis Type="Italic">Penicillium oxalicum</Emphasis> PJ3 phytase in <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

The 1,332 bp phytase gene of Penicillium oxalicum PJ3 was inserted into the expression vector, pPICZαA and expressed in the methylotrophic yeast, Pichia pastoris as an active, extracellular phytase. The recombinant phytase reached a maximum yield of 12 U/ml of medium at 120 h of cultivation after methanol induction under shake-flask conditions. The enzyme was glycosylated, with a molecular mass of about 62.5 kDa. The Michaelis constant (K _m) and maximum reaction rate (V _max) for sodium phytate was 0.37 mM and 526.3 U/mg of protein, respectively. The optimal activity occurred at pH 4.5 and 55°C. Jaecheon Lee and Yunjaie Choi contributed equally to this work.     

Mn<Superscript>2+</Superscript> enhances theanine-forming activity of recombinant glutamine synthetase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn²⁺ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence of 10 mM Mn²⁺ at optimal pH 7.5. The activity is markedly higher activated by Mn²⁺ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn²⁺ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1 g/L by paper chromatography and HPLC, respectively.     

High production of laccase B from <Emphasis Type="Italic">Trametes</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The coding sequence of a laccase isozyme from Trametes sp. AH28-2 was cloned in pPIC9K vector and heterologously overexpressed in the yeast Pichia pastoris strain GS115. In the minimal medium containing 0.3 mM CuSO₄ and 0.6% alanine, the maximum yield of the recombinant laccase rLacB reached 32,000 U/l (1,012 U/mg), slightly higher than that of the native enzyme nLacB (∼30,000 U/l, 1,356 U/mg). The enzymatic properties of rLacB were different from those of nLacB as well. Regardless of the inferior thermal stability, rLacB had much better stability at both neutral and basic pH range compared to nLacB. In addition, the dye decolorization potential of rLacB was similar to that of nLacB.     

Production of a thermophilic maltooligosyl-trehalose synthase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The thermoacidophilic archaeon Sulfolobus solfataricus MT4 encodes a maltooligosyltrehalose synthase (MTS), that catalyzes an intramolecular transglycosylation process converting the glycosidic linkages at the reducing end of dextrins from alpha-1,4 into alpha-1,1. In this research the gene encoding MTS was cloned and expressed in Lactococcus lactis NZ9000 using the so-called NICE system. Growth conditions of the recombinant strain were optimized in flask experiments in relation to enzyme production. Batch experiments in 2 L-fermenters were performed on the best identified semidefined medium and 256 U L(-1) of recombinant MTS were produced. Purified recombinant MTS shows its optimal activity at 70 degrees C and pH 5.5, prefers maltoheptaose and maltohexaose as substrates, and demonstrates minimal side hydrolytic activity.     

High level expression of kringle 5 fragment of plasminogen in <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">pastoris</Emphasis>

Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl^-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.Revisions requested 9 November 2004; Revisions received 2 December 2004     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.     

Optimization of culturing conditions of <Emphasis Type="Italic">Porphyridium cruentum</Emphasis> using uniform design

The red microalga Porphyridium contains many valuable compounds such as polysaccharides, polyunsaturated fatty acids, and phycoerythrin (PE). In this study, a uniform design method and regression analysis were used to investigate the effects of initial pH, light intensity, inoculation ratio, and liquid volume in flask on the optimal biomass, exopolysaccharides (EPS), and PE production of Porphyridium cruentum in a batch culture at laboratory scale. Using regression analysis, we obtained the models to clarify the effects of individual factors and their interactions on the biomass, EPS, and PE production of P. cruentum. The optimal condition for the biomass was the following: pH 5.0, light intensity 7098.0 lx, inoculation ratio 1:17.2, and liquid volume 100.0 ml; for EPS was pH 5.0, light intensity 4501.0 lx, inoculation ratio 1:20, and liquid volume 100.3 ml; while pH 8.0, light intensity 7100.0 lx, inoculation ratio 1:20, and liquid volume 100.3 ml was the best for PE production. The maximum biomass 3.27 g/l, EPS production 543.1 mg/l, and PE production 132.0 mg/l were demonstrated by confirmatory experiment to the optimum culture conditions in a reciprocal shaker. The statistical methods used in the present study are useful strategies for optimizing of culture conditions for other microalgae.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Characterization and large-scale production of recombinant <Emphasis Type="Italic">Streptoverticillium platensis</Emphasis> transglutaminase

Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was ~33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.     

Acid phosphatase production by recombinant <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett–Burman design, three fermentation variables (pH, sucrose concentration, and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production was achieved in shake flasks (17,054 U g⁻¹ DYB) and laboratory fermenter (18,465 U g⁻¹ DYB), respectively.     

Production of fructosyltransferase by <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. ATCC 20524 in batch and two-step batch cultures

A comparison of fructosyltransferase (EC 2.4.1.9) production by Aureobasidium sp. ATCC 20524 in batch and two step batch cultures was investigated in a 1-l stirred tank reactor using a sucrose supply of 200 g/l. Results showed that the innovative cultivation in two step of Aureobasidium sp. produced more fructosyltransferase (FFase) than the single batch culture at the same sucrose concentration with a maximal enzyme production of 523 U/ml, which was 80.5% higher than the one obtained in the batch culture. The production of fructooligosaccharides (FOSs) was also analyzed; their concentration reached a maximum value of 160 g/l the first day in the two-step culture and 127 g/l in the single-batch mode. The use of the two-step batch culture with Aureobasidium sp. ATCC 20524 in allowing the microorganism to grow up prior to the induction of sucrose (second step), proved to be a powerful method for producing fructosyltransferase and FOSs.     

Production of bifunctional single-chain antibody-based fusion proteins in <Emphasis Type="Italic">Pichia </Emphasis><Emphasis Type="Italic">pastoris</Emphasis> supernatants

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

Efficient extracellular expression of <Emphasis Type="Italic">Bacillus deramificans</Emphasis> pullulanase in <Emphasis Type="Italic">Brevibacillus choshinensis</Emphasis>

In this study, the pullulanase gene from Bacillus deramificans was efficiently expressed in Brevibacillus choshinensis. The optimal medium for protein expression was determined through a combination of single-factor experiments and response surface methodology. The initial pH of the medium and the culture temperature were optimized. The pullulanase yield increased 10.8-fold through medium and condition optimization at the shake-flask level. From the results of these experiments, the dissolved oxygen level was optimized in a 3-L fermentor. Under these optimized conditions, the pullulanase activity and the specific pullulanase productivity reached 1005.8 U/mL and 110.5 × 10³ U/g dry cell weight, respectively, with negligible intracellular expression. The Brevibacillus choshinensis expression system has proven to be valuable for the extracellular production of pullulanase.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.