Cloning of the Thermostable Cellulase Gene from Newly Isolated Bacillus subtilis and its Expression in Escherichia coli

A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated strain. The optimum temperature of the enzyme reaction was 50°C, and CelDR retained 70% of its maximum activity at 75°C after incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain.     

Screening and Identification of New Isolate: Thermostable <Emphasis Type="Italic">Escherichia coli</Emphasis> with Novel Thermoalkalotolerant Cellulases

A cellulase-producing bacterium strain was isolated from soil that produced novel thermoalkalotolerant cellulases after growth on CMC-Na agar screening plate at 37°C. It was identified as Escherichia coli using the method of 16S rRNA and intergenic spacer gene analysis combined with morphological, physiological, and biochemical tests. Three major components of the cellulases [carboxymethyl cellulase (CMCase), filter paper cellulase, and β-glucosidase] were produced with maximal activities (0.23, 0.08, and 0.15 U/ml) and maximum specific activities 4.13, 0.56, and 0.50 U/mg protein after 72, 96, and 120 h growth, respectively. Maximum CMCase activity was measured at 50°C and pH 6.0, respectively, and it also retained more than 60% of its maximal activity for at least 20 min at 50–70°C and 10 min at 80°C, respectively, and retained approximately 50% of its maximal activity after incubating at 90°C for 10 min. The enzyme could be applied in bioconversion of lignocellulosic agricultural wastes.     

Gene Cloning of Endoglucanase Cel5A from Cellulose-Degrading <Emphasis Type="Italic">Paenibacillus xylanilyticus</Emphasis> KJ-03 and Purification and Characterization of the Recombinant Enzyme

The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.     

Optimization of cellulase production by <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>: application in the biosoftening of cotton fibers

The enhancement of the cellulase activity of Aspergillus nidulans by combinational optimization technique and the usage of cellulase for the biofinishing of cotton fibers were investigated in this study. The strain isolated from decayed, outer shell of Arachis hypogaea was compared for the first time for its ability to produce cellulolytic enzyme in shaken cultures using the optimized media formulated by combinational statistical approach using one factor at a time methodology (OFAT), Plackett Burmann Methodology (PB) and response surface methodology (RSM). A four-factor-five-level central composite design (CCD) was employed to determine the maximum activity of cellulase at optimum levels of carboxy methyl cellulose (CMC), ammonium nitrate and potassium dihydrogen phosphate at varying pH values. The cellulase activity is the best so far obtained with this strain of Aspergillus nidulans. The optimum values of the parameters studied were found to be 0.75 mg/l, 1.5 mg/l, 0.01 mg/l, and 2.15 g/l for KH₂PO₄, NH₄NO₃, Thiamine HCl and CMC, respectively at pH 6.0. This optimization led to the fine tuning of the cellulase production, thereby enhancing the cellulase activity from 4.91 to 60.54 U/ml. This cellulase of higher activity was employed in the biofinishing of the cotton fibers. The results of the scanning electron microscope (SEM) analysis after the treatment favored the fact that maximum surface finishing was achieved at a cotton fiber concentration of 15% (w/v) at 45°C and pH 5.0 using cellulase (60.54 U/ml) at 16th hour of the treatment. A probable mechanism of enzymatic finishing of cotton fibers has also been represented.     

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.     

An effective extracellular protein secretion by an ABC transporter system in <Emphasis Type="Italic">Escherichia coli</Emphasis>: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.     

Signal peptide of <Emphasis Type="Italic">Aureobasidium</Emphasis><Emphasis Type="Italic">pullulans</Emphasis> xylanase: use for extracellular production of a fungal xylanase by <Emphasis Type="Italic">Escherichia coli</Emphasis>

An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia coli. The A. pullulans xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-d-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which were twofold higher than that of the native enzyme.     

Cloning of the thermostable α-amylase gene from Pyrococcus woesei in Escherichia coli

Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Expression of Arginine Deiminase from <Emphasis Type="Italic">Pseudomonas plecoglossicida</Emphasis> CGMCC2039 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and Its Anti-Tumor Activity

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent in clinical trials for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. The arcA gene encoding ADI was cloned from a recently isolated strain Pseudomonas plecoglossicida CGMCC2039. The nucleotide sequence of ADI comprises an ORF of 1,254 bp encoding 417 amino acids. The deduced ADI protein sequence has a calculated molecular weight of 46.5 kDa and shows 97% and 85% identity to ADIs from P. putida and P. aeruginosa, respectively. The arcA from P. plecoglossicida CGMCC2039 was expressed in Escherichia coli BL21 with a N-terminal His₆-tag, and purified to homogeneity. A molecular mass of approximate 49 kDa was confirmed by SDS-PAGE analysis and specific activity was determined to be 4.76 U/mg (pH 6.0 and 37°C). In vivo activity study showed that the rADI could effectively inhibit H22 tumor growth at a total dose of 5 U/mouse over a 2-week dosing period.     

Characterization,gene cloning,and heterologous expression of β-mannanase from a thermophilic <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis BCC41051 producing a thermostable β-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular β-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pi value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0–11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg²⁺ (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).     

Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus

D‐alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3) cells, which were prepared through embedding and cross‐linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700 mM with an enantiomeric excess of 99.99% and e.e._p of 99.50%. The immobilized recombinant B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N‐acetyl‐D‐alanine methyl esters.     

Heterologous expression of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP _w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.     

Xylanase production using inexpensive agricultural wastes and its partial characterization from a halophilic <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TPSV 101

A halophilic and alkali-tolerant Chromohalobacter sp. TPSV 101 with an ability to produce extracellular halophilic, alkali-tolerant and moderately thermostable xylanase was isolated from solar salterns. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. The culture conditions for higher xylanase production were optimized with respect to NaCl, pH, temperature, substrates and metal ions and additives. Maximum xylanase production was achieved in the medium with 20% NaCl, pH-9.0 at 40°C supplemented with 1% (w/v) sugarcane bagasse and 0.5% feather hydrolysate as carbon and nitrogen sources. Sugarcane bagasse (250 U/ml) and wheat bran (190 U/ml) were the best inducer of xylanase when used as carbon source as compared to xylan (61 U/ml). The xylanase that was partially purified by protein concentrator had a molecular mass of 15 kDa approximately. The xylanase from Chromohalobacter sp. TPSV 101 was active at pH 9.0 and required 20% NaCl for optimal xylanolytic activity and was active over a broad range of temperature 40–80°C with 65°C as optimum. The early stage hydrolysis products of sugarcane bagasse were xylose and xylobiose, after longer periods of incubation only xylose was detected.     

Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation

The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, M_r 23 kDa) and 218 amino acids (β subunit, M_r 24 kDa) and the NHase activator of 413 amino acids (M_r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.     

Production and biochemical characterization of a novel cellulase-poor alkali-thermo-tolerant xylanase from <Emphasis Type="Italic">Coprinellus disseminatus</Emphasis> SW-1 NTCC 1165

Fungi producing xylanases are plentiful but alkali-thermo-tolerant fungi producing cellulase-poor xylanase are rare. Out of 12 fungal strains isolated from various sources, Coprinellus disseminatus SW-1 NTCC 1165 yielded the highest xylanase activity (362.1 IU/ml) with minimal cellulase contamination (0.64 IU/ml). The solid state fermentation was more effective yielding 88.59% higher xylanase activity than that of submerged fermentation. An incubation period of 7 days at 37°C and pH 6.4 accelerated the xylanase production up to the maximum level. Among various inexpensive agro-residues used as carbon source, wheat bran induced the maximum xylanase titres (469.45 IU/ml) while soya bean meal was the best nitrogen source (478.5 IU/ml). A solid substrate to moisture content ratio of 1:3 was suitable for xylanase production while xylanase titre was repressed with the addition of glucose and lactose. The xylanase and laccase activities under optimized conditions were 499.60 and 25.5 IU/ml, respectively along with negligible cellulase contamination (0.86 IU/ml). Biochemical characterization revealed that optimal xylanase activity was observed at pH 6.4 and temperature 55°C and xylanase is active up to pH 9 (40.33 IU/ml) and temperature 85°C (48.81 IU/ml). SDS–PAGE and zymogram analysis indicated that molecular weight of alkali-thermo-tolerant xylanase produced by C. disseminatus SW-1 NTCC 1165 was 43 kDa.     

Overexpression of methyl parathion hydrolase and its application in detoxification of organophosphates

The coding region of mpd gene corresponding to mature methyl parathion hydrolase (MPH) was heterologously overexpressed in Escherichia coli BL21 (DE3) by using pET expression system. The lactose-induced expression yield of MPH is increased 2-fold compared with IPTG as inducer. Furthermore, it was found that specific activity of MPH increased 48% by reducing the induction temperature to 22°C. The addition of 25 mM lactose at 22°C, the MPH activity of fermentation broth had a specific activity of 1.4 × 10⁴ U/mg protein. Plasmid was no significant decrease in the modified medium. The optimal pH and temperature of MPH were 8.0 and 30°C, respectively. Over a period of 5 months, the dried cells showed no significant decrease in the activity of the detoxifying enzymes. The crude enzymes in 50 mM citrate-phosphate buffer (pH 8.0) were able to degrade about 98% of the organophosphate pesticides sprayed on cabbage. The detoxification efficiency was superior to that of the treatments of water, detergent, and a commercially available enzyme product. Additionally, the products of pesticide hydrolysis generated by treatment with the enzyme extract were determined to be virtually nontoxic.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

Characterization of two soil metagenome-derived lipases with high specificity for <Emphasis Type="Italic">p</Emphasis>-nitrophenyl palmitate

Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters. P. Wei and L. Bai contributed equally to this work.