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ABSTRACT: We cloned a Paenibacillus sp. E18 5.3-kb xylanolytic gene cluster that contains three open reading frames encoding two family-43 α-l-arabinofuranosidases (Abf43A and Abf43B) and one family-10 xylanase (XynBE18). The deduced amino acid sequences of Abf43A and Abf43B were at most 68% and 63% identical with two putative family-43 proteins from Clostridium sp. DL-VIII (EHI98634.1 and EHI98635.1), respectively, but were only 11% identical with each other. Recombinant Abf43A and Abf43B had similar activities at 45°C and pH 6.0, but varied in thermostabilities and substrate specificities. Abf43B was active against only 4-nitrophenyl α-l-arabinofuranoside, whereas Abf43A acted on 4-nitrophenyl α-l-arabinofuranoside, wheat arabinoxylan, 4-nitrophenyl α-d-xylopyranoside, and sugar beet arabinan. The sequential and combined effects on xylan degradation by XynBE18, Abf43A, and Abf43B were characterized. For beechwood, birchwood, and oat spelt xylans as substrates, synergistic effects were found when XynBE18 and Abf43A or Abf43B were incubated together and when the substrates were first incubated with Abf43A or Abf43B and then with XynBE18. Further HPLC analysis showed that the amounts of xylobiose and xylose increased sharply in the aforementioned reactions. For water-soluble wheat arabinoxylan as substrate, Abf43A not only released arabinose but also had synergistic effect with XynBE18. Synergy may arise as the result of removal of arabinose residues from xylans by α-l-arabinofuranosidases, which eliminates steric hindrance caused by the arabinose side chains and which allows xylanases to then degrade the xylan backbone, producing short xylooligosaccharides.
Applied and environmental microbiology 01/2013; · 3.69 Impact Factor
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ABSTRACT: A novel β-mannanase gene, man5XZ7, was cloned from thermophilic fungus Thielavia arenaria XZ7, and successfully expressed in Pichia pastoris. The gene (1,110 bp) encodes a 369-amino acid polypeptide with a molecular mass of approximately 40.8 kDa. The deduced sequence of Man5XZ7 consists of a putative 17-residue signal peptide and a catalytic module belonging to glycoside hydrolase (GH) family 5, and displays 76 % identity with the experimentally verified GH 5 endo-β-1,4-mannanase from Podospora anserina. Recombinant Man5XZ7 was optimally active at 75 °C and pH 5.0 and exhibited high activity at a wide temperature range (>50.0 % activity at 50-85 °C). Moreover, it had good adaptability to acidic to basic pH (>74.1 % activity at pH 4.0-7.0 and 25.6 % even at pH 9.0) and good stability from pH 3.0 to 10.0. These enzymatic properties showed that Man5XZ7 was a new thermophilic and alkali-tolerant β-mannanase. Further amino acid composition analysis indicated that Man5XZ7 has several characteristic features of thermophilic enzymes.
Applied Microbiology and Biotechnology 01/2013; · 3.42 Impact Factor
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ABSTRACT: A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85 % with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0-9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg(-1)). The K (m) and V (max) values towards p-nitrophenyl β-D-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol min(-1) mg(-1), respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-D-cellobioside (pNPC) and p-nitrophenyl-β-D-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.
Applied biochemistry and biotechnology 01/2013; · 1.94 Impact Factor
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ABSTRACT: Mannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.
The man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.
This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.
PLoS ONE 01/2013; 8(2):e56146. · 4.09 Impact Factor
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ABSTRACT: Three xylanase genes (xynA, xynB, xynC) of glycosyl hydrolase family 10 were identified in Humicola insolens Y1. The deduced protein sequences showed the highest identity of ⩽83% to known fungal xylanases and of ⩽38% with each other. Recombinant XynA-C produced in Pichia pastoris showed optimal activities at pH 6.0-7.0 and at high temperature (70-80°C), and exhibited good stability over a broad pH range and temperatures at 60°C. The gene xynC produced by H. insolens Y1 (named XynW) was similar in enzyme properties with XynC expressed by Pichia. XynA exhibited better alkaline adaptation and thermostability, and had higher catalytic efficiency and wider substrate specificity. Under simulated mashing conditions, addition of XynA-C showed better performance on filtration acceleration (37.4%) and viscosity reduction (13.5%) than Ultraflo from Novozyme. Thus the three xylanases represent good candidates for application in the brewing industry.
Bioresource technology 12/2012; 130C:161-167. · 4.25 Impact Factor
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ABSTRACT: A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K (m) and V (max) values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.
MIRCEN Journal of Applied Microbiology and Biotechnology 11/2012; · 1.08 Impact Factor
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ABSTRACT: Alkaline pectate lyases are favorable for the textile industry. Here, we report the gene cloning and expression of a low-temperature-active alkaline pectate lyase (PL D) from Xanthomonas campestris ACCC 10048. Deduced PL D consists of a putative 27-residue signal peptide and a catalytic domain of 320 residues belonging to family PF09492. Recombinant PL D (r-PL D) produced in Escherichia coli was purified to electrophoretic homogeneity with a single step of Ni(2+)-NTA affinity chromatography and showed an apparent molecular weight of ~38 kDa. The pH and temperature optima of r-PL D were found to be 9.0 °C and 30 °C, respectively. Compared with its microbial counterparts, r-PL D had higher activity over a wide pH range (>45 % of the maximum activity at pH 3.0-12.0) and at lower temperatures (>35 % of activity even at 0 °C). The K (m) and V (max) values of r-PL D for polygalacturonic acid were 4.9 g l(-1) and 30.1 μmol min(-1) mg(-1), respectively. Compared with the commercial compound pectinase from Novozymes, r-PL D showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (35.1 % vs. 36.5 %) and in bioscouring of jute (10.25 % vs. 10.82 %). Thus, r-PL D is a valuable additive candidate for the textile industry.
Applied biochemistry and biotechnology 09/2012; · 1.94 Impact Factor
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ABSTRACT: Polygalacturonases are important feed and food additives. In the present study an exo-polygalacturonase gene (pgu B) was cloned from Klebsiella sp. Y1 CGMCC 4433 and expressed in Escherichia coli BL21 (DE3). pgu B encodes a 658-amino acid polypeptide belonging to Glycoside Hydrolase Family 28. The optimal pH and temperature of exo-PGU B activity were 6.0 and 40-50°C, respectively. The enzyme exhibited >35% of maximum activity within the pH range of 2.0-12.0. Exo-PGU B or an exo-PGU B/ endo-polygalacturonase mixture reduced the viscosity of polygalacturonic acid (1.0%, w/v) by 15.6 and 39.4%, respectively. Under simulated alimentary tract conditions, exo-PGU B was very stable (>25% activity from pH 1.5 to 6.8) and active, releasing 53.7 and 109.6μg of galacturonic acid from 400 to 800μg of polygalacturonic acid, respectively. These properties make exo-PGU B a potentially valuable additive for applications in feed and food.
Bioresource technology 07/2012; 123:171-6. · 4.25 Impact Factor
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ABSTRACT: Two novel cellulase genes, cbh6A and egGH45, were cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. The putative polypeptide of CBH6A consists of a family 1 CBM and a catalytic domain of glycosyl hydrolase family 6 cellobiohydrolases, while deduced EgGH45 only contains a catalytic domain of family 45 endoglucanases. CBH6A and EgGH45 were optimally active at pH 7.0 and 65°C, and pH 6.0 and 60°C, respectively. Both enzymes exhibited high activities and stabilities over a wide pH range and had good thermostability at 70°C. CBH6A and EgGH45 had significant resistance to SDS (10mM), remaining 35% and 54% activities, respectively. These enzymes had synergic effect on the hydrolysis of filter paper, showing the highest efficiency in the ratio of CBH6A to EgGH45 at 80:20. The properties make this enzyme combination potential for application in textile and detergents industries.
Bioresource technology 07/2012; 121:404-10. · 4.25 Impact Factor
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ABSTRACT: Using degenerate polymerase chain reaction (PCR), thermal asymmetric interlaced (TAIL)-PCR, and reverse transcription (RT)-PCR
techniques, a new β-mannanase gene, denoted as man5C6, was obtained from Penicillium sp. C6. The gene has an open reading frame of 1,155bp, and codes for a polypeptide (Man5C6) of 384 amino acids including
a putative 26-residue signal peptide. The deduced amino acid sequence showed highest identity (59.2%) with an experimentally
verified β-mannanase from Podospora anserine belonging to glycoside hydrolase family 5. Man5C6 was successfully expressed in Pichia pastoris, and secreted up to 2.5g in 1l medium. Recombinant Man5C6 was easily purified to electrophoretic homogeneity by a sing-step
chromatography. The purified recombinant enzyme exhibited optimal activity at pH 4.5, and remained>55% of its maximum activity
at pH 3.0–7.0. The temperature optimum was found to be 70°C. The specific activity, and K
m and V
max values were 226.5 U mg−1, 12.3mgml−1 and 2,400.2μmolmin−1 mg−1, respectively, for locust bean gum, and 78.7Umg−1, 0.2mgml−1 and 894.6μmolmin−1 mg−1, respectively, for konjac flour. These properties make Man5C6 a potential candidate for high-level production of β-mannanase
with low cost and simple processing technology.
Keywords
Penicillium sp. C6–β-Mannanase–
Pichia pastoris
–Over-expression
World Journal of Microbiology and Biotechnology 05/2012; 27(12):2813-2819. · 1.53 Impact Factor
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ABSTRACT: A 37.5-kb chromosomal segment with 41.5% G+C content was cloned using genome walking method from Sphingobacterium sp. TN19, a symbiotic strain isolated from the gut of Batocera horsfieldi (Coleoptera, Cerambycidae) larvae. Twenty-three complete and two partial open reading frames (ORFs) were found in this region,
and shared highest identities of 31.6–80.0% (11 of them <60%) to known sequences from the sources shared the common feature—fluid
with low dissolved oxygen levels. These ORFs were organized uniquely in chromosome compared to that from other bacteria, and
putatively involved in xylan and xylose metabolism. To confirm their putative functions, one xylanase gene (xynB19) and one arabinosidase gene (gh43A19), were hetero-expressed in Escherichia coli BL21 (DE3), respectively, and both the recombinant enzymes showed significant enzymatic activities. This result indicates
that xylan degradation is a complex process involving various enzymes, and genome walking is an efficient method to obtain
multiple genes.
Keywords
Batocera horsfieldi
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Sphingobacterium sp.-TN19-Gut-Xylan and xylose metabolism
World Journal of Microbiology and Biotechnology 04/2012; 26(4):761-765. · 1.53 Impact Factor
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ABSTRACT: A xylanase gene, xynA4-2, was obtained from the genome sequence of thermoacidophilic Alicyclobacillus sp. A4 and expressed in Escherichia coli BL21 (DE3). xynA4-2 encodes a mature protein of 411 residues with a calculated molecular weight of 46.8kDa. Based on the amino acid sequence
similarities (highest identity of 61%), the enzyme was confined into glycoside hydrolase family 10. The purified recombinant
XynA4-2 exhibited maximum activity at pH 6.2 and 55°C. The enzyme was stable over a broad pH range, retaining more than 90%
of the original activity at pH 5.8–12.0, 37°C for 1h. The substrate specificity of XynA4-2 was relatively narrow, exhibiting
100, 93, and 35% of the relative activity towards birchwood xylan, oat spelt xylan, and wheat arabinoxylan, respectively.
Supplementation of XynA4-2 to mash caused the reduction of mash filtration rate (5.6%) and viscosity (4.0%). When combined
with the commercial glucanase from Sunson, higher reduction was detected in the filtration rate (12.0%) and viscosity (17.2%).
These favorable properties make XynA4-2 a good candidate in the brewing industry.
Keywords
Alicyclobacillus sp. A4–Xylanase–pH stability–Mash–Viscosity–Filtration rate
World Journal of Microbiology and Biotechnology 04/2012; 27(2):207-213. · 1.53 Impact Factor
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ABSTRACT: A gene, aga-MJ11, encoding an α-galactosidase (EC 3.2.1.22) was cloned from Pedobacter nyackensis MJ11 CGMCC 2503, expressed in Escherichia coli, and biochemically characterized. The gene consisted of 2,163 nucleotides encoding a 720 amino acid–protein with a theoretical
molecular weight of 82.6kDa. The deduced amino acid sequence of Aga-MJ11 shared the highest identity of 51% to an α-galactosidase
from Parabacteroides distasonis (YP_001301506), which belongs to glycoside hydrolase (GH) family 36. Purified recombinant Aga-MJ11-H showed optimal activity
at pH 5.5 and 40°C, was stable at pH 4.0–10.0, retained ~80% of the maximum activity at 30°C (the optimum temperature for
freshwater fish), exhibited tolerance to some proteases, and had a wide substrate specificity (pNPG, melidiose, stachyose
and raffinose). All these features make Aga-MJ11 potentially useful for applications in aquaculture. The enzyme studied in
the present work may represent a novel GH-36 α-galactosidase from the genus Pedobacter.
World Journal of Microbiology and Biotechnology 04/2012; 25(9):1633-1642. · 1.53 Impact Factor
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ABSTRACT: A xylanase gene, xynE2, was cloned from thermoalkaline Anoxybacillus sp. E2 and was expressed in Escherichia coli BL21 (DE3). The gene consisted of 987bp and encoded a 328-residue xylanase with a calculated molecular weight of 38.8kDa.
On the basis of amino acid sequence similarities, this enzyme was assigned as a member of glycoside hydrolase family 10. Purified
recombinant XynE2 showed maximal activity at pH 7.8 and 65°C, and was thermostable at 60°C. The enzyme was highly active and
stable over a broad pH range, showing more than 90% of maximal activity at pH 6.6–pH 8.6 and retaining more than 80% of activity
at pH 4.6–pH 12.0, 37°C for 1h, respectively. These favorable properties make XynE2 a good candidate in the pulp and paper
industries. This is the first report on gene cloning, expression and characterization of a xylanase from the genus Anoxybacillus.
Keywords
Anoxybacillus sp. E2-Xylanase-pH stability-Thermostability
World Journal of Microbiology and Biotechnology 04/2012; 26(5):917-924. · 1.53 Impact Factor
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ABSTRACT: A novel method is proposed to produce both phytase and single-cell protein in recombinant Pichia pastoris fermentation using monosodium glutamate wastewater (MSGW) as the basal medium. Recombinant P. pastoris MR33 transformed with a phytase gene (AppA-m) from Escherichia coli was constructed and showed capability to utilize ammonium as the only nitrogen source. The fermentation medium was optimized
in shake flasks by single-factor test and response surface methodology. A fed-batch system containing 30% MSGW, 50g/l glucose,
1.58g/l CaSO4, 5.18g/l MgSO4 and 6.67g/l KH2PO4 was developed in a 3.7-l bioreactor. The maximum phytase activity in the MSGW medium reached 3,380U/ml, 84.2% of that in
chemically defined medium, and the dry cell weight was 136g/l. The single-cell protein (SCP; 46.66% dry cell weight) contains
a variety of amino acids and is low in fat, which is ideal for utilization in animal feed. Thus, it is feasible to use MSGW
medium for the production of enzymes that can be expressed in P. pastoris.
World Journal of Microbiology and Biotechnology 04/2012; 25(9):1643-1649. · 1.53 Impact Factor
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ABSTRACT: An endo-β-1,4-mannanase encoding gene, man5, was cloned from Bispora antennata CBS 126.38, which was isolated from a beech stump. The cDNA of man5 consists of 1,299 base pairs and encodes a 432-amino-acid protein with a theoretical molecular mass of 46.6 kDa. Deduced MAN5 exhibited the highest amino acid sequence identity of 58% to a β-mannanase of glycoside hydrolase family 5 from Aspergillus aculeatus. Recombinant MAN5 was expressed in Pichia pastoris and purified to electrophoretic homogeneity. The specific activity of the final preparation towards locust bean gum was 289 U mg(-1). MAN5 showed optimal activity at pH 6.0 and 70 °C and had good adaptation and stability over a broad range of pH values. The enzyme showed more than 60% of peak activity at pH 3.0-8.0 and retained more than 80% of activity after incubation at 37 °C for 1 h in both acid and alkaline conditions (pH 4.0-11.0). The K (m) and V (max) values were 1.33 mg ml(-1) and 444 μmol min(-1) mg(-1) and 1.17 mg ml(-1) and 196 μmol min(-1) mg(-1) for locust bean gum and konjac flour, respectively. Of all tested metal ions and chemical reagents, Co(2+), Ni(2+), and β-mercaptoethanol enhanced the enzyme activity at 1 mM, whereas other chemicals had no effect on or partially inhibited the enzyme activity. MAN5 was highly resistant to acidic and neutral proteases (trypsin, α-chymotrypsin, collagenase, subtilisin A, and proteinase K). By virtue of the favorable properties of MAN5, it is possible to apply this enzyme in the paper and food industries.
Applied biochemistry and biotechnology 03/2012; 166(6):1442-53. · 1.94 Impact Factor
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ABSTRACT: A glycosyl hydrolase family 5 endo-β-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 gl(-1) in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60 °C and exhibited good stability over a broad pH range from acidic to alkaline (>85.0% activity at pH 4.0-9.0, >70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co(2+), Cu(2+), Mn(2+) and β-mercaptoethanol and was strongly inhibited by Hg(2+) and SDS. The specific activity, K(m) and V(max) values were 47.5 U mg(-1), 7.8 mg ml(-1) and 70.4 μmol min(-1)mg(-1), respectively, for locust bean gum, and 40.3 U mg(-1), 2.3 mg ml(-1) and 61.7 μmol min(-1)mg(-1), respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.
Journal of Bioscience and Bioengineering 02/2012; 113(6):710-4. · 1.79 Impact Factor
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ABSTRACT: A novel endo-1,3(4)-β-D-glucanase gene (bgl16C1) from Penicillium pinophilum C1 was cloned and sequenced. The 945-bp full-length gene encoded a 315-residue polypeptide consisting of a putative signal peptide of 18 residues and a catalytic domain belonging to glycosyl hydrolase family 16. The deduced amino acid sequence showed the highest identity (82%) with the putative endo-1,3(4)-β-glucanase from Talaromyces stipitatus ATCC 10500 and 60% identity with the characterized β-1,3(4)-glucanase from Paecilomyces sp. FLH30. The gene was successfully overexpressed in Pichia pastoris. Recombinant Bgl16C1 constituted 95% of total secreted proteins (2.61 g l⁻¹) with activity of 28,721 U ml⁻¹ in a 15-l fermentor. The purified recombinant Bgl16C1 had higher specific activity toward barley β-glucan (12,622 U mg⁻¹) than all known glucanases and also showed activity against lichenan and laminarin. The enzyme was optimally active at pH 5.0 and 55°C and exhibited good stability over a broad acid and alkaline pH range (>85% activity at pH 3.0-7.0 and even 30% at pH 11.0). All these favorable enzymatic properties make it attractive for potential applications in various industries.
Journal of Industrial Microbiology 02/2012; 39(6):869-76. · 1.80 Impact Factor
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ABSTRACT: A pectate lyase gene (pl-str) was cloned from Streptomyces sp. S27 and expressed in Escherichia coli Rosetta. The full-length pl-str consists of 972 bp and encodes for a protein of 323 amino acids without signal peptide that belongs to family PF00544. The recombinant enzyme (r-PL-STR) was purified to electrophoretic homogeneity using Ni²⁺-NTA chromatography and showed apparent molecular mass of ~35 kDa. The pH optimum of r-PL-STR was found to be 10.0, and it exhibited >70% of the maximal activity at pH 12.0. After incubation at 37°C for 1 h without substrate, the enzyme retained more than 55% activity at pH 7.0-12.0. Compared with the commercial complex enzyme Scourzyme(@)301L from Novozymes, purified r-PL-STR showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (49.0 vs. 49.7%). When combined with cellulase and α-amylase, r-PL-STR had comparable performance in bioscouring of jute fabric (22.39 vs. 22.99%). Thus, r-PL-STR might represent a good candidate for use in alkaline industries such as textile.
Journal of Industrial Microbiology 01/2012; 39(6):909-15. · 1.80 Impact Factor
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ABSTRACT: Efficient degradation of pectin in the rumen is necessary for plant-based feed utilization. The objective of this study was to characterize the diversity, abundance, and functions of pectinases from microorganisms in the sheep rumen.
A total of 103 unique fragments of polygalacturonase (PF00295) and pectate lyase (PF00544 and PF09492) genes were retrieved from microbial DNA in the rumen of a Small Tail Han sheep, and 66% of the sequences of these fragments had low identities (<65%) with known sequences. Phylogenetic tree building separated the PF00295, PF00544, and PF09492 sequences into five, three, and three clades, respectively. Cellulolytic and noncellulolytic Butyrivibrio, Prevotella, and Fibrobacter species were the major sources of the pectinases. The two most abundant pectate lyase genes were cloned, and their protein products, expressed in Escherichia coli, were characterized. Both enzymes probably act extracellularly as their nucleotide sequences contained signal sequences, and they had optimal activities at the ruminal physiological temperature and complementary pH-dependent activity profiles.
This study reveals the specificity, diversity, and abundance of pectinases in the rumen ecosystem and provides two additional ruminal pectinases for potential industrial use under physiological conditions.
PLoS ONE 01/2012; 7(7):e40940. · 4.09 Impact Factor
