Production, purification and characterization of a novel antithrombotic and anticoagulant serine protease from food grade microorganism Neurospora crassa.

A novel thrombolytic enzyme was produced by food grade microorganism Neurospora crassa using agro-industrial by-products as substrates. Process parameters were optimized using Plackett-Berman and Box-Benhken design. Under the optimized fermentation conditions, high fibrinolytic activity of 403.59 U/mL was obtained. It was purified with a specific activity of 3572.4 U/mg by ammonium sulfate precipitation and SP Sepharose chromatography. The molecular weight of the enzyme was approximately 32 kDa. It exhibited maximum activity at 40 °C and pH 7.4. Its activity was enhanced by Cu2+, Na+, Zn2+, and completely inhibited by phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, which indicates it could be a serine protease. The enzyme could degrade fibrin clot directly without the need of plasminogen activator, and effectively cleaved Aα, Bß, γ chains of fibrinogen. It could inhibit the formation of blood clots in vitro and acts as an anticoagulant. Compared to heparin the purified enzyme showed extended anticoagulant activity. Blood clots were dissolved effectively and dissolution rate was increased with time. Based on these results, this novel enzyme has the potential to be developed as a thrombolytic agent.

Gene cloning, expression and homology modeling of first fibrinolytic enzyme from mushroom (Cordyceps militaris).

Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding "pre-pro-protein" of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The "oxyanion hole" is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents.

Enhanced elimination of non-digestible oligosaccharides from soy milk by immobilized α-galactosidase: A comparative analysis.

This study compared two immobilization matrices like calcium-alginate and chitosan for immobilization of α-galactosidase and evaluated their potential for the removal of non-digestible raffinose family oligosaccharides from soy milk which cause abdominal discomfort. The pH optima of the free and immobilized enzymes were found to be similar at pH 4.0. The chitosan-immobilized α-galactosidase displayed higher optimal temperature (60°C) compared to alginate-immobilized enzyme (45°C) and free enzyme (50°C). The chitosan-immobilized and alginate-immobilized α-galactosidases displayed 93.7% and 97.6% hydrolysis of raffinose family oligosaccharides, respectively, while the free enzyme hydrolyzed only 30.3% oligosaccharides present in soy milk in 4 hr. Remarkably, both the immobilized enzymes showed complete removal of raffinose family oligosaccharides in 8 hr. Moreover, reusability studies indicate that even after five cycles of reuse, the chitosan and alginate-immobilized enzymes displayed 99% and 60% hydrolysis, respectively. PRACTICAL APPLICATIONS: In this study, we have used two inexpensive and non-toxic matrices for immobilizing α-galactosidase. We report that entrapment of α-galactosidase with chitosan significantly improved the optimal temperature of α-galactosidase, which is advantageous in food industry. The hydrolysis of raffinose family oligosaccharides in soy milk was also greatly enhanced after immobilization with chitosan and alginate. Thus, the results described in this study have relevance for development of safe, cost-effective and efficient method for removal of non-digestible soy oligosaccharides in food industry.

Enhanced Properties and Lactose Hydrolysis Efficiencies of Food-Grade ß-Galactosidases Immobilized on Various Supports: a Comparative Approach.

In this study, a fungal and two yeast ß-galactosidases were immobilized using alginate and chitosan. The biochemical parameters and lactose hydrolysis abilities of immobilized enzymes were analyzed. The pH optima of immobilized fungal ß-galactosidases shifted to more acidic pH compared to free enzyme. Remarkably, the optimal temperature of chitosan-entrapped yeast enzyme, Maxilact, increased to 60 °C, which is significantly higher than that of the free Maxilact (40 °C) and other immobilized forms. Chitosan-immobilized A. oryzae ß-galactosidase showed improved lactose hydrolysis (95.7%) from milk, compared to the free enzyme (82.7%) in 12 h. Chitosan-immobilized Maxilact was the most efficient in lactose removal from milk (100% lactose hydrolysis in 2 h). The immobilized lactases displayed excellent reusability, and chitosan-immobilized Maxilact hydrolyzed > 95% lactose in milk after five reuses. Compared to free enzymes, the immobilized enzymes are more suitable for cost-effective industrial production of low-lactose milk due to improved thermal activity, lactose hydrolysis efficiencies, and reusability.

A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila.

In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10-5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by ß chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.

Biotechnological potential of microbial α-galactosidases.

The enzyme α-galactosidase (α-D-galactoside galactohydrolase; EC 3.2.1.22) catalyzes the hydrolysis of α-1,6-linked galactose residues in oligosaccharides and polymeric galactomannan. The α-galactosidases are of particular interest in view of their many potential biotechnological and medical applications. These enzymes have found wide use in various industries such as food and feed, sugar and paper and pulp for the removal of raffinose and stachyose. They are also important medically for blood group conversion and in the treatment of Fabry disease. Most of the research on α-galactosidases has focused on their isolation from various microbial sources. In the last decade, cloning of novel α-galactosidase genes and their heterologous expression has gained momentum. The present review focuses on the production of α-galactosidases from bacteria, fungi and yeast, and discusses their properties. Recent progress on cloning and heterologous expression in various hosts is summarized with special emphasis on their application in various fields.

Gene cloning and enzymatic characterization of an alkali-tolerant endo-1,4-ß-mannanase from Rhizomucor miehei.

An endo-1,4-ß-mannanase gene (RmMan5A) was cloned from the thermophilic fungus Rhizomucor miehei for the first time and expressed in Escherichia coli . The gene had an open reading frame of 1330 bp encoding 378 amino acids and contained four introns. It displayed the highest amino acid sequence identity (42%) with the endo-1,4-ß-mannanases from glycoside hydrolase family 5. The purified enzyme was a monomer of 43 kDa. RmMan5A displayed maximum activity at 55 °C and an optimal pH of 7.0. It was thermostable up to 55 °C and alkali-tolerant, displaying excellent stability over a broad pH range of 4.0-10.0, when incubated for 30 min without substrate. The enzyme displayed the highest specificity for locust bean gum (K(m) = 3.78 mg mL⁻¹), followed by guar gum (K(m) = 7.75 mg mL⁻¹) and konjac powder (K(m) = 22.7 mg mL⁻¹). RmMan5A hydrolyzed locust bean gum and konjac powder yielding mannobiose, mannotriose, and a mixture of various mannose-linked oligosaccharides. It was confirmed to be a true endo-acting ß-1,4-mannanase, which showed requirement of four mannose residues for hydrolysis, and was also capable of catalyzing transglycosylation reactions. These properties make RmMan5A highly useful in the food/feed, paper and pulp, and detergent industries.

High-level expression of a xylanase gene from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris.

A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5 l fermentor culture, the total extracellular protein was 8.1 g l(-1) with an activity of 52,940 U ml(-1). The enzyme was purified to homogeneity with a recovery of 48 %. The recombinant xynA was optimally active at 75 °C, as measured over 10 min, and at pH 7. The enzyme was stable up to 80 °C for 30 min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.

Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides.

The α-galactosidase gene, RmGal36, from Rhizomucor miehei was cloned and expressed in Escherichia coli. The gene has an open reading frame of 2256bp encoding 751 amino acid residues. RmGal36 was optimally active at pH 4.5 and 60°C, but is stable between pH 4.5 and 10.0 and at a temperature of up to 55°C for 30min retaining more than 80% of its relative activity. It displayed remarkable resistance to proteases and its activity was not inhibited by galactose concentrations of 100mM. The relative specificity of RmGal36 towards various substrates is in the order of p-nitrophenyl α-galactopyranoside>melibiose>stachyose>raffinose, with a K(m) of 0.36, 16.9, 27.6, and 47.9mM, respectively. The enzyme completely hydrolyzed raffinose and stachyose present in soybeans and kidney beans at 50°C within 60min. These features make RmGal36 useful in the food and feed industries and in processing of beet-sugar.

High level expression of a novel ß-mannanase from Chaetomium sp. exhibiting efficient mannan hydrolysis.

A novel ß-mannanase gene (CsMan5A) was cloned from Chaetomium sp. CQ31 and expressed in Pichia pastoris. It had an open reading frame of 1251bp encoding 416 amino acids and contained two introns. The deduced amino acid sequence shared the highest similarity (73%) with the ß-mannanase from Emericella nidulans and belongs to glycosyl hydrolase family 5. The recombinant ß-mannanase (CsMan5A) was secreted at extremely high levels of 50,030UmL-1 and 6.1mgmL-1 in high cell density fermentor. The purified enzyme was optimally active at pH 5.0 and 65°C and displayed broad pH stability (pH 5.0-11.0) and exhibited specificity towards locust bean gum (Km=3.1mgmL-1), guar gum (Km=9.3mgmL-1) and konjac powder (Km=10.5mgmL-1). It efficiently degraded mannan polysaccharides into mannose and mannooligosacccharides, and also hydrolyzed mannotriose and mannotetraose. These properties make CsMan5A highly useful in food, feed and paper/pulp industries.

Purification and properties of a psychrotrophic Trichoderma sp. xylanase and its gene sequence.

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5-9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K(m) values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.

High-level expression of a specific beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris.

In this study, a novel beta-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-beta-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular beta-1,3-1,4-glucanase. The recombinant beta-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l(-1) with an activity of 55,300 U ml(-1) in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS-PAGE. It was optimally active at pH 7.0 and a temperature of 70 degrees C. Furthermore, the enzyme exhibited strict specificity for beta-1,3-1,4-D: -glucans. This is the first report on the cloning and expression of a beta-1,3-1,4-glucanase gene from Paecilomyces sp.