| Penicillium can secrete a variety of important industrial enzymes, and produces kinds of excellent hemicellulases, including xylanase,β-mannanase andβ-glucanase etc. In this study, three Penicillium strains (C1, C6 and C7) were isolated from the acidic wastewater (pH 3.0) of a tin mine in Yunnan province, China, and used as donors for gene cloning and heterologous expression of hemicellulases.By combining TAIL-PCR and RT-PCR techniques with designing of degenerate primers, five full-length glycoside hydrolase genes encoding for three kinds of enzymes were cloned from Penicillium strains C1, C6 and C7. They are two xylanase genes (xyn10C1 and xyn11C1), twoβ-mannanse genes (man5C1 and man5C6) and oneβ-glucanase gene (bgl7C7), respectively. Homology searches in Genbank and sequence alignments indicated that their deduced amino acid sequences shared 49–83% identities with known proteins. Further homology modeling and catalytic residue analysis revealed that these six genes have certain sequence and structure novelty.xyn10C1 from Penicillium sp. C1 was expressed in Pichia pastoris, and the recombinant enzyme was purified and characterized. In shaker flask XYN10C1 was produced with the activity of 477 U/mL. XYN10C1 showed the optimal activity at pH 4.0–5.5 and 75°C, and exhibited good adaptability to broad ranges of acidic pHs and temperatures. It retained >69.0% of the maximal activity at pH 2.5–6.5 and >91.0% of the activity at 70–80°C, and 22.2% even at 90°C. The enzyme was stable at 65°C. The specific activity, Km and Vmax values towards soluble wheat arabinoxylan were 137 U/mg, 6.9 mg/mL and 209μmol/min/mg, respectively. Using birchwood xylan as the substrate, the specific activity, Km and Vmax values were 101 U/mg, 4.3 mg/mL and 195μmol/min/mg, respectively. XYN10C1 was strongly resistant to most metal ions and proteases (pepsin and trypsin). Under simulated mashing conditions, addition of XYN10C1 (80 U) to the brewery mash significantly increased the filtration rate by 27% and reduced the viscosity by 9.8%, respectively. All these favorable properties make XYN10C1 attractive for potential applications in food and animal feed industries.Twoβ-mannanases Man5C1 and Man5C6 were produced in P. pastoris with the activity of 162 U/mL and 575 U/mL, respectively. The yield of Man5C6 was up to 2.5 g/L after 5-day fermentation in a 3.7 L fermentor, which was higher than that of most knownβ-mannanases. Man5C6 was easily purified, and its yield might be further improved by molecular and protein engineering methods, thus Man5C6 was potential in low-cost industrial applications.The optimum pHs of Man5C1 and Man5C6 were 4.0 and 4.5, respectively. Their temperature optima were 70°C. MAN5C1 had strong resistance to pepsin and trypsin, retaining 93–116% activities at 37°C for 60 min. Man5C6 only retained 86.6% activity after trypsin treatment under the same conditions. Most metal ions and chemical reagents enhanced or had no effects on their enzyme activities. Using locust bean gum as the substrate, the specific activities, Km and Vmax values for Man5C1 and Man5C6 were 1035 U/mg, 5.6 mg/mL, 2785μmol/min/mg; 226.5 U/mg, 12.3 mg/mL and 2400μmol/min/mg, respectively. Under simulated gastric conditions, Man5C1 released more reducing sugars from locust bean gum in the presence of pepsin. Based on the enzymatic properties, Man5C1 have good prospects in industrial applications. Man5C6 was produced with high yield, and showed good thermal stability, pH adaptability and pH stability under acidic conditions. These properties make it more valuable in the animal feed industry.In conclusion, five hemicellulase genes were cloned from three Penicillium strains, and three of them were expressed and characterized. The three recombinant enzymes were all acidic, tolerant to high temperatures, and had excellent resistance to metal ions, chemical reagents, and proteases. Moreover, these enzymes had significant effects in application tests. All these properties indicate that they have good prospects for application in food and feed industries.
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