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Cloning, expression and characterization of metalloproteinase HypZn from Aspergillus niger.

ABSTRACT: A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics of recombinant HypZn were investigated after separation and purification. The results showed that the specific activity of the purified HypZn reached 1859.2 U/mg, and the optimum temperature and pH value of HypZn were 35°C and 7.0, respectively. HypZn remained stable both at 40°C and at pH values between 5.0 and 8.0. The preferred substrate of HypZn was soybean protein isolates, and the Km and Vmax values were 21.5 μmol/mL and 4926.6 μmol/(mL∙min), respectively. HypZn was activated by Co2+ and Zn2+ and inhibited by Cu2+ and Fe2+. The degree of soybean protein isolate hydrolysis reached 14.7%, and the hydrolysates were of uniform molecular weight. HypZn could tolerate 5000 mM NaCl and completely lost its activity after 30 min at 50°C. The enzymological characterizations indicated that HypZn has great application potential in the food industry, especially in fermented food processing.

SUBMITTER: Song P

PROVIDER: S-EPMC8584677 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Cloning, expression and characterization of metalloproteinase HypZn from Aspergillus niger.

Song Peng P Xu Wei W Wang Kuiming K Zhang Yang Y Wang Fei F Zhou Xiuling X Shi Haiying H Feng Wei W

PloS one 20211111 11

A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics of recombinant HypZn were investigated after separation and purification. The results showed that the specific activity of the purified HypZn reached 1859.2 U/mg, and the optimum temperature and pH value of HypZn were 35°C and 7.0, respectively. HypZn remained stable both at 40°C and at pH values between 5.0 and 8.0. The preferr ...[more]

PMID: 34762700

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Project description:Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger beta -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed.This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-beta-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant beta-mannanase will be valuable in various biotechnological applications.

Project description:BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn???) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp???-Val??? peptide was shortened to Asp???-Arg??? in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (?86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

Dataset Information

Cloning, expression and characterization of metalloproteinase HypZn from Aspergillus niger.

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Cloning, expression and characterization of metalloproteinase HypZn from Aspergillus niger.

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