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1.
Food Chem ; 305: 125447, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499289

RESUMO

A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2 U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9 kDa via SDS-PAGE. RmAmyA was optimally active at 75 °C and pH 6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16 h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.

2.
Food Chem ; : 125709, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31708343

RESUMO

To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 ß-mannanase gene (mRmMan134A) from Rhizopus microsporus var. rhizopodiformis F518 was expressed in Pichia pastoris and a high expression level (3680 U mL-1) was obtained through high cell density fermentation. mRmMan134A exhibited maximum activity at pH 5.5 and 50 °C. It was then subjected to hydrolyze cassia gum with 70.6% of overall yield of manno-oligosaccharides. From the hydrolysate, seven components (F1-F7) were separated and identified as mannose, mannobiose, galactose, mannotriose, mannotetraose, 61-α-d-galactosyl-ß-d-mannobiose, and mannopentaose, respectively. According to in vitro fermentation, the manno-oligosaccharides were able to promote the growth of three Bifidobacterium strains and six Lactobaillus strains with 3.0-fold increment in culture absorbance, and these strains preferred manno-oligosaccharides with degree of polymerization (DP) 2-3 rather than those with DP 4-5. Novel manno-oligosaccharides from cassia gum with promising prebiotic activity were provided in the present study.

3.
Int J Biol Macromol ; 127: 683-692, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30703426

RESUMO

A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.


Assuntos
Proteínas Fúngicas , Maltose/química , Pichia , Amido/química , alfa-Amilases , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trissacarídeos/química , alfa-Amilases/biossíntese , alfa-Amilases/química , alfa-Amilases/genética
4.
Biochim Biophys Acta Gen Subj ; 1862(6): 1376-1388, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29550433

RESUMO

Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. ß-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-ß-mannanase from Rhizopus microsporus (RmMan134A) was cloned and expressed. The recombinant RmMan134A showed maximal activity at pH 5.0 and 50 °C, and exhibited high specific activity towards locust bean gum (2337 U/mg). To gain insight into the substrate-binding mechanism of RmMan134A, four complex structures (RmMan134A-M3, RmMan134A-M4, RmMan134A-M5 and RmMan134A-M6) were further solved. These structures showed that there were at least seven subsites (-3 to +4) in the catalytic groove of RmMan134A. Mannose in the -1 subsite hydrogen bonded with His113 and Tyr131, revealing a unique conformation. Lys48 and Val159 formed steric hindrance, which impedes to bond with galactose branches. In addition, the various binding modes of RmMan134A-M5 indicated that subsites -2 to +2 are indispensable during the hydrolytic process. The structure of RmMan134A-M4 showed that mannotetrose only binds at subsites +1 to +4, and RmMan134A could therefore not hydrolyze mannan oligosaccharides with degree of polymerization ≤4. Through rational design, the specific activity and optimal conditions of RmMan134A were significantly improved. The purpose of this paper is to investigate the structure and function of fungal GH family 134 ß-1,4-mannanases, and substrate-binding mechanism of GH family 134 members.


Assuntos
Glicosídeos/metabolismo , Mananas/metabolismo , Rhizopus/enzimologia , beta-Manosidase/química , beta-Manosidase/metabolismo , Sequência de Aminoácidos , Catálise , Clonagem Molecular , Cristalografia por Raios X , Conformação Proteica , Homologia de Sequência , Especificidade por Substrato
5.
Bioresour Technol ; 256: 30-37, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29428611

RESUMO

An engineered ß-mannanase (mRmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris. Through high cell density fermentation, the expression level of mRmMan5A reached 79,680 U mL-1. The mRmMan5A showed maximum activity at pH 4.5 and 65 °C, and exhibited high specific activities towards mannans. To produce manno-oligosaccharides, palm kernel cake (PKC) was pretreated by steam explosion at 200 °C for 7.5 min, and then hydrolyzed by mRmMan5A. As a result, the total manno-oligosaccharide yield reached 34.8 g/100 g dry PKC, indicating that 80.6% of total mannan in PKC was hydrolyzed. Moreover, the kilo-scale production of manno-oligosaccharides was carried out to verify the feasibility of mass production. A total of 261.3 g manno-oligosaccharides were produced from 1.0 kg of dry PKC. An effective ß-mannanase for the bioconversion of mannan-rich biomasses and an efficient method for the production of manno-oligosaccharides from PKC are provided in this paper.


Assuntos
Oligossacarídeos , Pichia , beta-Manosidase , Explosões , Mananas , Vapor
6.
Int J Biol Macromol ; 105(Pt 1): 1171-1179, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28756198

RESUMO

Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel ß-mannanase gene (RmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200UmL-1 after 168h high cell density fermentation. The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65°C. RmMan5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5×104Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization<7. PHGG was further fractionated (F1-F4) by gradual ethanol precipitation. PHGG F1 with an Mw value of 3.6×104Da and a mannose/galactose (M/G) ratio of 1.47 was precipitated initially, followed by PHGG F2 and F3 which showed lower Mw and higher M/G ratio. According to the structure analysis, the distribution of α-d-galactose of PHGG F1 was compact and regular, and that of other fractions was more random. A suitable ß-mannanase for PHGG production and some useful information of PHGG are provided in this paper.


Assuntos
Galactanos/biossíntese , Mananas/biossíntese , Pichia/genética , Gomas Vegetais/biossíntese , Rhizomucor/enzimologia , beta-Manosidase/genética , beta-Manosidase/metabolismo , Fermentação , Galactanos/química , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Mananas/química , Peso Molecular , Gomas Vegetais/química , Rhizomucor/genética , Temperatura Ambiente
7.
Biotechnol Biofuels ; 10: 143, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28588644

RESUMO

BACKGROUND: ß-Mannanase randomly cleaves the ß-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of ß-mannanase have tremendously expanded in recent years, the wild-type ß-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 ß-mannanase (RmMan5A) from Rhizomucor miehei shows many outstanding properties, such as high specific activity and hydrolysis property. However, owing to the low catalytic activity in acidic and thermophilic conditions, the application of RmMan5A to the biorefinery of mannan biomasses is severely limited. RESULTS: To overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated ß-mannanase (mRmMan5A) with high catalytic activity in acidic and thermophilic conditions was obtained, and then characterized. The mutant displayed maximal activity at pH 4.5 and 65 °C, corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 °C in optimal temperature. The catalytic efficiencies (kcat/Km) of mRmMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of RmMan5A were inherited by the mutant mRmMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mRmMan5A, namely Tyr233His, Lys264Met, and Asn343Ser. To identify the function of each substitution, four site-directed mutations (Tyr233His, Lys264Met, Asn343Ser, and Tyr233His/Lys264Met) were subsequently generated, and the substitutions at Tyr233 and Lys264 were found to be the main reason for the changes of mRmMan5A. CONCLUSIONS: Through directed evolution of RmMan5A, two key amino acid residues that controlled its catalytic efficiency under acidic and thermophilic conditions were identified. Information about the structure-function relationship of GH family 5 ß-mannanase was acquired, which could be used for modifying ß-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a ß-mannanase from zygomycete engineered by directed evolution.

8.
Food Chem ; 213: 708-713, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27451238

RESUMO

In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.


Assuntos
Frutas/enzimologia , Peptídeo Hidrolases/metabolismo , Verduras/enzimologia , Actinidia/enzimologia , Brassica/enzimologia , Capsicum/enzimologia , Caseínas/metabolismo , Eletroforese em Gel de Poliacrilamida , Gengibre/enzimologia , Concentração de Íons de Hidrogênio , Cebolas/enzimologia , Extratos Vegetais/química , Inibidores de Proteases/metabolismo
9.
Food Chem ; 179: 290-5, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25722167

RESUMO

Multiple proteases were optimized to hydrolyze the rice residue protein (RRP) to produce novel antioxidant peptides. An antioxidant peptide fraction (RRPB3) with IC50 of 0.25 mg/ml was purified from the RRP hydrolysate using membrane ultrafiltration followed by size exclusion chromatography and reversed-phase FPLC. RRPB3 was found to include four peptides (RRPB3 I-IV) and their amino acid sequences were RPNYTDA (835.9 Da), TSQLLSDQ (891.0 Da), TRTGDPFF (940.0 Da) and NFHPQ (641.7 Da), respectively. Furthermore, four peptides were chemically synthesized and their antioxidant activities were assessed by DPPH radical scavenging, ABTS radical scavenging assay and FRAP-Fe(3+) reducing assay, respectively. Both RRPB3 I and III showed synergistic antioxidant activity compared to each of them used alone. All four synthetic peptides showed excellent stability against simulated gastrointestinal proteases. Therefore, the peptides isolated from RRP may be used as potential antioxidants in the food and drug industries.


Assuntos
Antioxidantes/química , Oryza/metabolismo , Peptídeo Hidrolases/química , Peptídeos/química , Hidrólise , Oxirredução
10.
Appl Biochem Biotechnol ; 174(1): 174-85, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25035104

RESUMO

Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis were investigated in the present study. A newly isolated R. microsporus var. rhizopodiformis F518 produced a high level of milk-clotting activity (1,001 SU/mL). A chymosin from the fungus was purified 3.66-fold with a recovery yield of 33.2 %. The enzyme appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 37.0 kDa. It was optimally active at 60 °C and was stable up to 40 °C. The purified enzyme was an acid protease with an optimum pH of 5.2 and retained 80 % of residual activity within pH 2.0-8.0. The inhibition of 96 and 100 % by pepstatin A at 0.01 and 0.02 mM, respectively, revealed that the enzyme is an aspartic protease. Thus, high milk-clotting activity of the chymosin with good stability will strengthen the potential use of the chymosin as a substitute for calf rennet in cheese manufacturing.


Assuntos
Quimosina/química , Quimosina/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Rhizopus/enzimologia , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio
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