Research progresses in microbial 1,3-1,4-β-glucanase: protein engineering and industrial applications / 生物工程学报

1,3-1,4-β-glucanase (E.C.3.2.1.73) is an important industrial enzyme which cleave β-glucans into oligosaccharides through strictly cutting the β-1,4 glycosidic bonds in 3-O-substituted glucopyranose units. Microbial 1,3-1,4-β-glucanase belongs to retaining glycosyl hydrolases of family 16 with a jellyroll β-sandwich fold structure. The present paper reviews the industrial application and protein engineering of microbial β-glucanases in the last decades and forecasts the research prospects of microbial β-glucanases.

Molecular cloning and biochemical characterization of an α-amylase family from Aspergillus niger

Background: α-Amylase is widely used in the starch processing, food and paper industries, hydrolyzing starch, glycogen and other polysaccharides into glucose, maltose and oligosaccharides. An α-amylase gene family from Aspergillus niger CBS513.88 encode eight putative α-amylases. The differences and similarities, biochemical properties and functional diversity among these eight α-amylases remain unknown. Results: The eight genes were cloned and expressed in Pichia pastoris GS115 by shaking-flask fermentation under the induction of methanol. The sequence alignment, biochemical characterizations and product analysis of starch hydrolysis by these α-amylases were investigated. It is found that the eight α-amylases belonged to three different groups with the typical structure of fungal α-amylase. They exhibited maximal activities at 30­40°C except AmyG and were all stable at acidic pH. Ca2+ and EDTA had no effects on the activities of α-amylases except AmyF and AmyH, indicating that the six amylases were Ca2+ independent. Two novel α-amylases of AmyE and AmyF were found. AmyE hydrolyzed starch into maltose, maltotriose and a small amount of glucose, while AmyF hydrolyzed starch into mainly glucose. The excellent physical and chemical properties including high acidic stability, Ca2+-independent and high maltotriose-forming capacity make AmyE suitable in food and sugar syrup industries. Conclusions: This study illustrates that a gene family can encode multiple enzymes members having remarkable differences in biochemical properties. It provides not only new insights into evolution and functional divergence among different members of an α-amylase family, but the development of new enzymes for industrial application.

Co-production of hydrogen and ethanol by Escherichia coli SS1 and its recombinant

Background: The development of a potential single culture that can co-produce hydrogen and ethanol is beneficial for industrial application. Strain improvement via molecular approach was proposed on hydrogen and ethanol co-producing bacterium, Escherichia coli SS1. Thus, the effect of additional copy of native hydrogenase gene hybC on hydrogen and ethanol co-production by E. coli SS1 was investigated. Results: Both E. coli SS1 and the recombinant hybC were subjected to fermentation using 10 g/L of glycerol at initial pH 7.5. Recombinant hybC had about 2-fold higher cell growth, 5.2-fold higher glycerol consumption rate and 3-fold higher ethanol productivity in comparison to wild-type SS1. Nevertheless, wild-type SS1 reported hydrogen yield of 0.57 mol/mol glycerol and ethanol yield of 0.88 mol/mol glycerol, which were 4- and 1.4-fold higher in comparison to recombinant hybC. Glucose fermentation was also conducted for comparison study. The performance of wild-type SS1 and recombinant hybC showed relatively similar results during glucose fermentation. Additional copy of hybC gene could manipulate the glycerol metabolic pathway of E. coli SS1 under slightly alkaline condition. Conclusions: HybC could improve glycerol consumption rate and ethanol productivity of E. coli despite lower hydrogen and ethanol yields. Higher glycerol consumption rate of recombinant hybC could be an advantage for bioconversion of glycerol into biofuels. This study could serve as a useful guidance for dissecting the role of hydrogenase in glycerol metabolism and future development of effective strain for biofuels production.

A biotechnology perspective of fungal proteases

Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.