Caldicellulosiruptor Bescii Thermophilic Alkaline Pectinase System Activity Research

Pectin material consists of a series of complex polysaccharide, is the nature of the most widely used polysaccharide widely found in higher plants(such as apples,onions, grapefruit, etc.) of the primary cell wall and intercellular space, pectin /pectinase degradation usually located in the root of most microorganisms and higher plants related to a variety of plants, stems, leaves, fruits.Pectinase is an enzyme complex containing a variety of ingredients, are a class of enzymes break down pectin substances in general. Pectinase optimum p H D- Multi-galacturonic acid, depending on their role in the substrate can be divided into acidic pectinase and alkaline pectinase. Currently on the industrial application of more acidic pectinase, acid pectinase application merely on the initial level of beverage processing,the enzyme optimum p H in the acidic range; as opposed to having a high alkaline pectinase enzyme activity in the alkaline range, domestic and foreign research pectinase also continue to make new progress, but mostly the research phase, rarely applied to the industry, thus thermophilic properties and application of alkaline pectinase industrial prospects are still very broad.We successfully constructed Cb Pel C, Cb Pel C-CD two thermophilic alkaline pectinase, which Cb Pel C wild-type alkaline pectinase, Cb Pel C-CD for the mutant alkaline pectinase, primarily designed to study mutants of unknown structure the relationship between the domain and the catalytic domain. The results showed that:unknown domains have a guiding role, protected from other protease hydrolysis;During the enzyme reaction hydrolysis sites hide folded portion alkaline pectinase unknown domain; alkaline pectinase for correct folding of some help; influence on theenzyme activity.After Blast sequence analysis pectin lyase Cb Pel C, with the reported amino acid sequence homology with the family pectinase up to 54%, respectively, after the nickel affinity chromatography to obtain 46 k D size protein band, and theoretical sequencing results prediction same. Characterization results show, Cb Pel C and Cb Pel C-CD enzymatic properties are basically the same, the maximum absorption assay by 235 nm pectinase Cb Pel C and vitality Cb Pel C-CD, which is the optimum temperature was 70℃; the optimum Ca2+ concentration to 0.8m M; at 70 ℃ incubation, half-life of 6h;substrate is 0.2%(w / v)D- Multi-galacturonic acid, p H 9.5. The optimum p H 9.5,Ca2+ 0.8m M, 70 ℃ when Cb Pel C activity of 900 U / mg; Cb Pel C-CD activity was5300 U / mg. No presence of Ca2+, Cb Pel C and Cb Pel C-CD no vitality, Ca2+concentration 0.6m M-1.0m M, enzyme activity is maintained at more than 80%relative activity. The alkaline pectinase can be stored for long periods at 4 ℃ or room temperature and relative activity remained above 85%.Summing up the appeal, we successfully obtained two strains of novel thermophilic alkaline pectinase Caldicellulosiruptor bescii DSM 6725 Department,showed higher enzyme activity and good thermal stability, good enzymatic properties used in industrial applications, so that resources can be used again, reducing the energy crisis and environmental damage, with great economic effect is a subject worthy of further study.