Cellobiose Dehydrogenase Production By Pycnoporus Sanguineus And Its Primary Research In The Synthesis Of Lactobionic Acid

Lactobionic acid is a novel lactose derivative, and has been widely applied in the food, pharmaceutical and cosmetics industries. Lactobionic acid is currently produced by chemical synthesis which associated with the use of costly catalysts and production of undesirable compound. The enzymatic synthesis of lactobionic acid displays several advantages, such as easy products separation, high specificity and mild reaction conditions. But enzyme purification approaches are costly and time-consuming. The bio-production of lactobionic acid has become a feasible way to overcome certain drawbacks associated with chemical or enzymatic approaches, and aroused great interest of researchers.In this study, a fungal strain secreting cellobiose dehydrogenase(CDH) and laccase was isolated. Production, purification and characterization of this CDH as well as its application for synthesis of lactobionic acid were researched and the results were shown as follows:1) A fungal strain SYBC-L10, secreting CDH and laccase, was isolated and identified as Pycnoporus sanguineus base on its morphological characteristics and gene sequence analysis.2) The optimized fermentation medium component was composed as follows: microcrystalline cellulose 7.0 g·L-1, glucose 5.0 g·L-1, soybean meal 6.1 g·L-1,(NH4)2HPO4 2.0 g·L-1, FeSO4 19.6 μmol·L-1, micronutrient 0.7%(v·v-1), VB3 0.3 mmol·L-1, Tween40 0.6 g·L-1. The optimized fermentation medium conditions were as follows: temperature 30 oC, pH 4.5, seed age 2 d, inoculum concentration 8%(v·v-1), liquid volume 50 m L/250 m L. Under this condition, the enzyme activity reached 2569±48 U·L-1 which was 65.8 folds of the initial activity(39±5.8 U·L-1).3) The CDH and laccase were purified by anion exchange column and gel filtration. The enzyme was determined as CDH by the protein profiling assay and Blast analysis. The isoelectric point of CDH was 3.79 and the molecour weight was 90.70 kDa. Enzymatic synthesis of lactobionic acid by CDH was determined.4) P. sanguineus SYBC-L10 had a lactobionic acid synthesis capacity. When the wastewater was added to the culture broth at 5 d, the maximum lactobionic acid of 14.6 g·L-1 was obtained within 12 h. The lactose conversion rate was above 80% with the lactose in waste water was less than 40 g·L-1. The lactobionic acid was reaching the maximum(35.3 g·L-1) and the lactose conversion rate was 75% with the lactose final concentration of 45 g·L-1.