An efficient system for pre-delignification of gramineous biofuel feedstock in vitro: Application of a laccase from Pycnoporus sanguineus H275

An efficient system is presented for pre-delignifying cereal straw in vitro using laccase produced by Pycnoporus sanguineus H275. Under our experimental conditions, over 97% of the klason lignin was removed from 1 g wheat straw lignocellulose, without loss of Somogyi carbohydrate (reducing sugar). Microscopic observation of birefringence changes further indicated that the structure of the cellulose building blocks (microfibrils) of wheat straw scattered after pre-degradation, compared with the tightly arranged microfibrils in untreated control straw. The P. sanguineus H275-laccase-encoding gene was then cloned and successfully expressed in Pichia pastoris X-33, with a production of 3000 U/L after 6 days of cultivation. The identity of the expressed laccase was further confirmed by using MALDI-TOF-MS/MS. Homology modeling revealed that the native H275-laccase, predicted with five possible N-glycosylation sites, has extra cupredoxine domains and three common Cu-oxidase domains. Both kinetic characterization on the substrate ABTS and pre-delignification investigation on wheat straw lignocellulose showed that the activity of the recombinant laccase was nearly the same as that of the native laccase.