Digging And Characterization Of Neutral β-1,4-endoglucanase And The Fundamental Study On Application Of It In Biostoning

Neutral (3-1,4-endoglucanase (EG) has a high application value in biostoning of denim, but domestic neutral EG doesn’t have independent intellectual property rights. As a result, it is significantly meaningful to dig new neutral EG in order to break the monopoly of foreign neutral EG. Complete neutral EGs and truncated neutral EGs have different performances in biostoning of denim, which is concerned with cellulose-binding domain of cellulase, but the deep-seated reason has not been found. The paper focuses on digging, heterologous expression and characterization of neutral EG, and fundamentally explores the application of denim in biostoning.In the first section, the study about gene digging, efficient heterologous expression and characterization of a neutral EG from Bacillus subtilis LH, and the fundamental study on application of it in biostoning of denim were carried out.1) B. subtilis LH producing the neutral EG was screened from the strain bank of Newworld Institute of Biotechnology, and the neutral EG gene from B. subtilis LH was amplified. The mature EG was expressed in Escherichia.coli Rosetta (DE3) and Pichia pastoris GS115(named as MBSEG-1and MBSEG-2, respectively), respectively, and the result showed that the expression level of MBSEG-2is much higher than that of MBSEG-1. By using the optimized condition, the EG activity of MBSEG-2without concentration is25686U/mL, which is the highest expression level having been reported at present.2) MBSEG-1and MBSEG-2have some similar properties such as the optimum temperature of65℃and the optimum pH of6.86, and possess β-1,4-endoglucanase and β-1,4-exoglucanase activities, but N-glycosylation produces the differences in thermal stability and the limited papain hydrolysis of the two enzymes.3) On the basis of the experiment about treating denim with the truncated MBSEG-2and the complete MBSEG-1and MBSEG-2, and according to the analysis of their3D models, it was found out that neutral EGs with the higher percentage of surface hydrophobic amino acids and aromatic amino acids regardless of whether neutral EGs (must have catalytic domain) help denim to enhance the indigo dye removal and the weight loss.In the second section, identification of Petriella setifera LH, characterization of neutral EG, and the fundamental study on application of neutral EG in biostoning of denim were carried out.The fungus producing the neutral EG was screened from the preserved soil of Newworld Institute of Biotechnology. The fungus was identified as Pe. Setifera and named as Pe. setifera LH on the basis of the analysis of the phylogenetic tree of the partial elongation factor-1a gene (EF-la) combined with its morphological characteristics. The EG from Pe. setifera LH has an above80%relative activity at40℃and the optimum pH of6-7. Compared with denim treatment by a commercial neutral cellulase, the EG exhibited a similar fabric weight loss and indigo dye removal, revealing that the enzyme has a potential application in denim biostoning.In the third section, gene digging, sequence analysis and characterization of two HGs from Phaeosphaeria sp LH21were carried out.1) The fungus was screened from the extreme environment deep-sea mud, identified as Phaeosphaeria sp. and named as Phueosphaeria sp. LH21on the basis of the alignment analysis of the partial sequences of EF-1a and18S rDNA.2) The two new EG (GH45EG-1and EG45EG-2) genes from Phaeosphaeria sp. LH21were first cloned by genome walking and RT-PCR. GH45EG-1and GH45EG-2consists of235and233amino acids, respectively. The alignment analysis of the amino acid sequences was performed by NCBI BLAST, indicating that GH45EG-1and GH45EG-2belong to glycoside hydrolase family45, possess catalytic domain without cellulose-binding domain and share the maximum amino acid sequence identities of71%and63%with the known EGs. respectively.3) On the basis of the amino acid sequences from the two mature EGs (mGH45EG-1and mGH45EG-2) and Humicola insolens EG V, and according to their3D models, it was speculated that one of the active central sites of mGH45EG-1could be Asp16, that the active central sites of mGH45EG-2could be Asp122and Aspl1and that their catalytic mechanisms could be the inversion mechanism.4) mGH45EG-1and mGH45EG-2were expressed in P. pastoris GS115, respectively. The substrate spectra of the recombinant enzymes mGH45EG-1and mGH45EG-2confirmed that they belong to endonuclease. They have the highest activities on CMC, and the relative activities on crystalline cellulose and filter paper are0.1-0.3%.10mM Ca2+and Mg2+increase8-27%of relative activities of the recombinant enzymes mGH45EG-1and mGH45EG-2, and most metal ions inactivate them. The recombinant enzymes mGH45EG-1and mGH45EG-2have the optimum pH of8, above75%relative activities at pH (?)10, above90%relative residual activities after incubating at50℃and pH3-10for1h, and the optimum temperatures of65℃and60℃, respectively.