This study aims to screen a strain from Armillaria for the cultivation of Gastrodia elata. Specifically, Armillaria strains were isolated from different producing areas of G. elata and identified. Based on the growth characteristics of the strains and the experiment on the cultivation of G. elata, an optimal A. gallica strain was screened out. The specific process is as follows. The fungus-gro-wing materials of G. elata were collected from four producing areas and the Armillaria strains were isolated(G,Y,S,H). The strains were then identified based on morphological observation and phylogeny analysis and the commonly used strains were determined. The sucrase genotypes of the strains were identified according to our previous research findings, and the growth characteristics of the strains, such as growth rate, diameter, dry weight, and polysaccharide content of the rhizomorphs, were measured. According to the biological characteristics and sucrase genotypes, two strains were selected for the cultivation of G. elata. The tuber yield and the content of gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata were measured to select the optimal strain. The results showed that the four strains were all A. gallica. The rhizomorphs of strains G and H of the same sucrase genotype had larger/higher length, growth rate, diameter, branch number, dry weight, and polysaccharide content than those of strains S and Y of the same sucrase genotype. The tuber yield and the total content of gastrodin and p-hydroxybenzyl alcohol in tuber of G. elata cultivated with strain H were 6.528 kg·m~(-2) and 0.566%, respectively, which were 4.58 and 1.30 folds those of G. elata cultivated with strain S. Strains H and S were screened out from four strains of A. gallica based on the growth characteristics and sucrase genotype. According to the tuber yield and content of total gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata, strain H was identified as the optimal one. The findings in this study are expected to lay a basis for cultivating G. elata with high yield and quality of tubers.
Armillaria/genetics* ; Gastrodia ; Polysaccharides

We used reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) techniques to obtain the full-length cDNA of beta-mannanase (EC 3.2.1.78) from Armillariella tabescens EJLY2098 (an edible fungus). Sequence analysis of the 1481 bp full-length cDNA encoding 445 amino acid residues indicated that the gene contained two structural domains, cellulose-binding domains (CBD) and glycoside hydrolase family 5 (GHF5) domains, other than the conserved beta-mannanase domain. Thus, we classified this gene as a member of glycoside hydrolase family 5. Next, we cloned a 1308 bp fragment encoding the beta-mannanase mature peptide (re-atMAN47) into the expression vector pPICZalphaA and expressed it in Pichia pastoris. The yield was 440 mg/L. Enzyme activity reached a maximum of 1.067 IU/mL after 72 h of methanol induction. The re-atMAN47 had an optimal temperature of 60 degrees C and an optimal pH of 5.5. It manifested broad thermostability from 30 degrees C-65 degrees C, and was stable between pH 4.5-7.0. This study represents the first record of a beta-mannanase from Armillariella tabescens EJLY2098 and provides a new source of carbohydrate hydrolysis enzyme with good biosafety, thermostability and wide pH stability. It is a good approach for the industrial needs of feed, food and pharmaceutical manufacturers.
Armillaria ; classification ; enzymology ; genetics ; Cloning, Molecular ; Enzyme Stability ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; beta-Mannosidase ; biosynthesis ; chemistry ; genetics

Firstly, We used error-prone PCR to induce mutations on Armillariella tabescens MAN47 beta-mannanase gene, Secondly, we cloned the mutated fragments into secreted expression vector pYCalpha, Then the recombinant plasmids were transformed into Saccharomyces cerevisiae BJ5465 after amplified and extracted in DH5alpha cells. Through three cycles of error-prone PCR we built a mutant database, Then we screened one optimum (named M262) from about 104 mutants. The evoluted MAN47 beta-mannanase displayed both higher thermal stability and activity than wide type. The evoluted enzyme M262 retained high activity after treatment at 80 degrees C for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of M262 can reach to 25 U/mL, which is 4.3 times as wide type under optimum temperature. In addition, pH stability and pH range of evoluted enzyme M262 were both improved compared with wild-type enzyme. The optimum pH was estimated to be similar to that of wild-type enzyme. The sequence comparison illustrated that there were three nucleotide substitutions (T343A/C827T/T1139C) which carried corresponding amino acid changes (Ser115Thr/Thr276Met/Val380Ala). According to homologous modeling by SWISS-MODEL Repository, three mutated amino acids located at the sixth amino acid of the fourth beta-sheet, the first amino acid of the sixth alpha-helix, the turn between the tenth and eleventh beta-sheet, respectively.
Armillaria ; classification ; enzymology ; genetics ; Directed Molecular Evolution ; Enzyme Stability ; Escherichia coli ; enzymology ; genetics ; Hot Temperature ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering ; Recombinant Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; genetics ; beta-Mannosidase ; chemistry ; genetics ; metabolism