Studies On Alkaline β-mannanase Produced By Alkaliphilic Bacillus Sp. N16-5

β-mannanase has extensive applications in the food, pharmaceutical, textile,detergent, paper-making, feedstock and petroleum industry. Alkaline β-mannanases provideobvious advantages for the applications in the processes that demand extreme conditions,such as in laundry detergents, paper pulp bleaching and hydraulic fracturing of oil well. Itwill be with great potential to discovery the alkaline β-mannanase from alkaliphilicmicroorganisms in soda lakes. This study describes the alkaliphilic resources in soda lakes ofChina, isolation and identification of alkaliphiles producing mannanase, optimization offermentation conditions, purification and properties of the mannanase, cloning and expressionof the mannanase gene, alkaline adaptation of the mannanase and evaluation ofbiotechnological potential of the mannanase.(1)The bacterial diversity of the soda lakes in China was investigated usingculture-dependent and culture-independent approaches. Phylogenetic analysis of 16S rRNAgene sequences cloned showed the presence of members of the α and β subdivision ofProteobacteria, which were not found previously among cultivated soda lake isolates, and thepresence of novel taxa, which have not been recognized before. Some 106 alkaliphilic isolateswere got under aerobic conditions and grew optimally between pH 9 and 10. Thephylogenetic analysis of 16S rDNA sequences from thirty-two isolates revealed that over 60%of soda lake isolates represent potentially novel species or genera (<97% sequence similarity).Of them, strain N10 was differentiated from currently recognized genera and proposed as thetype species of the new genus Alkalimonas within the gamma subdivision of theProteobacteria, named Alkalimonas amylolytica sp. nov. The members of Halomonas andBacillus rRNA group 7 were identified for the first time as the main polysaccharide-hydrolaseproducers in soda lakes. Twenty-two alkaliphilic isolates, exhibiting extracellular mannanaseactivity at pH 10, were screened from all of soda lake isolates. The results of polyphasictaxonomy revealed that β-mannanase producing strain N16-5 represented a new specieswithin the genus Bacillus, named Bacillus mannanolyticus sp. nov.(2)The effects of nutritional and environmental factors on the production of alkalineβ-mannanase by N16-5 were investigated and optimized, the enzyme activity can reach 470U/mL in 250L and 1000L fermentor. The pH and temperature optima of the crude mannanaseof strain N16-5 were 9.5 and 70 oC, respectively. The hydrolysates of Konjac powder andlocust bean gum by this enzyme were a series of oligosaccharides.(3)Three extracellular β-mannanase (M1, M2, M3) were purified to homogeneity fromthe culture broth of strain N16-5. Their molecular weights were estimated to be 51, 38 and 35kD by SDS-PAGE, respectively. These enzymes exhibited maximal activity at pH 9.0 and70oC (M1) and pH 10.0 and 70oC (M2 and M3), and the enzymes were stable at the range ofpH 8.0 to 10.0. The enzymes were resistant to some metals and surfactants. The Km values ofthe three β-mannanases for konjac β-glucomannan were 2.9, 1.7 and 12.5 mg ml-1,respectively.(4)The gene of alkaline β-mannanase A(M1) was cloned from the genome DNA ofstrain N16-5, the full length of the gene is 1479bp encoding 493 amino acids residue with adeduced molecular weight 54215 Da. M1 belongs to glycosyl hydrolase Family 5, SubfamilyA8. M1 gene is the firstly reported alkaline β-mannanase gene which belongs to Family 5.The amino acid sequence of strain N16-5 ManA deduced from the manA ORF showed highhomology to family 5 β-mannanases: 59% to ManG of Bacillus circulans, 42% to ManA ofThermobifida fusca, 36% to ManA of Vibrio sp. MA-138 and 35% to ManA of Streptomyceslividans 66. No significant similarity was found to the alkaline β-mannanases from Bacillussp AM001(19%), only one extensively characterized among alkaline β-mannanases reportedto date. The manA has been expressed extracellularly in Pichia pastoris. The recombinantstrain secreted 100.8 U/ml of active ManA after 96 h of growth in a complex medium, and theratio of extracellular enzyme is 73%.(5)The analysis of the amino acid sequence of M1 showed that M1 has high content ofA and G. Its C-terminal region (less than 160aa) showed no significant relevant with itsalkaline-adaptation characteristics by deletion mutation analysis. A pH-acid-shifting mutantwas obtained by Error-prone PCR technique. The optimal pH of the mutant is shift down from9.5 to 8.5, and it shows no activity at pH 10.0, while the wild type M1 retains 75% activity atpH 10.0. Compared the nucleotide sequence of the mutant with that of the wild type, threesites of the nucleotide acid sequence changed, which lead to two amino acid residues changed:133rd amino acid residue, Ala, was substituted by Val, 327th amino acid residue, Thr, wassubstituted by Ala. Only Ala-133→Val mutation was responsible for the shift of optimal pH,indicating that the Ala-133 residue was essential for catalytic activity of the β-mannanase inalkaline conditions.(6)The conditions for the products of enzymatic hydrolysis from konjac polysaccharidewere optimized and the hydrolysate were analyzed. The enzyme efficiently hydrolyzed konjacpolysaccharide producing a series of manno-oligosaccharides. The contents of mannobiose,mannotriose, and mannotetraose in the hydrolysate were 14%, 25% and 21.7%, respectively.In pilot-scale experiment of 10 M3 tank, the rate of substrate hydrolysis can reach more than90%, oligomer recovery rate more than 80% and the 1 to 6mer range is from 60% to 80%.The alkaline β-mannanase from the strain N16-5 was effective to degrade thepolysaccharide in hydraulic fracturing fluids within a certain pH and temperature range. Theresidual viscosity and percent residue-after-break of the fracturing fluids were reduced to lessthan 10 mPa.s and about 6-7 %. It suggested the β-mannanase, as a alkaline and thermostableenzyme breaker, can be advantageously employed during enhancing oil recovery operations.