Isolation And Identification Of Bacillus Altitudinis SYBC Hb4 And Its Catalases Properties And Applications

An emerging trend is biotechnological application of Bacillus isolated from honey or bee bread. So far, many Bacillus strains of honey origin have been exploited for production of probiotics, antimicrobial substance and industrial enzymes, etc. Given diversity of Bacillus strains of honey origin, their functional development and biotechnological application deserve extension. Especially, the biotechnological applications of Bacillus strains of honey origin have not yet received enough attention in domestic. Bacillus strains of honey origin may develop special mechanisms to survive in unique conditions of honey and bee bread, such as hyperosmosis, low pH and existence of hydrogen peroxide（H₂O₂）. These Bacillus strains may produce some osmophilic or acid-stable proteins and enzymes. Some catalases with unique properties may also be produced for disposal of H₂O₂. However, so far, properties study and functional development of catalases from Bacillus strains of honey origin have not yet been conducted. In addition, catalase is also a common functional enzyme affecting soil fertility. Catalase activity generally decreases in polluted soil, especially in acidic soil. The decrease of catalase activity can negatively affect plant growth. In the past, Bacillus strains of honey origin were mainly exploited as bio-control bacteria. In constrast, the exploitation of plant growth-promoting properties（PGPP） received less attention. The Bacillus strains of honey origin, which are characterized with high production of catalases and tolerance to low pH, may supplement to lack of catalase activity in acidic soil. In addition to bio-control function, the Bacillus strains of honey origin may have other PGPP.In this study, Bacillus strains were isolated and identified from honey and bee bread produced by the local bee（Apis cerana）. A strain with a much higher production of CAT was selected and identified. Its catalses genes were cloned and heterologously expressed in host of E. coli BL21（DE3）. The recombinant catalases were purified for enzymatic assays. Its catalases isoenzymes were also identified and purified for enzymatic assays and applications. In addition, PGPP of these Bacillus strains were analyzed by standard protocols. They were also exploited for promoting growth of barley seedlings in rare earth tailings soil（RETS）. The topic of above work was committed to opening an avenue for functional exploitation and biotechnological application of the Bacillus strains of honey origin. The main findings and contents were summarized as follows:（1） A total of 62 bacteria were isoalted and classified into genera of Bacillus, Fictibacillus, Lysinibacillus and Paenibacillus. 48 bacteria could grow in double stresses of 30% glucose and pH 4.5. Their growth was not inhibited apparently by 18 mmol?L^-1 H₂O₂ under above stresses. Instead, the strains of hb 4, 47 and 58 grew better. These Bacillus strains harbor a much higher resistance to H₂O₂.（2） A strain hb4 was selected because it had a much higher production of catalases. The strain hb4 was identified and designated as Bacillus altitudinis SYBC hb4. Four catalase genes of katB, kat X, katN1 and katN2 were cloned from genome of B. altitudinis SYBC hb4. These four genes were cloned and transformed into E. coli BL21（DE3）, respectively. The four recombinant strains could express the recombinant catalases under induction of 0.5 mmol?L^-1 IPTG at 15oC. The four recombinant catalases of r KatB, rKatX, r KatN1 and r KatN2 exhibited different molecular mass, in order, 61.8, 57.6, 31.9 and 34.2 KDa（fusion with His tag）. Their optima of pH were, in that order, 9.0, 6.0, 6.0 and 9.0. Their optima of temperature were, in that order, 30, 45, 40 and 45oC. The values of Km were, in that order, 61.6, 64.3, 27.7 and 25.1 mmol?L^-1; The values of kcat/Km were, in that order, 643, 597, 50 and 67 L?mmol^-1 s^-1. The results suggest that B. altitudinis SYBC hb4 is a potent strain with high production of catalases. It has four genes encoding catalases with different enzymatic properties.（3） Without induction of Mn2+, B. altitudinis SYBC hb4 produced three catalase isoenzymes. One of them was identified as KatB by technologies of SDS-PAGE and MALDI-TOF MS. B. altitudinis SYBC hb4 produced a new catalase different from above three catalases under induction with 0.15 mmol?L^-1 Mn2+. This catalase was identified as the sporulation-related manganese catalase（YjqC）, which was the production of katN1（yjqC）. KatB and YjqC were purified via ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The molecular mass of KatB and YjqC was about 57 and 31 KDa, respectively. The optima of KatB were 30oC and pH 5.0. Its apparent values of Km、Vmax and kcat/Km were 62 mmol?L^-1, 33.4 mol?min^-1?mg^-1 and 2047 L?mmol^-1?s^-1. The optima of YjqC were 35oC and pH 7.0. Its apparent values of Km, Vmax and kcat/Km were 23.5 mmol?L^-1, 3195 U?mg^-1 and 407 L?mmol^-1?s^-1. KatB displayed higher activity and stability at pH 5.0. YjqC was more stable than KatB in higher values of pH and temperature.（4） Besides catalase activity of YjqC, it could also catalyze oxidation of SA and SNP with a certain amount of H₂O₂ as a co-substrate. In addition, YjqC could endow spore with catalytic activity toward SA and SNP. Compared with the free YjqC, Km values of spores for SA and SNP increased, and the catalytic efficiencies also lowered. But the stability of spore-catalyzed oxidation of SA and SNP was much higher than that of the free YjqC. After treating rapeseed meal with spore suspension plus 15 mmol?L^-1 H₂O₂ at 45oC for 18 h, the content of SA and SNP extraction decreased by 51 and 41%, respectively. Therefore, spores of B. altitudinis SYBC hb4 could be regarded as the potent biocatalyst used for degradation of SA and SNP in rapeseed meal.（5） A total of nine Bacillus strains were selected due to their higher tolerance to stresses of acid（pH 4.5） and 1.5 mmol?L^-1 Al3+. These nine strains could all dissolve phosphate and produce organic acid, auxin, siderophore, CAT and SOD. But only six strains produced ACC deaminase. These nine strains could all promote growth of barley seedlings in RETS. The possible growth-promoting mechanisms were shown as follows: a) accumulation of Al in seedlings was reduced by organic acid secreated by the nine strains. b) accumulation of malondialdehvde in seedlings was also reduced. c) accumulation of ethylene was reduced by ACC deaminase. d) CAT and SOD could relieve oxidative stress to roots of barley seedlings. Therefore, the Bacillus strains of honey origin also had similar plant growth-promoting properties to the rhizospheric Bacillus strains. Their catalases might have promotion on plant growth.