Efficient biosynthesis of exopolysaccharide from Jerusalem artichoke using a novel strain of Bacillus velezensis LT-2.

This study focused on the non-grain biorefining of Jerusalem artichoke (JA) for exopolysaccharide (EPS) efficient production by using Bacillus velezensis LT-2. Results showed that LT-2 could directly utilize JA tuber power (JATP) for EPS production, and its EPS yield reached 11.47 ± 0.33 g/L in the simultaneous saccharification and fermentation (SSF) mode. Furthermore, the SSF mode shortened the fermentation period by 26.67% and reduced the fermentation cost by 79.41% due to the improved substrate utilization and the avoidance of inhibition effects of a high fructose concentration. Transcriptome sequencing results showed that inulin could accelerate nucleotide-sugars biosynthesis, induce EPS synthetic gene cluster transcription, and strengthen the electron transport respiratory chain and the transporter systems, thereby ensuring EPS efficient synthesis. This work exhibited a productive non-grain and environmentally friendly fermentation strategy for EPS biosynthesis, which promoted the JA industry development and created new prospects for high-value industrial products biosynthesis by using JATP.

Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel.

This study reported simultaneously improved thermostability and hydrolytic pattern of α-amylase from Bacillus subtilis CN7 by rationally engineering the mostly conserved central beta strands in TIM barrel fold. Nine single point mutations and a double mutation were introduced at the 2nd site of the ß7 strand and 3rd site of the ß5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of α-amylase. All the five active mutants changed the compositions and percentages of maltooligosaccharides in final hydrolytic products compared to the product spectrum of the wild-type. A mutant Y204V produced only maltose, maltotriose, and maltopentaose without any glucose and maltotetraose, indicating a conversion from typical endo-amylase to novel maltooligosaccharide-producing amylase. A mutant V260I enhanced the thermal stability by 7.1 °C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of α-amylase by engineering central beta strands of TIM barrel and the novel "beta strands" strategy proposed here may be useful for the protein engineering of other TIM barrel proteins.

New gene cluster from the thermophile Bacillus fordii MH602 in the conversion of DL-5-substituted hydantoins to L-amino acids.

The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-alpha-amino acids. Since the reaction at higher temperature, the advantageous for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile was firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2 kb DNA fragment by Restriction Site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein hyuP, hyperprotein hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-alpha-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 comparing respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.

[Screening and identification of an organic solvent-stable protease producer].

An organic-solvent-tolerant bacterium strain YP1 producing organic-solvent-stable protease was isolated from crude oil contaminant soil. Strain YP1 was strictly aerobic, motile, gram positive, spore-forming, and rod shaped. The YP1 strain was identified as Bacillus licheniformis using culture system BIOLOG analysis (SIM = 0.62, 16-24h). The 16S rDNA sequence analysis (GenBank accession number EF105377) suggested that strain YP1 was clustered together with B. licheniformis in phylogenetic tree. Based on all the taxonomy,strain YP1 was identified as B. licheniformis. YP1 strain could tolerant organic solvents at different levels, especially it can grow well in the presence of water-miscible solvents dimethylformamide (DMF, logP = -1.0) and dimethylsulphoxide (DMSO, logP = -1.35) at a concentration of 10% [V/V]. Strain YP1 can also tolerant middle concentrations of NaCl and extra alkaline conditions (pHl2). More than 80% of the biomass remained at pH range 10.5-12. However strain YP1 was sensitive to antibiotics such as ampicillin, tetracycline, kanamycin and chloromycetin. The protease production could be enhanced by acetone and repressed by alkanols such as dodecylalcohol and octanol during the fermentation. Compared to trypsin, the YP1 protease had a wider tolerance for organic solvents. YP1 protease tolerated up to at least 11 organic solvents with logP ranging from -1.35 to 5.6 including benzene, toluene, DMSO and DMF etc at 50% (V/V) concentration. Moreover, when solvents such as decane and dodecyl alcohol with log P values above 4.0 were added to the crude protease, the enzyme activity levels were 1.08 and 1.21 times higher than the control respectively. Its high tolerance for water-miscible solvents DMF and DMSO makes it an ideal catalyst for kinetic- and equilibrium-controlled synthesis. This organic solvent stable protease could be used as a biocatalyst for enzymatic synthesis in the presence of organic solvents.

Biochemical properties and potential applications of a solvent-stable protease from the high-yield protease producer Pseudomonas aeruginosa PT121.

An organic solvent-stable protease from Pseudomonas aeruginosa PT121 was purified in a single step with 55% recovery by hydrophobic interaction chromatography on a Phenyl Sepharose High Performance matrix. The purified protease was homogenous on SDS-PAGE and had an estimated molecular mass of 33 kDa. The optimal pH and temperature conditions for enzyme activity were 8.0 and 60 degrees C, respectively. The enzyme was classified as a metalloprotease based on its strong inhibition by EDTA and 1,10-phenanthroline and exhibited good stability across a broad pH range (6.0-11.0). The protease was quite stable in the presence of various water-miscible organic solvents. This is a unique property of the protease which makes it an ideal choice for application in aqueous-organic phase organic synthesis including peptides synthesis. The synthetic activity of the protease was tested using N-carbobenzoxy-L-asparagine (Z-Asp) and L-phenylalaninamide (Phe-NH2) as substrate in the presence of various water-miscible organic solvents for aspartame precursor synthesis. The highest yield was obtained in the presence of 50% DMSO (91%). The synthesis rate in the presence of DMSO was also much higher than the rates in the other tested organic solvents, and the initial rates of Z-Asp-Phe-NH2 synthesis in mixtures of various water-miscible organic solvents, with the exception of ethanol, correlated with the yields of Z-Asp-Phe-NH2. Furthermore, the PT121 protease was able to use various carboxyl components (Z-AA) and Phe-NH2 as substrates to catalyze the syntheses of the dipeptides, indicating that this protease has a broad specificity for carboxylic acid residue.

Breeding of D(-)-lactic acid high producing strain by low-energy ion implantation and preliminary analysis of related metabolism.

The low-energy nitrogen ion beam implantation technique was used in the breeding of mutant D(-)-lactic-acid-producing strains. The wild strain Sporolactobacillus sp. DX12 was mutated by an N(+) ion beam with energy of 10keV and doses ranging from 0.4 x 10(15) to 6.60 x 10(15) ions/cm(2). Combined with an efficient screening method, an efficient mutant Y2-8 was selected after two times N(+) ion beam implantation. By using the mutant Y2-8, 121.6g/l of D-lactic acid was produced with the molar yields of 162.1% to the glucose. The yield of D-lactic acid by strain Y2-8 was 198.8% higher than the wild strain. Determination of anaerobic metabolism by Biolog MT2 was used to analyze the activities of the concerned enzymes in the lactic acid metabolic pathway. The results showed that the activities of the key enzymes responded on the substrates such as 6-phosphofructokinase, pyruvate kinase, and D-lactate dehydrogenase were considerably higher in the mutants than the wild strain. These might be affected by ion beam implantation.