Chengtuo Niu

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis

1,3–1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C) were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm) compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

Production of a thermostable 1,3-1,4-β-glucanase mutant in Bacillus subtilis WB600 at a high fermentation capacity and its potential application in the brewing industry

1,3-1,4-β-glucanase was an important biotechnological aid in the brewing industry. In a previous research, a Bacillus BglTO mutant (BglTO) with high tolerance towards high temperature and low-pH conditions was constructed and expressed in Escherichia coli. However, E. coli was not a suitable host for enzyme production in food industry. Therefore, the present work aimed to achieve the high-level expression of BglTO in Bacillus subtilis WB600 and to test its effect in Congress mashing. The β-glucanase mutant was successfully expressed in B. subtilis WB600 and favorable plasmid segregation and structural stability were observed. The maximal extracellular activity of β-glucanase in recombinant B. subtilis WB600 reached 4840.4UmL-1 after cultivation condition optimization, which was 1.94-fold higher than that before optimization. The fermentation capacity of recombinant B. subtilis reached 242.02UmL-1h-1, which was the highest among all reported β-glucanases. The addition of BglTO in Congress mashing significantly reduced the filtration time and viscosity of mash by 29.7% and 12.3%, respectively, which was superior to two commercial enzymes. These favorable properties indicated that B. subtilis WB600 was a suitable host for production of BglTO, which was promising for application in the brewing industry.

Simultaneous determination of diethylacetal and acetaldehyde during beer fermentation and storage process: Determination of diethylacetal and acetaldehyde

BACKGROUND Acetaldehyde is an important flavor component in beer which is possibly carcinogenic to humans. Owing to the limitations of present detection methods, only free-state acetaldehyde in beers has been focused on, while acetal in beers has hardly been reported so far. RESULTS A sensitive headspace gas chromatography method was developed for the determination of diethylacetal and acetaldehyde in beer. The column DB-23 was chosen with a total run time of 22.5 min. The optimal addition amount of NaCl, equilibrium temperature and equilibrium time were 2.0 g, 70 °C and 30 min respectively. For both diethylacetal and acetaldehyde analyses, the limit of detection was 0.005 mg L-1 with relative standard deviation < 5.5%. The recoveries of acetaldehyde and diethylacetal were 95-110 and 95-115% respectively. The diethylacetal and acetaldehyde average contents in 24 beer products were 11.83 and 4.36 mg L-1 respectively. The Pearson correlation coefficient between diethylacetal and acetaldehyde was the highest (0.963). Both diethylacetal and acetaldehyde contents increased to a peak value after fermentation for 3 days and then decreased to a lower value. During both normal and forced aging storage, the diethylacetal content decreased and the acetaldehyde content increased gradually over time. When beers were forced aged for 4 days, the increased ratio of acetaldehyde could be above 40.00%. CONCLUSION The newly established method can be used to assess acetaldehyde level and flavor quality in beer more scientifically.

The FKS family genes cause changes in cell wall morphology resulted in regulation of anti-autolytic ability in Saccharomyces cerevisiae

The aim of this study was to discuss the functions of FKS family genes which encode β-1, 3-glucan synthase regarding the viability and autolysis of yeast strain. Loss of FKS1 gene severely influences the viability and anti-autolytic ability of yeast. Mutation of FKS1 and FKS2 genes led to cell reconstruction, resulting in a sharp shrinkage of cell volume and decreased stress resistance, viability, and anti-autolytic ability. Deletion of FKS3 gene did not clearly influence the synthesis of β-1, 3-glucan of yeast but increased the strains stress resistance, viability, and anti-autolytic ability. It is suggested that FKS3 would be the potential target for improving the stress resistance of yeast. The results revealed the relationship among FKS family genes and demonstrated their functions on yeast cell wall construction and anti-autolytic ability.

Cell wall polysaccharides: before and after autolysis of brewer’s yeast

Brewer’s yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of β-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.

Genome Analysis of the Yeast M14, an Industrial Brewing Yeast Strain Widely Used in China

Abstract The lager brewing yeast M14 is the most widely used yeast strain in the high gravity brewing process in China. To investigate the characteristics of this strain, the genome of the yeast M14 was sequenced and the genome annotation information is presented in this study. The current assembly contained 133 scaffolds and its total size was around 23 Mb with a GC content of 38.98%. The brewing yeast M14 is a hybrid Saccharomyces cerevisiae × Saccharomyces uvarum at the genomic level and its genome is comprised of one circular mitochondrial genome originating from S. uvarum. Furthermore, the functions of the 9,796 protein coding genes were annotated and their functions were analyzed using the Swiss-Prot database. Among them, the key genes responsible for typical lager brewing yeast characteristics, such as maltotriose uptake and sulfite production, were annotated and analyzed. Interestingly, nine specific genes present in the brewing yeast M14 were not found in the genome of either S. uvarum CBS 7001 or S. cerevisiae S288C, which are very close to strain M14 in the phylogenetic relationship. These nine genes encoding proteins were melibiase, DNA replication protein, fructose symporter, hypothetical protein, hypothetical protein M773_09155, LIF1, minor spike protein H, ribosomal protein S27, and mitochondrial chaperones, respectively. The genome sequence of the yeast strain M14 provides a new tool to better understand brewing yeast behavior in industrial beer production.

Monitoring the Microbial Conditions in Breweries in Yangtze River Delta Region, China

ABSTRACT This study aimed to investigate the contamination degree and quality control of seven breweries from the Yangtze River Delta region, China. A total of 205 spoilage bacteria strains were isolated and no wild yeast was observed. The vast majority of the spoilage bacteria belonged to Lactobacillus, Lactococcus, and Leuconostoc species and 28.8% of them could grow in beer. Among the seven sampling points, fermented beer, finished beer, and equipment contained larger amounts of spoilage bacteria. The most abundant of the spoilage bacteria that could grow in beer existed in fermented beer and the number of spoilage bacteria was significantly reduced in the finished beer. The equipment in fermentation and packaging workshops showed high percentages of spoilage bacteria that could grow in beer. This indicated that more attention should be paid in controlling the spoilage bacteria in the fermentation and packaging processes. The hop tolerance related gene analysis showed that the majority of spoilage bacteria contained at least one of the HorA, HorC and HitA genes. None of these genes were observed in the two spoilage bacteria strains that could grow in the beer. This information could be useful for breweries in the Yangtze River Delta region for removing risks in order to produce high-quality products in good microbial condition.

Rationally designed perturbation factor drives evolution in Saccharomyces cerevisiae for industrial application

Saccharomyces cerevisiae strains with favorable characteristics are preferred for application in industries. However, the current ability to reprogram a yeast cell on the genome scale is limited due to the complexity of yeast ploids. In this study, a method named genome replication engineering-assisted continuous evolution (GREACE) was proved efficient in engineering S. cerevisiae with different ploids. Through iterative cycles of culture coupled with selection, GREACE could continuously improve the target traits of yeast by accumulating beneficial genetic modification in genome. The application of GREACE greatly improved the tolerance of yeast against acetic acid compared with their parent strain. This method could also be employed to improve yeast aroma profile and the phenotype could be stably inherited to the offspring. Therefore, GREACE method was efficient in S. cerevisiae engineering and it could be further used to evolve yeast with other specific characteristics.

Comparative analysis of the effect of protein Z4 from barley malt and recombinant Pichia pastoris on beer foam stability: Role of N-glycosylation and glycation

This study aimed to elaborate the effect of N-glycosylation and glycation of protein Z4 from barley malt and recombinant Pichia pastoris on beer foam stability. The malt protein Z4 and recombinant protein Z4 showed similar N-glycosylation patterns while recombinant protein Z4 was glycosylated at a higher degree. In the simulated mashing and boiling, malt protein Z4 and deglycosylated malt protein Z4 preferred to glycate with glucose and maltose while recombinant protein Z4 and deglycosylated recombinant protein Z4 showed preference towards fructose. The addition of protein Z4 and protein Z4-saccharide complexes in finished beer showed that the addition of glycosylated protein Z4 only slightly enhanced the beer foam stability while the addition of glycated protein Z4 and protein Z4 with both glycation and glycosylation could significantly increase the beer foam stability. Therefore, glycation instead of N-glycosylation of protein Z4 played important roles in maintaining beer foam stability.

Process optimization of the extraction condition of β‐amylase from brewer's malt and its application in the maltose syrup production

β‐Amylase is of important biotechnological aid in maltose syrup production. In this study, the extraction condition of β‐amylase from brewers malt and the optimal dosage of β‐amylase in maltose syrup production were optimized using response surface methodology and uniform design method. The optimal extraction condition of β‐amylase from brewers malt was composed of 1:17 (g/v) material/liquid ratio, 44°C extraction temperature, pH 6.4 buffer pH, 2.3 H extraction time, and 1.64 g L−1 NaSO3 dosage with a predicted β‐amylase activity of 1,290.99 U g−1, which was close to the experimental β‐amylase activity of 1,230.22 U g−1. The optimal dosages of β‐amylase used in maltose syrup production were 455.67 U g−1 starch and its application in maltose syrup production led to a 68.37% maltose content in maltose syrup, which was 11.2% and 28.9% higher than those using β‐amylases from soybean and microbe (P < 0.01). Thus, β‐amylase from brewers malt was beneficial for production of high maltose syrup.