Haiquan Yang

Structure-based engineering of histidine residues in the catalytic domain of α-amylase from Bacillus subtilis for improved protein stability and catalytic efficiency under acidic conditions.

This work aims to improve the protein stability and catalytic efficiency of α-amylase from Bacillus subtilis under acidic conditions by site-directed mutagenesis. Based on the analysis of a three dimensional structure model, four basic histidine (His) residues His(222), His(275), His(293), and His(310) in the catalytic domain were selected as the mutation sites and were further replaced with acidic aspartic acid (Asp), respectively, yielding four mutants H222D, H275D, H293D, H310D. The mutant H222D was inactive. Double and triple mutations were further conducted and four mutants H275/293D, H275/310D, H293/310D, and H275/293/310D were obtained. The acidic stability of enzyme was significantly enhanced after mutation, and 45-92% of initial activity of mutants was retained after incubation at pH 4.5 and 25°C for 24h, while that for wild-type was only 39.5%. At pH 4.5, the specific activity of wild-type and mutants H275D, H293D, H310D, H275/293D, H275/310D, H293/310D, and H275/293/310D were 108.2, 131.8, 138.9, 196.6, 156.3, 204.6, and 216.2U/mg, respectively. The catalytic efficiency for each active mutant was much higher than that of wild-type at low pH. The kcat/Km values of the mutants H275D, H293D, H310D, H275/293D, H275/310D, H293/310D, and H275/293/310D at pH 4.5 were 3.3-, 4.3-, 6.5-, 4.5-, 11.0-, 14.5-, and 16.7-fold higher, respectively, than that of the wild-type. As revealed by the structure models of the wild-type and mutant enzymes, the hydrogen bonds and salt bridges were increased after mutation, and an obvious shift of the basic limb toward acidity was observed for mutants. These changes around the catalytic domain contributed to the significantly improved protein stability and catalytic efficiency at low pH. This work provides an effective strategy to improve the catalytic activity and stability of α-amylase under acidic conditions, and the results obtained here may be useful for the improvement of acid-resistant ability of other enzymes.

How to achieve high-level expression of microbial enzymes: Strategies and perspectives

Microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. The enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. Recombinant enzymes have been expressed in bacteria (e.g., Escherichia coli, Bacillus and lactic acid bacteria), filamentous fungi (e.g., Aspergillus) and yeasts (e.g., Pichia pastoris). The favorable and very advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. Bacterial hosts (e.g., E. coli) can be used to quickly and easily overexpress recombinant enzymes; however, bacterial systems cannot express very large proteins and proteins that require post-translational modifications. The main bacterial expression hosts, with the exception of lactic acid bacteria and filamentous fungi, can produce several toxins which are not compatible with the expression of recombinant enzymes in food and drugs. However, due to the multiplicity of the physiological impacts arising from high-level expression of genes encoding the enzymes and expression hosts, the goal of overproduction can hardly be achieved, and therefore, the yield of recombinant enzymes is limited. In this review, the recent strategies used for the high-level expression of microbial enzymes in the hosts mentioned above are summarized and the prospects are also discussed. We hope this review will contribute to the development of the enzyme-related research field.

Molecular engineering of industrial enzymes: recent advances and future prospects

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.

Structure-based engineering of methionine residues in the catalytic cores of alkaline amylase from Alkalimonas amylolytica for improved oxidative stability

ABSTRACT This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H2O2 at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the k cat values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 × 104 to 5.6 × 104 min−1. The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.

Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

ABSTRACT In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) from Alkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (k cat) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK: k cat/Km increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H2O2, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.

Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis.

BackgroundAlkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase.ResultsThe alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21.ConclusionsThis is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis.

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

ABSTRACT High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (k cat/Km ) increased from 1.8 × 104 to 2.4 × 104 liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

Recent advances in recombinant protein expression by Corynebacterium , Brevibacterium , and Streptomyces : from transcription and translation regulation to secretion pathway selection

Gram-positive bacteria are widely used to produce recombinant proteins, amino acids, organic acids, higher alcohols, and polymers. Many proteins have been expressed in Gram-positive hosts such as Corynebacterium, Brevibacterium, and Streptomyces. The favorable and advantageous characteristics (e.g., high secretion capacity and efficient production of metabolic products) of these species have increased the biotechnological applications of bacteria. However, owing to multiplicity from genes encoding the proteins and expression hosts, the expression of recombinant proteins is limited in Gram-positive bacteria. Because there is a very recent review about protein expression in Bacillus subtilis, here we summarize recent strategies for efficient expression of recombinant proteins in the other three typical Gram-positive bacteria (Corynebacterium, Brevibacterium, and Streptomyces) and discuss future prospects. We hope that this review will contribute to the development of recombinant protein expression in Corynebacterium, Brevibacterium, and Streptomyces.

Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline α-amylase as a case study

In this study, atmospheric and room temperature plasma (ARTP), a promising mutation breeding technique, was successfully applied to generate Bacillus subtilis mutants that yielded large quantities of recombinant protein. The high throughput screening platform was implemented to select those mutants with the highest yield of recombinant alkaline α-amylase (AMY), including the preferred mutant B. subtilis WB600 mut-12#. The yield and productivity of recombinant AMY in B. subtilis WB600 mut-12# increased 35.0% and 8.8%, respectively, the extracellular protein concentration of which increased 37.9%. B. subtilis WB600 mut-12# exhibited good genetic stability. Cells from B. subtilis WB600 mut-12# became shorter and wider than those from the wild-type. This study is the first to report a novel powerful mutagenesis tool (ARTP) that significantly improves the yield of recombinant proteins in B. subtilis and may therefore play an important role in the high expression level of proteins in recombinant microbial hosts.

Comparative analysis of heterologous expression, biochemical characterization optimal production of an alkaline α‐amylase from alkaliphilic Alkalimonas amylolytica in Escherichia coli and Pichia pastoris

An alkaline α‐amylase gene from alkaliphilic Alkalimonas amylolytica was synthesized based on the preferred codon usage of Escherichia coli and Pichia pastoris, respectively, and then was expressed in the according heterologous host, E. coli BL21 (DE3) and P. pastoris GS115. The alkaline α‐amylase expressed in E. coli was designated AmyA, whereas that produced by P. pastoris was designated AmyB. The specific activity of AmyA and AmyB was 16.0 and 16.6 U/mg at pH 9.5 and 50°C, respectively. The optimal pH and pH stability of AmyA and AmyB were similar, whereas the optimum temperature and thermal stability of AmyB were slightly enhanced compared with those of AmyA. The AmyA and AmyB had a similar melting temperature of 64°C and the same catalytic efficiency (kcat/Km) of 2.0 × 106 L/(mol min). AmyA and AmyB were slightly activated by 1 mM Co2+, Ca2+, or Na+, but inhibited by all other metal ions (K+, Mg2+, Fe3+, Fe2+, Zn2+, Mn2+, and Cu2+). Tween 80 or Tween 60 (10% (w/v)) had little influence on the stability of AmyA and AmyB, while the 10% (w/v) sodium dodecyl sulfate caused the complete loss of AmyA and AmyB activities. The AmyA and AmyB were stable in the presence of solid detergents (washing powder), while were less stable in liquid detergents. Under the optimal conditions in 3‐L bioreactor, the extracellular AmyB activity reached 600 U/mL, which was about 10 times as that of AmyA. These results indicated that P. pastoris was a preferable host for alkaline α‐amylase expression and the produced alkaline α‐amylase had a certain application potential in solid detergents.