Fuping Lu

Improvement of Subtilisin-Like Serine Alkaline Protease by Directed Evolution for Cold-Adaptation

The activities in low temperatures of a mesophilic alkaline protease TC4 was improved by directed evolution. The process involved random mutagenesis by error-prone PCR and DNA shuffling followed by first screening on skim-milk plate and second screening by 96-well plate with AAPF as the substrate. It yielded a mutant T31, with a kcat 5 times and a catalytic efficiency kcat/Km 6.33 times greater than that of wild type TC4 at 20 ℃. The relative activity of T31 at 20 ℃ was improved as much as 25% compared to that of wild type enzyme TC4 with casein as the substrate. Four amino acid substitutions were found in T31 and the results showed that these amino acid substitutions were sufficient to generate a mutant whose low temperature activity was greater than that of TC4.

Cloning and Expression of a Thermostable Pullulanase Gene from Thermotoga maritima MSB8 in Bacillus subtilis WB600

The pulA gene enconding a thermostable pullulanase of 844 amino acids was cloned by PCR amplification from the genomic DNA of Thermotoga maritima MSB8. We successfully expressed the pulA gene in a protease-deficient strain Bacillus subtilis WB600, and the expressed pullulanase was secreted into the culture broth. Preliminary results showed that the pullulanase exhibited optimal activity at 90°C, and was stable at the temperature range from 30 to 80°C for 30 min. The stable pH range for this pullulanase was from 5.5 to 7.0, with an optimum at pH 6.0.

Optimization of Process for Optical Resolution of dl-Pantolactone Using Immobilized d-Lactonohydrolase

d-pantolactone is an important intermediate for the production of pantothenic acid, panthenol, and calcium d-pantothenate. It is an efficient method to obtain d-pantolactone by optical resolution of dl-pantolactone using d-lactonohydrolase. In order to improve the hydrolysis rate of dl-pantolactone using immobilized d-lactonohydrolase which showed excellent pH and temperature stability, the process for optical resolution of dl-pantolactone using immobilized d-lactonohydrolase was investigated by optimization of hydrolysis condition. The results showed that the optimum process for optical resolution of dl-pantolactone was: temperature 40°C, pH7.0, substrate concentration 20%, hydrolysis time 8 h. Under the optimum condition, the hydrolysis rate of dl-pantolactone reached 36.8%, and the hydrolysis rate still remained at 85.6% of the original hydrolysis rate after 20 batches of reuse, indicating that immobilized d-lactonohydrolase had excellent operational stability.

Construction of the Escherichia coli-Bacillus subtilis Shuttle Vector pBE2R and Identification of the Critical Residues Involved in the Autoprocessing of the Propeptide of the Alkaline Protease

A new Escherichia coli-Bacillus subtilis shuttle vector pBE2R was constructed to provide a convenient tool for the construction of the mutant library in the directed evolution of the alkaline protease. pBE2R was constructed by fusing P43 promoter with signal peptide as well as propeptide sequence of the apr E gene to the pBE2 vector. Alkaline protease can be expressed successfully in Bacillus subtilis WB600 by introducing the mature peptide encoding gene into the pBE2R vector with the ligation of BamH I which located at the downstream of the first amino acid of the alkaline protease mature peptide. Five insertional positions around the junction between propeptide and mature peptide were tested to detect the critical residues involved in the autoprocessing of the propeptide. Finally, it was deduced that the four critical amino acids “TTMA” which located around the 3′ terminal of the propeptide, as well as the maintenance of its integrity were necessary for the effective autoprocessing of the propeptide. Furthermore, the effect of autoprocessing of the propeptide on the formation of an active protease suggested that the propeptide was a potent inhibitor to the mature domain although it was needed in the folding of the protein before secreting.

An Unusual Pectate Lyase with Noticeable Effects on the Chinese Medicinal Herb Extract: Cloning and Characterization

Pectate lyase obtained the wide-spread application in the Chinese traditional medicine extraction because it can remarkably reduce impurity content and viscosity of the plant materials. However the low enzyme activity and monoicous species hindered the application of enzymatic method to the extraction process of the herbal medicine. In this paper, we constructed a engineering strain which can overexpressed pectate lyase. The pectate lyase activity of gene engineering bacteria was even 100 times of the wild strain. The optimal temperature and pH of recombinant enzyme were 60℃ and pH10.5.When the recombinant enzyme was used in the extraction process of flax seeds, the viscosity of the raw materials could be reduced by more than 45% and linseed oil content could be increased by 12%.The usage of pectate lyase in the extraction process showed to be a feasible way to promote the product development process of flax seeds or other medicinal herbs.

Heterologous Expression of ksdD Gene Encoding 3-Ketosteroid-Delta1-Dehydrogenase from Arthrobacter simplex in Escherichia coli

Arthrobacter simplex is extensively used in pharmaceutical industry to introduce C1-C2 double bond into steroid nucleus. This dehydrogenation reaction is known characterized by its 3-ketosteroid-△1-dehydrogenase(KSDH), which is encoded by ksdD gene. In this research, we report the expression of KSDH in Escherichia coli. The activity of the enzyme has been determined by spectrophotometric method. We found the activity of purified enzyme was about 5 fold more than the crude enzyme of cell extract. Additionally, compared with A. simplex, the transformation level by broken cell preparations of E. coli recombination cells used in the dehydrogenation reaction of 4-androstene-3, 17-dione was dramatically improved.

An Effective Electroporation Protocol for Bacillus alcalophilus TCCC11004

An electroporation protocol had been established for Bacillus alcalophilus TCCC11004. Parameters including growth condition of cells, electric field strength, electroporation medium, DNA concentration were evaluated in order to improve transformation efficiency. The transformation efficiency increased with electric field strength in the range of 15-20 kV/cm. The highest transformation efficiency of B. alcalophilus TCCC11004 was achieved at 20 kV/cm. The presence of osmoticums, sorbitol and mannitol, in the electroporation, growth and recovery media resulted in about 9.7 times increase in the transformation efficiency of B. alcalophilus TCCC11004, with a maximum value of 1.5×104 transformants per µg of pUB110. The results demonstrated that electroporation was an effective, simple and reproducible transformation method for the B. alcalophilus TCCC11004 compared with other methods.

Cloning, Characterization and Application of the Promoter Region of the Alkaline Protease Gene in Bacillus alcalophillus PB92

Promoter fragment of alkaline protease gene was cloned from Bacillus alcalophillus PB92 genome by TAIL-PCR. The fragment was sequenced and analyzed, then submitted to GenBank (EU130686). The results showed that it contained several typical promoter charactered regions and two reverse translation frames located in - 538 to - 370 bp and - 275 to - 128 bp region. Deletion analysis of the sequence indicated that 414 bp upstream of the TSS exhibited predominant promoter activity, while an 105 bp length could serve as this function. Additionally, our data demonstrated that a representative Sec- type signal peptide structure presented in PB92 alkaline protease signal peptide. The efficiency of P aprE - AprE signal peptide gene cassette was validated by its driving a plant sweet protein monellin gene highly expression in Bacillus subtilis 1A751.