• Journal of Microbiology and Biotechnology
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• v.32 no.6
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• pp.800-807
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• 2022

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.65-74
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• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.

• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.861-868
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• 2011

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at $55^{\circ}C$ and was thermostable at $50^{\circ}C$ and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent $K_m$ and $V_{max}$ values were 1.18 mg/ml and 1,961 ${\mu}mol$/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.

• Journal of Sericultural and Entomological Science
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• v.28 no.1
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• pp.66-71
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• 1986

The studies were carried out to screen the optimum conditions for enzymatic degumming of raw silk yarn and silk fabric by use of Alkalase, a protease produced by Bacteria, comparing with Papain and Trypsin representing natural proteolytic enzymes. 1. The optimum temperature and acidity of degumming solution were 70$^{\circ}C$, pH 5-6 for Papain degumming, 40$^{\circ}C$, pH 8 for Trypsin and 50-60$^{\circ}C$ pH 8-9 for Alkalase. 2. By increasing the Alkalase concentration in the range of 0.6 to 1.0 gram per liter, the time for enzymatic degumming of silk yarn could be reduced by 40 minutes. 3. In degumming of silk yarn by Alkalase, the pretreatment of 95$^{\circ}C$, 10 minutes at 0.1% sodium bicarbonate solution or posttreatent of 80$^{\circ}C$, 20 minutes at 2% (o.w.f.) sodium silicate solution improved the efficiency of enzymatic degumming, as compared to that of nontreatment. 4. The breaking strength, elongation and Lousiness results of enzymatically degummed silk yarn were apt to be improved more than those of soap-degummed one. 5. When the pretreatment of alkaline solution was done with over 20% of degumming ratio, the enzymatic degumming efficiency of both Havutae and Crepe de chine could be reached to the same level with those of soap-soda degummed. 6. As the pretreated silk fabric with 20% of degumming ratio was under action of three proteases, respectively, the deumming efficiency of Havutae and Crepe de chine were completed by Alkalase more than by Papain or Trpysin. 7. The stiffness of enzymatically degummed Crepe de chine was not only reduced by 17% more than that of soap-soda degummed one but also the Drape coefficient was decreased in enzymatically degummed fabrics, which was closely related with the soft touch of degummed fabrics.