• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.58-68
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• 2012

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.

• Journal of Microbiology and Biotechnology
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• 제34권2호
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• pp.436-456
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• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

• 생명과학회지
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• 제23권2호
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• pp.273-281
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• 2013

본 연구는 부산 인근의 해수욕장에서 얻은 해수에서 protease를 생산하는 균주를 분리하여 동정하고 균주의 배양학적인 특성과 protease의 효소학적 특성을 확인하였다. 해수에서 분리한 protease를 생산하는 미생물은 16S rDNA sequencing을 통해 Micrococcus sp. PS-1으로 동정하였다. Protease 생산의 최적조건은 2% skim milk와 1% NaCl이 포함된 pH 7.0의 LB배지에 48시간 배양이었다. 효소의 부분정제를 위해 ultrafiltration과 acetone 침전법을 사용하였고, zymography를 통해 분자량이 35.0 kDa과 37.5 kDa인 protease를 확인하였다. 또한 효소의 최적 활성은 pH 9.0와 $37^{\circ}C$에서 나타났고, 효소는 pH 8.0에서 11.0까지, $25^{\circ}C$에서 $37^{\circ}C$까지 80% 이상의 효소활성이 유지되어 안정한 것으로 확인되었으며 PMSF, EDTA 처리시 protease가 저해되는 것을 통해 alkaline metallo-serine protease로 확인되었다.

• 한국염색가공학회지
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• 제5권3호
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• pp.206-215
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• 1993

In the prior study, wool gabardines were treated with alkaline proteases which were some kinds of enzyme to decompose protein, and their tensile strengths were determined, and the surface of the fibers were also observed using a scanning electron microscope. Enzylon ASN 30 and Alkalase 2.5L DX did not show much effect on the weight loss of wool, however, the weight loss of wool increased considerably with treating Esperase 8.0L. Pretreatment of wool with dichloloisocyanuric acid before protease-treatment increased the weight loss of wool to a great extent. In this study, the enzyme treated wools dyeing behaviors with acid dye, Milling Cyanine 5R, were mainly investigated. The protease-treatment remarkably increased not only the rate of dyeing but also the saturation dye uptake. From these results, it seemed likely that the structural relaxation of adhesive filler of interscale or intercellular cement facilitated the dye penetration into the fibers, at the same time, the change in the inner structure of the wool fibers by the protease made the fixation of the dyes more efficient.

• 한국염색가공학회지
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• 제3권4호
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• pp.7-12
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• 1991

Wool gabardines were treated with alkaline proteases, and their tensile strerigth and dyeing behavior were obtained. Enzylon ASA 30 and Alcalase 2.5L DX did not show much effect on the weight loss of wool, but Esperase 8.0L decreased the weight of wool to a great extent. Pretreatment of wool with dichloroisocyarturic acid before protease-treatment increased the weight loss of wool considerably. Weight loss was accompanied by serious strength decrease and the use of sodium sulfate in the protease-treatment had not effect on the strength retention, only lowering the weight loss of wool. Protease-treatment of wool increased dyeability considerably, which may be due to the change in the inner structure of wool fiber by protease.

• Journal of Ginseng Research
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• 제14권1호
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• pp.21-26
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• 1990

The chemical composition and stabilities of the purified ginseng invertase were investigated. The purified enzyme was found to be a glycoprotein composed of 80.2% protein and 19.7% total sugar. The protein component of the enzyme was composed of acidic amino acid (9.3%), basic amino acid (48.9%), nonpolar amino acid (21.4%), polar amino acid (20.4%) and 6.1% S-containing amino acid. It showed especially high contents of histidine and serine. The enzyme was inactivated almost completely by the treatment with some proteases (papain, pepsin. trypsin, pancreatin and microbial alkaline pretense) and protein denatllrants (8M urea and 6M guanidine-HC1), bolt not with glyrosidase (${\alpha}$-amylase, ${\beta}$-amylase. glcoamylese and cellullase). btonosaccharides sllch as glilrose, fructose, galactose and mannose did not exert any influence on the enzyme activity. The activity of the enzyme was inhibited by Ag+, Mn2+, Hg2+, Zn2+ and Al3+, whereas Ca2+, Mg2+, Ba2+ and Fe3+ gave rather activating effects on the enzyme activity. The enzyme was relatively stable in the VH range of VH 6 and 8, and at the temperatures below 35$^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.197-208
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• 2014

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.

• Journal of Animal Science and Technology
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• 제66권3호
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• pp.457-470
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• 2024

This study aimed to evaluate the effect of supplementing different protease enzymes on growth performance, intestinal morphology, and selected carcass traits in broilers fed diets reduced 3.5% in crude protein (CP) and amino acids (AA). One thousand one-day-old Ross 308 broilers (41 g) were assigned to five dietary treatments with ten replicates of 20 birds each: a positive control (PC) diet formulated to meet Ross 308 AA requirements, a negative control (NC) diet reformulated to provide 3.5% lower CP and AA compared to PC, NC supplemented with a multi-protease (PR1) solution, containing 3 different coated proteases produced from Aspergillus niger, Bacillus subtilis and Bacillus licheniformis, NC supplemented with a serine protease (PR2) produced from Bacillus licheniformis, and NC supplemented with an alkaline protease (PR3) produced from Bacillus licheniformis. At slaughter, 40 birds per treatment were used to assess the effect of the different treatments on carcass traits. At 32 days, samples of the duodenum, jejunum, and ileum of 10 birds per treatment were collected for intestinal morphology evaluation. Birds fed PC and NC supplemented with multi-protease exhibited better (p < 0.05) feed efficiency compared to NC and NC supplemented with all the other protease enzymes. Multi-protease supplementation was linked to the highest (p < 0.05) carcass weight and yield. There were significant differences (p < 0.05) between treatments in all gut segments, with PC, PR1, PR2, and PR3 exhibiting longer villi height (VH) compared to NC. This study demonstrates that 3.5% reduction of CP and AA negatively affected for the overall period feed efficiency, carcass yield, and intestinal morphology. The supplementation of the multi-protease restored feed efficiency and improved carcass yield.

• 농업생명과학연구
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• 제45권6호
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• pp.201-212
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• 2011

인천 연안의 심하게 오염된 갯벌로부터 강력한 세포외 알카리성 단백질 분해효소를 생산하는 호알카리성 Bacillus clausii I-52를 분리하였으며, 이 균주로부터 알카리성 단백질 분해효소의 유전자를 cloning하여 서열 분석을 하였다. Chromosome 서열이 완전히 밝혀진 Bacillus subtilis의 서열을 기초로 하여 알카리성 단백질 분해효소 및 promoter를 포함하도록 primer를 고안하여 PCR을 수행하여 2,277 bp의 DNA 단편을 얻었으며 BLAST 분석 결과 29 개의 아미노산으로 이루어진 signal peptide, 77 개의 아미노산으로 이루어진 propeptide 및 275 개의 아미노산을 갖는 활성형의 BCAP으로 구성된 총 381 개의 아미노산을 코딩하는 1,143 bp의 open reading frame을 확인하였다. 활성형 BCAP의 N-말단 아미노산은 Ala이며, 분자량 및 pI 값은 각각 27698.7 Da과 6.30으로 계산되었다. 아미노산 상동성을 분석한 결과, B. subtilis 유래의 nattokinase precursor 및 B. subtilis BSn5 유래의 subtilisin E precursor와 99%의 서열 상동성을 나타내어 B. clausii I-52 유래의 BCAP은 subtilisin 계열의 단백질 분해효소임을 확인하였다. E. coli BL21(DE3)에서 발현한 재조합 BCAP는 N-Suc-Ala-Ala-Pro-Phe-pNA 를 효율적으로 분해하였다. Refolding한 재조합 BCAP은 전형적인 serine protease inhibitor인 PMSF에 의하여 강하게 효소 활성이 억제됨으로써 serine protease 계열의 단백질 분해효소임을 알 수 있었다.

• Journal of Microbiology
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• 제45권2호
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.