DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability

Yao, Zhuang,Jeon, Hye Sung,Yoo, Ji Yeon,Kang, Yun Ji,Kim, Min Jae,Kim, Tae Jin,Kim, Jeong Hwan The Korean Society for Microbiology and Biotechnol 2022 Journal of microbiology and biotechnology Vol.32 No.6

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.

Degradation of low density polyethylene by Bacillus species

Yao Zhuang,Seong Hyeon Jeong,Jang Yu-Sin 한국응용생명화학회 2022 Applied Biological Chemistry (Appl Biol Chem) Vol.65 No.6

Since its invention, polyethylene (PE) has brought many conveniences to human production and life. In recent years, however, environmental pollution and threats to human health caused by insufficient PE recycling have attracted widespread attention. Biodegradation is a potential solution for preventing PE pollution. In this study, Bacillus subtilis and Bacillus licheniformis, which are widespread in the environment, were examined for their PE degradation abilities. Biodegradation of low-density polyethylene (LDPE) was assessed by weight loss, Fourier transform infrared spectroscopy (FTIR), and high performance liquid chromatography (HPLC) analyses. Weight losses of 3.49% and 2.83% were observed for samples exposed to strains B. subtilis ATCC6051 and B. licheniformis ATCC14580 for 30 days. Optical microscopy revealed obvious structural changes, such as cracks, pits, and roughness, on the surfaces of the microorganism-exposed LDPE sheets. Oxidation of the LDPE sheet surfaces was also demonstrated by the FTIR-based observation of carbon-unsaturated, –OH, –NO, –C=C, and –C–O bonds. These results support the notion that B. subtilis ATCC6051 and B. licheniformis ATCC14580 can degrade PE and could potentially be used as PE-biodegrading microorganisms. Further research is needed to examine potential relevant degradation mechanisms, such as those involving key enzymes. Since its invention, polyethylene (PE) has brought many conveniences to human production and life. In recent years, however, environmental pollution and threats to human health caused by insufficient PE recycling have attracted widespread attention. Biodegradation is a potential solution for preventing PE pollution. In this study, Bacillus subtilis and Bacillus licheniformis , which are widespread in the environment, were examined for their PE degradation abilities. Biodegradation of low-density polyethylene (LDPE) was assessed by weight loss, Fourier transform infrared spectroscopy (FTIR), and high performance liquid chromatography (HPLC) analyses. Weight losses of 3.49% and 2.83% were observed for samples exposed to strains B. subtilis ATCC6051 and B. licheniformis ATCC14580 for 30 days. Optical microscopy revealed obvious structural changes, such as cracks, pits, and roughness, on the surfaces of the microorganism-exposed LDPE sheets. Oxidation of the LDPE sheet surfaces was also demonstrated by the FTIR-based observation of carbon-unsaturated, –OH, –NO, –C=C, and –C–O bonds. These results support the notion that B. subtilis ATCC6051 and B. licheniformis ATCC14580 can degrade PE and could potentially be used as PE-biodegrading microorganisms. Further research is needed to examine potential relevant degradation mechanisms, such as those involving key enzymes.

Properties of a fibrinolytic enzyme secreted by Bacillus subtilis JS2 isolated from saeu (small shrimp) jeotgal

Zhuang Yao,김정아,김정환 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.3

Bacillus species were screened to be used as starters for jeotgals, salted and fermented Korean sea foods. A strain, JS2, showing strong fibrinolytic activity was isolated from saeu (small shrimp) jeotgal, and identified as Bacillus subtilis. Bacillus subtilis JS2 grew well at 20% (w/v) NaCl concentration. SDS-PAGE of culture supernatant from JS2 showed 3 major bands of 27, 29, and 60 kDa in size. Fibrin zymography showed that the 27 kDa band was the major fibrinolytic protein. The gene, aprEJS2, was cloned and introduced into B. subtilis WB600 using pHY300PLK. A B. subtilis transformant harboring pHYJS2 showed higher fibrinolytic activity than B. subtilis JS2. aprEJS2 was overexpressed in Escherichia coli BL21 (DE3). The optimum pH and temperature for AprEJS2 were pH 8.0 and 40 C, respectively. Km and Vmax values were determined. AprEJS2 has strong a-fibrinogenase activity and moderate b-fibrinogenase activity.

Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr

Zhuang Yao,Yu Meng,Huong Giang Le,이세진,전혜성,유지연,김현진,김정환 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.11

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40oC, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-pnitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

Gene Cloning, Expression, and Properties of a Fibrinolytic Enzyme Secreted by Bacillus pumilus BS15 Isolated from Gul (Oyster) Jeotgal

Zhuang Yao,김정아,김정환 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.3

A Bacillus strain, BS15, showing strong fibrinolytic activity, antibacterial activity, and salt tolerance was isolated from gul (oyster) jeotgal, a Korean fermented sea food. BS15 was identified as B. pumilus. B. pumilus BS15 was able to grow in LB broth with 18% (w/v) NaCl. When culture supernatant was analyzed by SDS-PAGE, 22, 27, 35, and 60 kDa proteins were observed. The 27 kDa protein was determined to be major fibrinolytic enzyme by fibrin zymography. The gene (aprEBS15) was cloned in pHY300PLK, a Bacillus-E. coli shuttle vector. A B. subtilis transformant (TF) harboring pHYBS15 showed higher fibrinolytic activity than B. pumilus BS15, and produced the same 27 kDa protein. aprEBS15 was overexpressed in E. coli BL21 (DE3), and recombinant enzyme (AprEBS15) was purified. The optimum pH and temperature of AprEBS15 were pH 8.0 and 40oC, respectively. Km and Vmax values were 0.26 mM and 21.88 μmol/L/min, respectively. B. pumilus BS15 can be used as a starter for jeotgals and other fermented foods with high salinities.

Properties of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens RSB34, Isolated from Doenjang

Zhuang Yao,Xiaoming Liu,JaeMin Shim,KangWook Lee,Hyun-Jin Kim,Jeong Hwan Kim 한국식품영양과학회 2016 한국식품영양과학회 학술대회발표집 Vol.2016 No.10

Properties of a fibrinolytic enzyme secreted by Bacillus subtilis JS2 isolated from saeu (small shrimp) jeotgal

Yao, Zhuang,Kim, Jeong A,Kim, Jeong Hwan Korean Society of Food Science and Technology 2018 Food Science and Biotechnology Vol.27 No.3

Bacillus species were screened to be used as starters for jeotgals, salted and fermented Korean sea foods. A strain, JS2, showing strong fibrinolytic activity was isolated from saeu (small shrimp) jeotgal, and identified as Bacillus subtilis. Bacillus subtilis JS2 grew well at 20% (w/v) NaCl concentration. SDS-PAGE of culture supernatant from JS2 showed 3 major bands of 27, 29, and 60 kDa in size. Fibrin zymography showed that the 27 kDa band was the major fibrinolytic protein. The gene, aprEJS2, was cloned and introduced into B. subtilis WB600 using pHY300PLK. A B. subtilis transformant harboring pHYJS2 showed higher fibrinolytic activity than B. subtilis JS2. aprEJS2 was overexpressed in Escherichia coli BL21 (DE3). The optimum pH and temperature for AprEJS2 were pH 8.0 and $40^{\circ}C$, respectively. Km and $V_{max}$ values were determined. AprEJS2 has strong ${\alpha}$-fibrinogenase activity and moderate ${\beta}$-fibrinogenase activity.

Novel DNAH1 Mutation Loci Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Literature Review

Zhuang Bao-Jun,Xu Su-Yun,Dong Liang,Zhang Pei-Hai,Zhuang Bao-Lin,Huang Xiao-Peng,Li Guang-Sen,You Yao-Dong,Chen Di'Ang,Yu Xu-Jun,Chang De-Gui 대한남성과학회 2022 The World Journal of Men's Health Vol.40 No.4

The protein encoded by dynein axonemal heavy chain 1 (DNAH1) is a part of dynein, which regulates the function of cilia and sperm flagella. The mutant of DNAH1 causes the deletion of inner dynein arm 3 in the flagellum, leading to multiple morphological abnormalities of the sperm flagella (MMAF) and severe asthenozoospermia. However, instead of asthenozoospermia and MMAF, the result caused by the mutation of DNAH1 remains unknown. Here we report a male infertility patient with severe asthenozoospermia and teratozoospermia. We found two heterozygous mutations in DNAH1 (c.6912C>A and c.7076G>T) and which were reported to be associated with MMAF for the first time. We next collected and analyzed 65 cases of DNAH1 mutation and found that the proportion of short flagella is the largest, while the bent flagella account for the smallest, and the incidence of head deformity is not high in the sperm of these patients. Finally, we also analyzed 31 DNAH1 mutation patients who were treated with intracytoplasmic sperm injection (ICSI) and achieved beneficial outcomes. We hope our research will be helpful in the diagnosis and treatment of male infertility caused by DNAH1 mutation.

Isolation of 2 Bacillus Strains with Strong Fibrinolytic Activities from Kimchi

( Zhuang Yao ),( Yu Meng ),( Huong Giang Le ),( Se Jin Lee ),( Hye Sung Jeon ),( Ji Yeon Yoo ),( Diana Nur Afifah ),( Jeong Hwan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2020 한국미생물·생명공학회지 Vol.48 No.4

Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis and B. velezensis, respectively. Tryptic soy broth (TSB) was found to best maintain both the growth and the fibrinolytic activity of these strains. Culture supernatants were analyzed by SDS-PAGE and fibrin zymography, and the results indicate that a 40 and 27 kDa band seem to be responsible for the fibrinolytic activities of these two isolates and the 27 kDa band was subsequently identified as the mature form of AprE, the major fibrinolytic enzyme. Thus the aprE genes were cloned and the translated amino acid sequences demonstrated 99.3% identity with each other, and 86.5% identity with BsfA, a fibrinolytic enzyme from B. subtilis ZA400 also isolated from Kimchi, and AprE2, a fibrinolytic enzyme from B. subtilis CH3-5 isolated from Cheonggukjang, a traditional Korean fermented soy. Given this B. subtilis K3 and B. velezensis K208 may be promising starter cultures in the production of fermented foods.

Characterization of a Fibrinolytic Enzyme Secreted by Bacillus velezensis BS2 Isolated from Sea Squirt Jeotgal

( Zhuang Yao ),( Jeong A Kim ),( Jeong Hwan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.3

Bacillus sp. BS2 showing strong fibrinolytic activity was isolated from sea squirt (munggae) jeotgal, a traditional Korean fermented seafood. BS2 was identified as B. velezensis by molecular biological methods. B. velezensis BS2 grows well at 15% NaCl and at 10℃. When B. velezensis BS2 was cultivated in TSB broth for 96 h at 37℃, the culture showed the highest fibrinolytic activity (131.15 mU/μl) at 96 h. Three bands of 27, 35 and 60 kDa were observed from culture supernatant by SDS-PAGE, and fibrin zymography showed that the major fibrinolytic protein was the 27 kDa band. The gene (aprEBS2) encoding the major fibrinolytic protein was cloned, and overexpressed in heterologous hosts, B. subtilis WB600 and E. coli BL21 (DE3). B. subtilis transformant showed 1.5-fold higher fibrinolytic activity than B. velezensis BS2. Overproduced AprEBS2 in E. coli was purified by affinity chromatography. The optimum pH and temperature were pH 8.0 and 37℃, respectively. K_m and V_max were 0.15 mM and 39.68 μM/l/min, respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA was used as the substrate. AprEBS2 has strong α-fibrinogenase and moderate β-fibrinogenase activity. Considering its high fibrinolytic activity, significant salt tolerance, and ability to grow at 10℃, B. velezensis BS2 can be used as a starter for jeotgal.