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Purification and Characterization of β-Glucosidase from Penicillium verruculosum
Chun, Soon Bai,Kim, Dong Ho,Kim, Kang Hwa,Chung, Ki Chul 한국미생물 · 생명공학회 1991 Journal of microbiology and biotechnology Vol.1 No.3
The β-glucosidase was purified to homogeneity from the culture filtrate of P. verruculosum by column chromatography. The enzyme was a glycoprotein with a relative size of approximately 220 kDa with an isoelectric point of 4.8, which was composed of dimeric protein of 105 kDa. The enzyme was stable up to 60℃ and the presence of glycerol significantly increased its thermostability. The enzyme was found to hydrolyze both β-aryl and β-alkyl-glucosides in addition to β-glucosyl glucose and catalyzed glucosyl transfer to cellobiose. The enzyme attacked laminarin in an exotype-like fashion. The apparent Km's of the enzyme toward cellobiose, laminaribiose, laminarin were 0.53mM, 0.35mM and 1.11mM, respectively. Glucose and glucono-δ-lactone were competitive inhibitors for the enzyme. Copper (Cu^2+), mercury (Hg^2+) and p-chloromercuribenzoate were strong inhibitors of the enzyme. The immunoblotting result revealed that one form of β-glucosidase was biosynthesized, irrespective of carbon sources used. Polyacrylamide gel electrophoresis analysis of the in vitro translated product of total RNA from avicel grown mycelium established that the P. verruculosum β-glucosidase precursor was approximately 95 kDa in size. The amino acid composition and N-terminal amino acid sequence are given.
Purification and Characterization of ${\beta}-Glucosidase$ from Penicillium verruculosum
Chun, Soon-Bai,Kim, Dong-Ho,Kim, Kang-Hwa,Chung, Ki-Chul The Korean Society for Microbiology and Biotechnol 1991 Journal of microbiology and biotechnology Vol.1 No.3
The ${\beta}-glucosidase$ was purified to homogeneity from the culture filtrate of P. verruculosum by column chromatography. The enzyme was a glycoprotein with a relative size of approximately 220 kDa with an isoelectric point of 4.8, which was composed of dimeric protein of 105 kDa. The enzyme was stable up to $60^{\circ}C$ and the presence of glycerol significantly increased its thermostability. The enzyme was found to hydrolyze both ${\beta}-aryl$ and ${\beta}-alkyl-glucosides$ in addition to ${\beta}-glucosyl$ glucose and catalyzed glucosyl transfer to cellobiose. The enzyme attacked laminarin in an exotype-like fashion. The apparent Km's of the enzyme toward cellobiose, laminaribiose, laminarin were 0.53 mM, 0.35 mM and 1.11 mM, respectively. Glucose and glucono-${\delta}-lactone$ were competitive inhibitors for the enzyme. Copper ($Cu^{2+}$), mercury ($Hg^{2+}$) and p-chloromercuribenzoate were strong inhibitors of the enzyme. The immunoblotting result revealed that one form of ${\beta}-glucosidase$ was biosynthesized, irrespective of carbon sources used. Polyacrylamide gel electrophoresis analysis of the in vitro translated product of total RNA from avicel grown mycelium established that the P. verruculosum ${\beta}-glucosidase$ precursor was approximately 95 kDa in size. The amino acid composition and N-terminal amino acid sequence are given.
Cloning of Autonomously Replicating Sequence from Phaffia rhodzyma
Chun, Soon Bai,Chun, Seung Hee 한국미생물 · 생명공학회 1995 Journal of microbiology and biotechnology Vol.5 No.6
A Phaffia rhodozyma chromosomal fragment (approximately 3.8 kb) capable of functioning as an origin for the replication of a kanamycin resistance (km') plasmid in S. cerevisiae was isolated by the use of origin search plasmid, pHN134. In S. cerevisiae, transformation frequencies using the plasmid pHN134 containing an autonomously replicating sequence of P. rhodozyma was 450-580 CFU/㎍ DNA. The stability of the recombinant plasmid were 16-19%.
Induction of Glucoamylasen in the Yeast Candida tsukubaensis
Chun, Soon-Bai,Chung, Hee-Young Korean Society for Biochemistry and Molecular Biol 1995 Journal of biochemistry and molecular biology Vol.28 No.4
The induction of glucoamylase biosynthesis from the yeast Candida tsukubaensis by different carbon sources was investigated by using either an enzyme activity assay or immunoblot analysis. The induction by C. tsukubaensis appears to be independent of the carbon sources, although the level of enzyme activity was lower in slowly utilizable carbon sources such as galactose. This glucoamylase is a constitutive enzyme and its biosynthesis is resistant to carbon catabolite repression. Glucose was more effective for the enzyme induction than starch, maltose or glycerol. In addition, this enzyme is regulated by both induction and repression.
Chun, Soon-Bai,Bai, Suk,Im, Suhn-Young,Choi, Won-Ki,Lee, Jin-Jong Korean Society for Biochemistry and Molecular Biol 1995 Journal of biochemistry and molecular biology Vol.28 No.5
Two forms of glucoamylase (GI and GII) from starch-grown Lipomyces kononenkoae CBS 5608 mutant were purified to apparent homogeneity by means of ultrafiltration, Sephacryl S-200 gel filtration and DEAE Sephadex A-50 chromatography. The apparent molecular weight was calculated as ca. 150 kDa for GI and ca. 128 kDa for GII, respectively. Both enzymes were glycoproteins with isoelectric points of 5.6 (GI) and 5.4 (GII). They had a pH optimun of 4.5 and were stable from pH 5 to 8. The temperature optimum for both enzymes was $60^{\circ}C$, but they were rapidly inactivated above $70^{\circ}C$. The $K_m$ values toward starch were estimated to be 6.57 mg per ml for GI and 4.52 mg per ml for GII, and the $V_{max}$ values were 16.28 ${\mu}M$ per mg for GI and 32.25 ${\mu}M$ per mg for GII, respectively. The $K_m$ and $V_{max}$ values of GII for ${\alpha}-$ or ${\beta}-cyclodextrin$ were estimated to be 0.15 mg per ml and 2.0 mg per ml, respectively ($K_m$) and 1.02 ${\mu}M$ per mg or 1.02 ${\mu}M$ per mg, respectively ($V_{max}$). Neither enzyme exhibited pullulanase activity but they released only glucose from starch or cyclodextrin. Amino acid analysis indicated that both glucoamylases were enriched in proline and acid amino acids. Glucoamylase GII strongly cross-reacted with a monoclonal antibody raised against GI enzymes, and the two enzymes shared very similar amino acid composition. Western blot analysis indicated that L. kononenkoae CBS 5608 mutant produced two forms of glucoamylase on starch, and that synthesis of them was subject to glucose repression.
Induction of Glucoamylasen in the Yeast Candida tsukubaensis
Soon Bai Chun,Hee Young Chung 생화학분자생물학회 1995 BMB Reports Vol.28 No.4
The induction of glucoamylase biosynthesis from the yeast Candida tsukubaensis by different carbon sources was investigated by using either an enzyme activity assay or immunoblot analysis. The induction by C. tsukubaensis appears to be independent of the carbon sources, although the level of enzyme activity was lower in slowly utilizable carbon sources such as galactose. This glucoamylase is a constitutive enzyme and its biosynthesis is resistant to carbon catabolite repression. Glucose was more effective for the enzyme induction than starch, maltose or glycerol. In addition, this enzyme is regulated by both induction and repression.
Cloning and Expression of Schwanniomyces castellii Starch Gene
Park, Jong-Chun,Bai, Suk,Chun, Soon-Bai 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.6
Schuamniomyces castelli CBS 2863의 glucoamylase 유전자를 Saccharomyces ccrevisiac에 cloning하고 발현시켰다. Southern blot 분석결과, 형질전환체의 glucoamylase 유전자는 Sch. castellii genomic DNA로부터 나온 것임을 확인하였고 5.1 혹은 1.3kb의 Sch. castellii 유전자에 해당되는 DNA 절편이 S. cercvisiac에서는 관찰되지 않았다. S. cervisine 형질전환체의 glucoamylase 활성은 Sch. castellii의 그것에 비해 2,000배 정도 낮았고 E. coli에서는 발현되지 않았다. S. cerevisiac 형질전환체가 생산한 glucoamylase는 Sch. castellii의 glucoamylase와 동일한 특성과 분자량을 가지고 있음을 알 수 있었다. The gene encoding glucoamylase from Schwanniomyces castellii CBS 2863 was cloned and expressed in Saccharomyces cerevisiae. Southern blot analysis confirmed that this glucoamylase gene was derived from the genomic DNA of Schwanniomyces castellii and that no DNA fragments corresponding to 5.1 or 1.3 kb of Sch. castellii DNA were detected in S. cerevisiae. The glucoamylase activity from S, cerevisiae transformant was approximately 2,000 times less than that of donor yeast. No expression was found in E. coli. The secreted glucoamylase from S. cerevisiae transformant was indistinguishable from that of Sch. castellii on the basis of molecular weight and enzyme properties.