Piyanun Harnpicharnchai - Academia.edu (original) (raw)

Papers by Piyanun Harnpicharnchai

Letters in Applied Microbiology

This study demonstrates a remarkably high level of microbial-induced calcium carbonate precipitat... more This study demonstrates a remarkably high level of microbial-induced calcium carbonate precipitation (MICP) using a mixed culture containing TBRC 1396 (Priestia megaterium), TBRC 8147 (Neobacillus drentensis) and ATCC 11859 (Sporosarcina pasteurii) bacterial strains. The mixed culture produced CaCO3 weights 1·4 times higher than those obtained from S. pasteurii, the gold standard for efficient MICP processes. The three strains were selected after characterization of various Bacillus spp. and related species for their ability to induce the MICP process, especially in an alkaline and high-temperature environment. Results showed that the TBRC 1396 and TBRC 8147 strains, as well as TBRC 5949 (Bacillus subtilis) and TBRC 8986 (Priestia aryabhattai) strains, could generate calcium carbonate at pH 9–12 and temperature 30–40°C, which is suitable for construction and consolidation purposes. The TBRC 8147 strain also exhibited CaCO3 precipitation at 45°C. The TBRC 8986 and TBRC 8147 strains a...

Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent... more Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes.

Microbiology and Biotechnology Letters, 2020

Bioscience, Biotechnology, and Biochemistry, 2016

A cDNA encoding β-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pas... more A cDNA encoding β-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high kcat/Km (461 mg−1 s−1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-β-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant β-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additi...

Applied biochemistry and biotechnology, Jan 30, 2015

The thermotolerant methylotrophic yeast Ogataea thermomethanolica is a host for heterologous prot... more The thermotolerant methylotrophic yeast Ogataea thermomethanolica is a host for heterologous protein expression via secretion to the culture medium. Efficient secretion is a major bottleneck for heterologous protein production in this strain. To improve protein secretion, we explored whether the use of a native signal peptide sequence for directing heterologous protein secretion and overexpression of native ER-resident chaperone genes could improve heterologous protein secretion in O. thermomethanolica. We cloned and characterized genes encoding α-mating factor (Otα-MF) and ER-resident chaperones OtBiP, OtCNE1, and OtPDI. The pre and pre-pro sequences of Otα-MF were shown to promote higher secretion of heterologous endoxylanase comparing with the classical pre-pro sequence of Saccharomyces cerevisiae. However, in the case of heterologous glycosylated phytase, only the Otα-MF pre-pro sequence significantly enhanced protein secretion. The effect of chaperone overexpression on heterolo...

Lipases are used as catalysts in synthetic organic chemistry and various important industries. In... more Lipases are used as catalysts in synthetic organic chemistry and various important industries. In this study, a lipase containing lipolytic activity (capable of using both triolein and tributyrin as substrates and producing clear halo zone around the colony) was isolated from 36,000 transformants of the metagenomic library of Jae-sawn hot spring. The identified lipase has predicted molecular weight of 32 kDa and is encoded by a gene with 876 nucleotides. The enzyme contains 39% homology to the phospholipase of Carboxydothermus hydrogenoformans. This phospholipase

RNA, 2004

The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein nec... more The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necsessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA3 to 27SBS pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA3 and 27SBL pre-ribosomal RNAs.

RNA, 2002

To identify new gene products that participate in ribosome biogenesis, we carried out a screen fo... more To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene N0P7 that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A 0 , A 1 , and A 2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A.

Protein Expression and Purification, 2009

A fungal strain, BCC2871 (Periconia sp.), was found to produce a thermotolerant beta-glucosidase,... more A fungal strain, BCC2871 (Periconia sp.), was found to produce a thermotolerant beta-glucosidase, BGL I, with high potential for application in biomass conversion. The full-length gene encoding the target enzyme was identified and cloned into Pichia pastoris KM71. Similar to the native enzyme produced by BCC2871, the recombinant beta-glucosidase showed optimal temperature at 70 degrees C and optimal pH of 5 and 6. The enzyme continued to exhibit high activity even after long incubation at high temperature, retaining almost 60% of maximal activity after 1.5h at 70 degrees C. It was also stable under basic conditions, retaining almost 100% of maximal activity after incubation for 2h at pH8. The enzyme has high activity towards cellobiose and other synthetic substrates containing glycosyl groups as well as cellulosic activity toward carboxymethylcellulose. Thermostability of the enzyme was improved remarkably in the presence of cellobiose, glucose, or sucrose. This beta-glucosidase was able to hydrolyze rice straw into simple sugars. The addition of this beta-glucosidase to the rice straw hydrolysis reaction containing a commercial cellulase, Celluclast 1.5L (Novozyme, Denmark) resulted in increase of reducing sugars being released compared to the hydrolysis without the beta-glucosidase. This enzyme is a candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.

Molecular and Cellular Biology, 2005

The essential, conserved yeast nucleolar protein Ytm1 is one of 17 proteins in ribosome assembly ... more The essential, conserved yeast nucleolar protein Ytm1 is one of 17 proteins in ribosome assembly intermediates that contain WD40 protein-protein interaction motifs. Such proteins may play key roles in organizing other molecules necessary for ribosome biogenesis. Ytm1 is present in four consecutive 66S preribosomes containing 27SA 2 , 27SA 3 , 27SB, and 25.5S plus 7S pre-rRNAs plus ribosome assembly factors and ribosomal proteins. Ytm1 binds directly to Erb1 and is present in a heterotrimeric subcomplex together with Erb1 and Nop7, both within preribosomes and independently of preribosomes. However, Nop7 and Erb1 assemble into preribosomes prior to Ytm1. Mutations in the WD40 motifs of Ytm1 disrupt binding to Erb1, destabilize the heterotrimer, and delay pre-rRNA processing and nuclear export of preribosomes. Nevertheless, 66S preribosomes lacking Ytm1 remain otherwise intact.

$Research paper thumbnail of An efficient purification and fractionation of genomic DNA from soil by modified troughing method$

Letters in Applied Microbiology, 2007

Aims: The aim of this study was to utilize a modified troughing method for purification of large ... more Aims: The aim of this study was to utilize a modified troughing method for purification of large genomic DNA obtained from microbiota in natural environment and for fractionation of genomic DNA into many size ranges that facilitates construction of metagenomic library. Methods and Results: Genomic DNA extracted from soil or termite gut was purified by the modified troughing method which utilized gel electrophoresis in the presence of 30% PEG8000. The method performed better than various purification kits and allowed no significant loss in the amount of DNA recovered. In addition, the efficiency of the modified troughing method for DNA size fractionation was investigated. DNA size fractionation was achieved with repetitive rounds of electrophoresis and DNA collection to obtain DNA with many size ranges. Conclusions: The modified troughing method is a simple and efficient method for purification of genomic DNA and for DNA size fractionation. Significance and Impact of the Study: The modified troughing method is a straightforward and inexpensive technique readily available for anyone working with environmental genomic DNA. It facilitates cloning of genomic DNA and enhances rapid discovery of useful bioactive compounds from microbial resources.

Journal of Biotechnology, 2008

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot sprin... more Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7-9 and 8-10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50-75 degrees C, with optimal activity at 70 degrees C and were stable at 70 degrees C for at least 120 min. Both PLP and Est1 exhibited high V(max) toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C(4) and C(5)) and long chain (C(14) and C(16)) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C(10). We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.

Genes & Development, 2007

More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors ... more More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors—e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs—remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing...

FEMS Microbiology Letters, 2009

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (T... more Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris. The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL À1 , respectively. Both recombinant phytases exhibited high affinity for phytate but not for p-nitrophenyl phosphate. Optimal phytase activity was observed at 50 1C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as 4 50% activity was retained after heating at 100 1C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.

FEMS Microbiology Letters, 2010

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell sur... more Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50-55 1C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.

Annals of Microbiology, 2013

ABSTRACT The glyceraldehyde-3-phosphate dehydrogenase (GAP) gene from the thermotolerant yeast st... more ABSTRACT The glyceraldehyde-3-phosphate dehydrogenase (GAP) gene from the thermotolerant yeast strain Pichia thermomethanolica BCC16875 was characterized. To investigate the efficiency of the GAP promoter for heterologous expression, especially at high temperature in various carbon sources, the promoter was employed for constitutive expression of a phytase reporter gene. The results showed that this promoter was able to drive efficient expression of phytase at 30 A degrees C; the native promoter was highly robust compared with the heterologous GAP promoter from Pichia pastoris. More importantly, the GAP promoter was shown to be able to function at higher temperatures up to 42 A degrees C, which could be useful for large-scale protein production to help reduce cooling costs in the fermenter. Expression in different carbon sources revealed that the GAP promoter was functional in glucose-, glycerol-, and methanol-containing media, with the highest level of expression in YPD medium. This strong promoter will help promote high expression of heterologous protein expression in P. thermomethanolica, especially in large-scale fermentation. In addition, a new tool for heterologous expression in yeast has been gained.