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Direct Cloning of a Xylanase Gene from Pawan-Riau Hot Spring IS HELIANTI
HAYATI Journal of Biosciences Vol. 14 No. 2 (2007): June 2007
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (137.564 KB) | DOI: 10.4308/hjb.14.2.54

Abstract

A functional gene containing an Open Reading Frame (ORF) encoding a ?-1, 4-endoxylanase glycosyl hydrolase family 11 was cloned directly using metagenomic PCR-cloning method from Pawan Hot Spring sample in Riau. The gene consisted of 642 nucleotides, encoded for 213 amino acids. The amino acid sequence analysis using BLAST showed that the gene has high homology (93%) with xylanase gene from Bacillus subtilis. The gene showed its function when it was subcloned into an expression vector and overexpressed in E. coli. The crude extract of the recombinant enzyme had activity for 170 U/ml at 50 oC. The result of this work showed that metagenomic approach was a powerful short cut method to obtain recombinant biocatalyst that was useful for industrial application. Key words: ?-1, 4-endoxylanase, metagenomic DNA, Pawan-Riau hot-spring
Recent Developments in the Bioconversion of Lignocelluloses into Ethanol . KOESNANDAR; IS HELIANTI; NIKNIK NURHAYATI
Microbiology Indonesia Vol. 2 No. 3 (2008): December 2008
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.2.3.1

Abstract

Ethanol has been commercially produced using sugars derived from sugarcane and corn. Recently, research has been focused on the development of thermotolerant and ethanol-tolerant yeast or bacteria that are able to produce ethanol efficiently, as well as the development of lignocellulosic materials as the carbon sources of fermentation. Utilization of lignocellulosic materials as fermentation substrate is promising since they are available in large amounts, renewable and relatively cheap. A lignocellulose biomass is a complex mixture of carbohydrate polymers. In order to develop an efficient process, there have been many attempts to obtain more efficient ways in the conversion of lignocelluloses to ethanol, including pretreatment, enzymatic hydrolysis of lignocelluloses and direct co-culture fermentation. This paper describes the production process of ethanol from starch-containing material, recent developments on the enzymatic bioconversion of lignocelluloses into sugars and their subsequent fermentation into ethanol and the possible recombination of microbes for the direct conversion of lignocelluloses into ethanol.
Cloning of α-L-arabinofuranosidase Genes and Its Expression in Escherichia coli: A Comparative Study of Recombinant Arabinofuranosidase Originatingin Bacillus subtilis DB104 and Newly Isolated Bacillus licheniformis CW1 MOCHAMAD NURCHOLIS; NIKNIK NURHAYATI; IS HELIANTI; MARIA ULFAH; BUDIASIH WAHYUNTARI; AGUSTIN KRISNA WARDANI
Microbiology Indonesia Vol. 6 No. 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1587.437 KB) | DOI: 10.5454/mi.6.1.1

Abstract

Arabinofuranosidase (Abfa) is one of the most important enzymes involved in degradation of lignocelullose biomass.  Two genes encoding α-L-Arabinofuranosidase (abfA), each from Bacillus subtilis DB104 (abfAa1) and an indigenous Indonesian B. licheniformis CW1 (abfAb3), were cloned by the PCR approach  and expressed in Escherichia coli. Sequences analysis of abfAa1 and abfAb3 revealed that each consists of 1721 and 1739 base pairs long DNA, respectively. Each clone contains a hypothetical open reading frame of 1503 and 1509 bp that encode an Abfa protein of 500 and 502 amino acids for abfAa1 and abfAb3, respectively. The deduced amino acid sequence of AbfaB3 shares 75% identity to that of AbfaA1. The recombinant enzymes were expressed constitutively in E. coli. Partial characterization of those enzymes revealed that the AbfaA1 and AbfaB3 were optimally active at 50 ºC and 60 ºC at pH 6, respectively. Thermostability studies of the recombinant enzymes with p-nitrophenyl α-L-arabinofuranoside at their optimal conditions showed that up to 50% AbfaA1 activity was lost after 5 h incubation at 50  ºC, whereas the AbfaB3 retained its activity over 75% after 12 h pre-incubation oat 60 ºC. This thermostability study of recombinant AbfaB3 showed for the first time that the arabinofuranosidase from B. licheniformis is a thermostable enzyme. The recombinant enzyme showed a higher optimal reaction temperature (60 ºC) in comparison to the previously reported thermostable arabinofuranosidase. The thermostable AbfaB3 has a potential to be applied to the degradation of lignocellulose biomass synergistically with thermostable xylanases, for instance in the production of xylo-oligosaccharides.
Cloning and Gene Expression of AnsZ Encoding L-Asparaginase Enzyme from Local Bacillus sp. RIMA AZARA; IS HELIANTI; JONI KUSNADI; YUNIANTA YUNIANTA
Microbiology Indonesia Vol. 8 No. 2 (2014): June 2014
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (822.691 KB) | DOI: 10.5454/mi.8.2.1

Abstract

L-asparaginase is an enzyme that catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. In medical aspect, L-asparaginase especially those came from E. coli and Erwinia chrysanthemi used as chemotherapy agent of acute lymphoblastic leukemia (ALL). However, new potential organisms possessing L-asparaginase production capacity with a similar therapeutic effect are still required . In Bacillus subtilis strain 168, there are two kinds of L-asparaginase gene, AnsA and AnsZ. The study of the later L-asparaginase (AnsZ) has not been conducted intensively. The aim of this study is, first, to isolate this gene of L-asparaginase (AnsZ) from local Bacillus sp. and then to express this gene in Escherichia coli. Using PCR-cloning method, an open reading frame (ORF) containing 1128 bp was obtained. The ORF has 99% homology with sequence of L-asparaginase from Bacillus subtilis Bsn5. The gene then was subcloned into pET 21d (+) with his6-tag in the C-terminal of the gene product and expressed in E.coli BL21. L-asparaginase activity analyses showed that recombinant E. coli containing recombinant plasmid with open reading frame (ORF) L-asparaginase (AnsZ) from Bacillus subtilis had higher activity than that is not containing ORF L-asparaginase (AnsZ). Purification with HisPur TM Ni-NTA Purification Kit increased the specific activity of L-asparaginase (AnsZ) enzyme to 29 fold.
Cloning, Sequencing, and Expression of the Gene Encoding a Family 9 Cellulase from Bacillus licheniformis F11 in Escherichia coli and Bacillus megaterium, and Characterization of the Recombinant Enzymes IS HELIANTI; MARIA ULFAH; NIKNIK NURHAYATI; LLINA MULYAWATI
Microbiology Indonesia Vol. 8 No. 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (913.463 KB) | DOI: 10.5454/mi.8.4.2

Abstract

 A gene encoding cellulase belonging to the glycosyl hydrolase family 9 along with its native promoter was isolated from Bacillus licheniformis F11, cloned in Escherichia coli DH5 α and subcloned by transconjugation to Bacillus megaterium MS941. Functionality of the encoded protein was proven both in heterologous hosts, E. coli and B. megaterium. In the latter, the gene product was found in the extracellular fraction expressing a high specific activity; whereas in E. coli the protein was not secreted into the medium, and rather, showed a lower specific activity. The optimum temperature of the recombinant enzyme expressed in the hosts range from 65-75 ºC; whereas the optimum pH is 6. The recombinant enzyme was stable between 50-60 ºC and in a broad pH range (pH 5 - 9). Addition of Ca2+ and Fe3+ enhanced the enzyme activity, whereas EDTA and Cu2+  had the opposite effect. Lichenin, rather than carboxyl methyl cellulose, is the preferred substrate.
Medium Optimization for Penicillin Acylase (PAc) Production by Recombinant B. megaterium MS941 Containing pac Gene from B. thuringiensis BGSC BD1 Using Response Surface Methodology FENTRI PARAMITHA PUTRI; ASTUTIATI NURHASANAH; NIKNIK NURHAYATI; IS HELIANTI; KHASWAR SYAMSU
Microbiology Indonesia Vol. 9 No. 2 (2015): June 2015
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1794.751 KB) | DOI: 10.5454/mi.9.2.3

Abstract

Penicillin G acylase (PAc) hydrolyses of the amide bond of benzylpenicillin (Pen-G) releasing PAA and 6-APA, key intermediate in the production of various semisynthetic penicillins. In this study, we optimised the production medium of PAc by RSM using two variables (xylose as inducer and CaCl2 as divalent cations) to obtain the optimum PAc specific activity from Bacillus megaterium btpacBD1. For this purpose, combinations of five different xylose concentrations (0.13 – 0.87 %) and five different CaCl2 concentrations (0.64 – 4.36 mM) were analysed, in a total of 22 experiments. CCD used for the analysis showed that in shake flask cultivations, xylose and CaCl2 showed significant effects on PAc volumetric activity and the quadratic model was in good agreement with the experimental results (R2= 0.86 (p-value < 0.0001)). The maximum specific activity (130.669 ± 50.241 units mg protein-1) was reached when xylose and CaCl2 concentrations were 0.49% and 2.4 mM, respectively, and medium pH was around 7. Under such conditions, the activity of PAc and protein concentration achieved were 1.318 ± 0.406 units mL-1 and  0.0101 ± 0.01 mg mL-1. The shake flask validation experiments demonstrated that with such medium composition the volumetric activity, protein concentration and specific activity achieved were 1.294 ± 0.171 units mL-1, 0.0102 ± 0.0003 mg mL-1 and 125.91 ± 13.309 units mg-1, respectively. When the optimum medium composition was applied in 10 L bioreactor, the optimum volumetric activity (2.0687 ± 0.0820 units mL-1) and protein concentration (0.0078 ± 0.0008 mg mL-1) were achieved 48 h after the start of the cultivation. However, the optimum PAc specific acivity (1260.52  ± 27.5711 units mg protein-1) was achieved 18 h after the start of the cultivation.
Application of Response Surface Method in Optimization of Medium Composition for Xylanase Production by Bacillus halodurans CM1 in Submerged Fermentation SARA GUSTIA WIBOWO; IS HELIANTI; ANI SURYANI; BUDIASIH WAHYUNTARI
Microbiology Indonesia Vol. 10 No. 3 (2016): September 2016
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (708.837 KB) | DOI: 10.5454/mi.10.3.5

Abstract

A two level factorial design was performed to optimized xylanase production by alkalothermophilic Bacillus halodurans CM1 using response surface method. The variables involved in this experiment were carbon (X), 1 nitrogen source (X) concentration, and pH (XCorn cob and fish powder were use as carbon and nitrogen source 2 3 respectively. Statistical analysis of the experimental results in the range studied, only carbon source gave significant effect on xylanase production.  A second-order model was proposed to represent the enzyme activity as a function of xylan concentration (X) and pH (X).  The optimum corn cobs concentration was 4.37% (w/v), 1 3 fish powder P concentration was 1.75% (w/v) and pH 9. These conditions were tested and validated experimentally since the maximum growth rate achieved with these parameters, and the highest xylanase activity.
Isolation, Characterization, and Production of Lipase from Indigenous Fungal for Enzymatic Interesterification Process LISA PRATAMA; IS HELIANTI; ANI SURYANI; BUDIASIH WAHYUNTARI
Microbiology Indonesia Vol. 11 No. 2 (2017): Juni 2017
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (750.463 KB) | DOI: 10.5454/mi.11.2.1

Abstract

Lipase catalyses hydrolysis and esterification of lipids. The purpose of this research was to  obtain lipase producing indigenous fungi, to identify the selected fungi, to study optimum temperature and pH of the enzyme activity, as well as the  enzyme ability in interesterification reaction. The isolates used in the experiment were isolated from tempeh, oncom and BPPT laboratory culture collection. The results showed that three fungal isolates which isolated, tempe and oncom and  an isolate of BPPT-CC were positive produced lipase after qualitative assay using Rhodamine B, olive oil and PVA. The morphology identification of the isolates, revealed that R isolate was  Aspergillus sp, T isolate was Neurospora sp. and O isolate was Rhizopus sp. Upon quantitative assay from determination of the media and time production, potato dextro broth (PDB) with olive oil 2% in 48 hours fermentation showed the highest specific activity of the enzymes. Lipase produced from three isolate have the optimum at pH 4, temperatures at 40-45 °C and stable in interesterification reaction (55 °C) for 30-40 min. HPLC analysis after interesterification enzymatic reaction in mixture palm kernel olein (PKOo) and palm stearin (POs) showed that the composition of triglycerides (TAG) do not change if compared with the commercial lipase (Lypozyme TL1M).
Cloning of Lipase Gene From Thermomyces langinosus into Pichia pastoris with its Original Signal Peptide MILANI ANGGIANI; IS HELIANTI; NIKNIK NURHAYATI; ABINAWANTO ABINAWANTO
Microbiology Indonesia Vol. 11 No. 2 (2017): Juni 2017
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (716.728 KB) | DOI: 10.5454/mi.11.2.4

Abstract

Lipase is one of the most important industrial enzymes, which is widely used in the preparation of food additives, cosmetics, and pharmaceutical industries. In the previous study, we have cloned synthetic Thermomyces lanuginosus lipase gene into Bacillus subtilis and Escherichia coli and resulting low expression for enzyme activity. The aim of this research was to construct the Thermomyces lanuginosus lipase (TLL) gene into Pichia pastoris vector expression with TLL original signal peptide. TLL gene was amplified by PCR and contained original signal peptide and then inserted into pPICZα A between XhoI and XbaI site, and transformed into competent cell E.coli DH5α. From the transformant, two of positive recombinants were analyzed by sequencing analysis. As the result,both of two recombinant have a positive target gene which has lipase gene. The correct plasmid was linearized and then was transformed in Pichia pastoris X-33 by electroporation method. Thermomyces lanuginosus synthetic gene lipase has been successfully integrated into chromosome of P. pastoris X-33, which revealed by clear zones arund the colony on Yeast extract Peptone Dextrose Tributyrin (YPD.TB) plate with zeocin. The Thermomyces lanuginosus lipase had an open reading frame of 916bp encoding TLL of 314 amino acids with theoretical molecular mass of 35 kDa. The recombinant enzyme, Thermomyces lanuginosus lipase had optimal temperature at 80˚C and optimal pH at pH 8.
Isolation of a Functional Gene Encoding Homologous Lysophospholipase from Indonesian Indigenous Bacillus halodurans CM1 SHANNI FERNANDA; ABINAWANTO ABINAWANTO; IS HELIANTI
Microbiology Indonesia Vol. 13 No. 1 (2019): March 2019
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (964.297 KB) | DOI: 10.5454/mi.13.1.2

Abstract

Lipase is a biocatalyst widely used in industry, for example detergent, pharmaceutical, food, or oil purification. One of the most widely lipase used for oil purification is lysophospholipase. As much as 50% of industrial enzyme needs are supplied from microorganisms. However, enzyme productivity from wild type microbial strain is usually limited and not applicable in industry, so that genetic engineering is necessary. Cloning gene encoding for lysophospholipase from Aspergillus niger and Cryptococcus neoformans have been conducted, but has never been conducted from alkalothermophilic bacteria, such as Bacillus halodurans. Bacillus halodurans CM1 is an alkalothermophilic bacterial strain isolated previously that has many industrially potential enzymes. This study aimed to isolate one of the gene encoding lipase from Bacillus halodurans CM1 and cloned into Escherichia coli DH5α using the pGEM-T easy vector. The gene fragment encoding lysophospholipase obtained with size 783 base pairs and had 100% similarity with gene encoding lysophospholipase from Bacillus halodurans C-125 (No access GenBank: BA000004.3). E. coli harbouring the recombinant plasmid with the gene also showed activity on trybutiryn medium compared to negative control.