LAPORAN PRAKTIKUM

MIKROBIOLOGI UMUM
ACARA 11
DRYLAB

Nama : Devinta Berliana P.

NIM : 19/446856/TP/12659
Kelompok :5
Golongan :I
Dosen Pengampu : Dr. rer. nat. Lucia Dhiantika Witasari S. Farm., Apt., M.
Biotech.
LABORATORIUM BIOTEKNOLOGI
DEPARTEMEN TEKNOLOGI PANGAN DAN HASIL PERTANIAN
FAKULTAS TEKNOLOGI PERTANIAN
UNIVERSITAS GADJAH MADA
YOGYAKARTA
2020

KELOMPOK 5
a. Protein
LSDPYHFTVNAAAETEPVDTAGDAADDPAIWLDPKNPQNSKLITTNKKSGLVVYS
LEGKMLHSYHTGKLN
NVDIRYDFPLNGKKVDIAAASNRSEGKNTIEIYAIDGKNGTLQSITDPDRPIASAIDE
VYGFSLYHSQKT
GKYYAMVTGKEGEFEQYELNADKNGYISGKKVRAFKMNSQTEGMAADDEYGSL
YIAEEDEAIWKFSAEPD
b. Nukleotida
CTGTCTGATCCTTATCATTTTACCGTGAATGCGGCGGCGGAAACGGAGCCGGTT
GATACAGCCGGTGATG
CAGCTGATGATCCTGCGATTTGGCTGGACCCCAAGAATCCTCAGAACAGCAAA
TTGATCACAACCAATAA
AAAATCAGGCTTAGTCGTGTACAGCCTAGAGGGAAAGATGCTTCATTCCTATC
ATACCGGGAAGCTGAAC
AATGTTGATATCCGTTATGATTTTCCGTTGAACGGAAAAAAAGTCGATATTGCG
GCGGCATCCAATCGGT

1. Nucleotide BLAST
ORIGIN
 1 ctgtctgatc cttatcattt taccgtgaat gcggcggcgg aaacggagcc ggttgataca
61 gccggtgatg cagctgatga tcctgcgatt tggctggacc ccaagaatcc tcagaacagc
121 aaattgatca caaccaataa aaaatcaggc ttagtcgtgt acagcctaga gggaaagatg
181 cttcattcct atcataccgg gaagctgaac aatgttgata tccgttatga ttttccgttg
241 aacggaaaaa aagtcgatat tgcggcggca tccaatcggt ctgaaggaaa gaataccatt
301 gagatttacg ccattgacgg gaaaaacggc acattacaaa gcattacgga tccagaccgc
361 ccgattgcat cagcaattga tgaagtatac ggtttcagct tgtaccacag tcaaaaaaca
421 ggaaaatatt acgcgatggt gacaggaaaa gaaggcgaat ttgaacaata cgaattaaat
481 gcggataaaa atggatacat atccggcaaa aaggtaaggg cgtttaaaat gaattctcag
541 acagaaggga tggcagcaga cgatgaatac ggcagtcttt atatcgcaga agaagatgag
601 gccatctgga agttcagcgc tgagccggac ggcggcagta acggaacggt tatcgatcgt
661 gccgatggca ggcatttaac ccctgatatt gaaggactga cgatttacta cgctgctgac
721 gggaaaggtt atctgcttgc ctcaagccag ggtaacagca gctatgcgat ttatgaaaga
781 cagggacaga acaaatatgt tgcggacttt caaataacag acgggcctga aacagacggc
841 acaagcgata cagacggaat tgacgttctg ggtttcgggc tggggcctga gtatccgttc
901 ggcctttttg tcgcacagga cggagagaat atagatcacg gccaaaaggc caatcaaaat
961 tttaaaatgg tgccatggga aagaatcgct gataaaatcg gctttcatcc gcaggtcaat
1021 aaacaggttg acccgagaaa aatgaccgac agaagcggaa aataa
Abstract
Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is
desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp.
MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and
expressed in Escherichia coli. The recombinant phytase exhibited high stability at
temperatures up to 100 degrees C. A higher enzyme activity was obtained when the gene
expression was done in the presence of calcium chloride. Production of the enzyme by batch-
and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved
oxygen at 20-30% saturation and depleting inorganic phosphate below 1 mM prior to induction
by IPTG resulted in over 10 U/ml phytase activity. For fed-batch cultivation, glucose
concentration was maintained at 2-3 g/l, and the phytase expression was increased to 327
U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an
increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production.
Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both
IPTG- and lactose-induced fed-batch cultivations.
Abstrak
Phytase digunakan sebagai aditif pakan untuk degradasi fitat antinutritional, dan enzim yang
diinginkan sangat termostabil agar tahan terhadap kondisi formulasi pakan. A Bacillus sp. MD2
menunjukkan aktivitas phytase diisolasi, dan gen encoding phytase diklon dan diekspresikan dalam
Escherichia coli. Phytase rekombinan menunjukkan stabilitas tinggi pada suhu hingga 100 derajat
C. Aktivitas enzim yang lebih tinggi diperoleh ketika ekspresi gen dilakukan dengan adanya
kalsium klorida. Produksi enzim oleh budidaya batch dan fed-batch dalam bioreaktor dipelajari.
Dalam budidaya batch, mempertahankan oksigen terlarut pada saturasi 20-30% dan menipiskan
fosfat anorganik di bawah 1 mM sebelum induksi oleh IPTG menghasilkan aktivitas phytase lebih
dari 10 U / ml. Untuk budidaya fed-batch, konsentrasi glukosa dipertahankan pada 2-3 g / l, dan
ekspresi phytase meningkat menjadi 327 U / ml. Induksi menggunakan laktosa selama budidaya
batch-batch menunjukkan fase lag 4 jam sebelum peningkatan aktivitas phytase menjadi 71 U / ml
selama periode yang sama dengan produksi yang diinduksi IPTG. Hingga 90% dari jumlah total
phytase yang diekspresikan bocor dari sel-sel E.coli di kedua kultur fed-batch yang diinduksi
laktosa dan laktosa.
2. Protein BLAST
 ORIGIN
1 lsdpyhftvn aaaetepvdt agdaaddpai wldpknpqns klittnkksg
lvvyslegkm
61 lhsyhtgkln nvdirydfpl ngkkvdiaaa snrsegknti eiyaidgkng
tlqsitdpdr
121 piasaidevy gfslyhsqkt gkyyamvtgk egefeqyeln adkngyisgk
kvrafkmnsq
181 tegmaaddey gslyiaeede aiwkfsaepd ggsngtvidr adgrhltpdi
egltiyyaad
241 gkgyllassq gnssyaiyer qgqnkyvadf qitdgpetdg tsdtdgidvl
gfglgpeypf
301 glfvaqdgen idhgqkanqn fkmvpweria dkigfhpqvn kqvdprkmtd rsgk
Abstract
Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is
desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp.
MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and
expressed in Escherichia coli. The recombinant phytase exhibited high stability at
temperatures up to 100 degrees C. A higher enzyme activity was obtained when the gene
expression was done in the presence of calcium chloride. Production of the enzyme by batch-
and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved
oxygen at 20-30% saturation and depleting inorganic phosphate below 1 mM prior to induction
by IPTG resulted in over 10 U/ml phytase activity. For fed-batch cultivation, glucose
concentration was maintained at 2-3 g/l, and the phytase expression was increased to 327
U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an
increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production.
Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both
IPTG- and lactose-induced fed-batch cultivations.
Abstrak
Phytase digunakan sebagai aditif pakan untuk degradasi fitat antinutritional, dan enzim yang
diinginkan sangat termostabil agar tahan terhadap kondisi formulasi pakan. A Bacillus sp. MD2
menunjukkan aktivitas phytase diisolasi, dan gen encoding phytase diklon dan diekspresikan dalam
Escherichia coli. Phytase rekombinan menunjukkan stabilitas tinggi pada suhu hingga 100 derajat
C. Aktivitas enzim yang lebih tinggi diperoleh ketika ekspresi gen dilakukan dengan adanya
kalsium klorida. Produksi enzim oleh budidaya batch dan fed-batch dalam bioreaktor dipelajari.
Dalam budidaya batch, mempertahankan oksigen terlarut pada saturasi 20-30% dan menipiskan
fosfat anorganik di bawah 1 mM sebelum induksi oleh IPTG menghasilkan aktivitas phytase lebih
dari 10 U / ml. Untuk budidaya fed-batch, konsentrasi glukosa dipertahankan pada 2-3 g / l, dan
ekspresi phytase meningkat menjadi 327 U / ml. Induksi menggunakan laktosa selama budidaya
batch-batch menunjukkan fase lag 4 jam sebelum peningkatan aktivitas phytase menjadi 71 U / ml
selama periode yang sama dengan produksi yang diinduksi IPTG. Hingga 90% dari jumlah total
phytase yang diekspresikan bocor dari sel-sel E.coli di kedua kultur fed-batch yang diinduksi
laktosa dan laktosa.