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Home > Glycosyl Hydrolases from Hyperthermophilic Archaeon Pyrobaculum Calidifontis: Cloning & Characterization

Glycosyl Hydrolases from Hyperthermophilic Archaeon Pyrobaculum Calidifontis: Cloning & Characterization

Thesis Info

Access Option

External Link

Author

Mehboob, Sumaira

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Biological Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/13131/1/thesis%20new%20title.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726341030

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4-α-glucanotransferases (4-α-GTases) play a vital role in starch modification for the production of resistant starches, cycloamyloses, prebiotics and thermoreversible starch gels. These products have a wide application in the development of starch based healthy foods. Synthesis of these products at industrial level requires highly thermostable enzymes. The present study describes the cloning and characterization of: 1) Pcal_0630, a β-glucosidase, 2) Pcal_0672, a glycosyl hydrolase and 3) Pcal_0768, a novel and highly thermostable 4-α-glucanotransferase from a hyperthermophilic aerobic archaeon Pyrobacculum calidifontis VA1. The genes encoding these enzymes were cloned in pET-21a(+) or pET-28a(+) and expressed in Escherichia coli. The blast search showed that Pcal_0630 belonged to family GH1 and exhibited a good sequence similarity with other uncharacterized enzymes from genus Pyrobaculum (upto 69%) while poor similarity was observed with the characterized thermophilic bacterial and archaeal enzymes like Pyrococcus furiosus (24%), Pyrococcus horikoshii (24%) and Halothermothrix orenii (22%). The gene sequence of Pcal_0630 is 1095 nucleotides in length which encoded a protein of 365 amino acids with a calculated molecular mass 42 kDa. Recombinant Pcal_0630 was produced as inclusion bodies in E. coli which were tried to be refold by denaturing in guanidine hydrochloride and urea but the refolded sample was inactive when assayed using cellobiose or pnitrophenol- α-D-glucopyranoside (PNPG) substrates. Pcal_0672, the second enzyme I studied, was composed of 1458 nucleotides which encoded a protein of 485 amino acids with an approximate molecular mass of 54 kDa. This gene was annotated as a glycosyl hydrolase belonging to family GH57 and showed a high homology of 81% with uncharacterized members of thermophilic bacteria and archaea. The sequence homology was very low with the characterized members of family GH57 like 4-α-GTase from T. litoralis (19%), branching enzyme from T. kodakarensis (18%), and α-amylase from P. furiosus (19%). Recombinant Pal_0672 was also produced as insoluble aggregates in E. coli. However, these insoluble aggregates exhibited the enzyme activity when analyzed by gel staining. Nucleotide analysis of the Pcal_0768 gene, the third enzyme of my study, showed that it consists of 1407 nucleotides encoding a protein of 468 amino acids with a calculated molecular mass of 53 kDa. Pcal_0768 was annotated as 4-α-GTase belonging to family GH77. The amino acid sequence of Pcal_0768 showed the highest homology of 75% with P. aerophilum amylomaltase. Four highly conserved regions characteristics of the α-amylase superfamily were also conserved in Pcal_0768. Amino acid residues constituting the catalytic triad were also conserved in Pcal_0768 at positions Asp270, Glu317 and Asp370. The gene was cloned and expressed in E.coli BL21 CodonPlus (DE3)-RIL and the recombinant protein was produced in soluble form which was further purified upto apparent homogeneity on SDS-PAGE. Recombinant Pcal_0768 exhibited maximum activity at 80 °C. Although the enzyme was active over the broad range of pH (3 – 10), its optimum pH was 7.0 and more than 50% residual activity was observed at pH 10. Pcal_0768 was highly stable at 80 °C. The half-life at 100 °C was 6.5 h. Pcal_0768 exhibited the highest ever reported disproportionation activity towards maltotriose (1951 U mg-1) as well as quite high transglycosylation activity (23 U mg-1). The activity of Pcal_0768 was independent of any metal ions. The recombinant enzyme was equally able to transglycosylate the high mass polysaccharides (in the presence or absence of an acceptor) to small linear chain glucans as well as disproportionate maltooligosaccharides (G2 – G7) to large amylaceous products. Pullulan, pannose and cyclodextrins (α, β and γ) were not the substrates of the enzyme. Pcal_0768 displayed transglycosylation activity on various maltooligosaccharides, among them the smallest donor and acceptor molecules were maltose and glucose, respectively whereas the smallest transferred unit was glucose. The enzyme was also efficient in disproportionating maltose into a mixture of maltooligosaccharides. These facts suggested that Pcal_0768 is a type V amylomaltase. Pcal_0768 was stable in the presence of ionic (1 mM SDS) as well as nonionic (2% Triton X-100 or Tween 20) detergents. However, loss of activity was observed in the presence of 5 mM SDS. When the protein was incubated with various concentrations of urea it was found that there was no loss of activity even at 8 M urea. The docked complex of urea and the enzyme active pocket revealed that due to the presence of charged/polar residue around the enzyme active pocket urea was unable to denature the enzyme. Pcal_0768 activity was markedly affected in the presence of DTT, p-chloromesrcuric benzoic acid and NBS which suggested that cysteine and tryptophan residues are critically involved in the enzyme catalysis. When Pcal_0768 was studied for its industrial applications it was found that it can successfully be used in the production of thermoreversible gels that are anticipated as gelatin/fat replacers in the food industry. Though cycloamyloses were not synthesized by the action of Pcal_0768 but there were strong indications for the synthesis of small linear chain maltooligosaccharides which can be used as prebiotics. However a detailed study on these aspects is needed. In conclusion, three starch processing enzymes have been studied. Among them, Pcal_0768 is highly efficient and a novel addition in type V amylomaltases. The enzyme is highly stable at high temperatures and in the presence of detergents and urea. Pcal_0768 is capable of forming white compact thermoreversible starch gels. These properties of Pcal_0768 make it a potential candidate for starch processing industry.
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ایرج افشار

جناب ایرج افشار کی رحلت
(رئیس احمد نعمانی)
پوسٹ بکس نمبر ۱۱۴،
علی گڑھ (ہند) ۲۰۲۰۰۱۔
برادر گرامی مراتب زید مناصبکم، السلام علیکم و رحمۃ اﷲ و برکاتہ!
۳؍ مارچ ۲۰۱۱؁ء کو ایران کے معروف اسکالر اور پبلشر جناب ایرج افشار دنیا سے رخصت ہوگئے، ایک مخلص کی اطلاع اور فرمائش پر یہ قطعۂ تاریخ لکھا ہے، امید ہے اس کو ’’معارف‘‘ میں جگہ دینے کی زحمت فرمائیں گے۔
جویائے خیر
رئیس احمد نعمانی
تاریخ در گذشت دکتر ایرج افشار
;دانشمند و پڑھشگر معروف ایران
یگانا مردِ دانا ایرج افشار
سخن گوی و سخن سنج و سخن یار
ادیبِ نامور، استادِ انشاء
چراغ بزمِ تحقیقات و جستار
رفیق رہروانِ راہ پارین
انیس ہمریانِ تازہ رفتار
کتاب و نامۂ اہلِ ادب را
ہمی بودہ امین و ہم نگہدار
مہارت داشت در تالیف و تدوین
علَم گردیدہ ہم در چاپ آثار
عزیز خاطر دانا و نادان
ستودہ از زبانِ خویش و اغیار
چو دل برداشتہ از کارِ دنیا
رمیدہ از ہجوم شہرو بازار
ہستہ رختِ جان و تن ز منزل
جہانیدہ بہ سوی گور رہوار
بجستم سالِ فوتش و ز دلِ من
صدا آمد: ’’دریغا ایرج افشار‘‘
۲۰۱۱ء
(۳ مارچ/۲۰۱۱ء)
( مئی ۲۰۱۱ء)

مروجہ جاگیردارانہ نظام کا تاریخی ارتقاء اور اسلامی تعلیمات کی روشنی میں تقابلی جائزہ

Islamic theory of possession is explicit. It is different from the contemporary feudal system. Islam does not believe in any tribe, nation and ancestry. Islam negates the concept of lordship and slavery. The history of Islam tells us that Muhammad (PBUH) awarded the property to the companion but it was a special gift and his purpose was not to rule. The purpose of feudal system is to land a certain class for political purpose. The Islamic concept of possession is different from feudal system. It is not correct to say that Islam allows the contemporary feudal system

Comparative Evaluation of Effect of Selenium Sources on Growth, Production and Reproduction in Different Varieties of Indigenous Aseel Chicken

This study was planned to evaluate the influence of selenium (Se) supplementation on growth, production and reproduction in Lakha, Mushki, Peshaweri, and Mianwali varieties of Aseel at Indigenous Chicken Genetic Resource Centre, University of Veterinary and Animal Sciences (UVAS), Lahore. The study was comprised of two main experiments. In the 1st experiment, 400 d-old birds, 100/variety (50 males and 50 females) were procured from Avian Research and Training center, UVAS, Lahore. The birds were given an adjustment period of 21 days. From the base population of 400 birds, a total of 240 birds were then selected randomly, divided into 4 groups, 60/variety (30 males and 30 females). The birds of either sex in each group were further subdivided into three treatment groups A, B, and C, 10/treatment. Each treatment was replicated 10 times with one bird/replicate. In this experiment, each bird was regarded as an experimental unit. Groups A and B were experimental, while C was control. Se-enriched yeast (SY) and sodium selenite (SS) were supplemented @ 0.3 ppm (mg/kg) in basal diets of group A and B, respectively, while, group C was fed without additional selenium. Birds were maintained individually in cages under uniform husbandry conditions from 4-21 weeks. Statistical analysis of data through Analysis of Variance procedures in a Randomized Complete Block Design under factorial arrangements and comparison of means through Duncan’s Multiple Range test showed the reduced feed intake, enhanced nutrient utilization for dry matter, crude protein, crude fiber, crude fat and ash; superior feed conversion ratio; higher live final body weight; lower mortality and rearing cost in SY fed birds, especially in the males of Lakha variety than the rest of the treatments. The dietary supplementation with SY increased the values of glucose, triglyceride, globulin, glutathione peroxidase (GPX) in blood serum but decreased the levels of urea, creatinine, uric acid, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol, and thyroxin (T4), especially in the birds of Lakha and Peshaweri varieties. Significant variations in slaughtering traits were observed. SY inclusion presented improved live final body weight, dressing weight, dressing%, eviscerated weight, eviscerated% and giblet weight. Selenium accumulation in the chest and thigh muscles was also significantly enhanced, especially in the birds of Lakha and Mushki exposed to SY supplemented diet. This study concluded that SY had a major influence in improving the overall growth performance of indigenous Aseel chicken. In the 2nd experiment, a total of 96 selenium-treated twenty-one-weeks-aged birds (84 females and 12 breeding males) from Lakha, Mushki, Peshaweri and Mianwali varieties of Aseel were selected randomly and distributed into four groups (21 females and 3 breeding males from each variety), subdivided into three treatment groups A, B and C with seven replicates and 7 females and 1 breeding male in each treatment. Each treatment was replicated 7 times with one bird in each replicate. Groups A and B were the experimental, while C was control group. Ration for the birds of group A and B was supplemented @ 0.3 ppm with SY and SS, while group C was fed the ration without Se additional supplement. The birds were maintained separately in battery cages from 22 to 42 weeks. Stud mating system was practiced for breeding by providing access to each of 7 females to respective males in which SY treated males were offered for mating to SY treated females, SS treated males were offered to SS treated females and non-treated males were offered to non-treated females once a week to obtain fertile eggs from hens. Results showed that SY fed Mianwali females exhibited higher feed intake, enhanced body weight and gained sexual maturity earlier, better egg production, higher egg weight and egg mass. FCR/dozen eggs, FCR/kg egg mass were observed higher in Peshaweri. Higher egg Se concentration was recorded in SY fed group compared to SS and control groups. Non-significant variations were observed in Haugh unit scores among all the four varieties. Interaction presented improvement in egg breadth, egg length, egg volume, egg weight, egg shape index, egg shell thickness, yolk index and Huagh unit scores of the eggs in all varieties with significantly higher values in SY-fed Mianwali females. The SY-fed females of Peshaweri showed decreased dead germ%, dead in shell% and clear egg% compared with the females receiving other treatments. Improved hatchability% and hatch of fertile% was also noticed in females of SY-fed group; the highest increase was recorded in Peshaweri. Similarly, superior body weights of the hatched chicks (next progeny) of Peshaweri and Mianwali varieties were detected on SY supplemented ration. It was therefore concluded that SY is the superior supplement that can improve the production and reproduction traits of Aseel as well as assist in manufacturing a quality functional food in the form of Se-enriched eggs.