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Production of Bacterial Pectinase Using Submerged Culture for Various Applications

Thesis Info

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Author

Bibi, Nowshad

Program

PhD

Institute

Government College University

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2018

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/12930/1/Nowshad%20Bibi%20Phd%20Thesis.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727024592

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Pectinases are pectin degrading enzymes and are naturally produced by plants, animals and microorganisms. Their major source of production at industrial scale is from microorganisms especially Bacillus sp, Aspergillus sp. and yeast sp. which are generally regarded as safe. Aspergillus sp. generally produce acidic pectinases which are used in the food and beverage industry for the extraction and clarification of fruit juices and maceration of vegetables for production of purees and pastes. Bacillus sp. are usually capable of producing alkaline pectinases which have diverse functions and are in use in many industrial processes, successfully substituting the use of harsh chemicals which not only causes the deterioration of product quality but also the deterioration of environment. Pectinases from Bacillus sp. are generally active at broad ranges of pH and temperature and due to this reason, they are multi-functional enzymes. Pectinases account for more than 10% of the industrial enzymes market and they constitute 25% of the global food enzymes market. The present study was concerned with the search for a novel bacterial isolate for the lab scale production of pectinase (Polygalacturonase). Keeping in view the increasing demand of pectinase, specially its need in Faisalabad, a textile city of Pakistan, isolation of new hyper producer bacterial strains locally is an easy and cheap way of getting the desirable products at low cost. Therefore, isolation of new strains for industrial enzyme production has been, and will be, a part of research. This method alone can also provide raw material for further research such as enzyme engineering or molecular directed evolution. Pectinase positive cultures were isolated using PSAM, the medium that is able to grow and differentiate pectin consuming bacteria from others. The pectinase producing bacteria form clear halos around their colonies while others do not form any clear zones. For the identification of hyper producer strains, colony PCR was done for 16S rRNA analysis. The reason to use the 16S rRNA gene for identification purposes is that there is a large database of DNA sequences available for the gene from the widest range of microbial species as compared with any other genetic target. The selected bacterial isolate NS1 (source of pectinase enzyme) was identified based on PCR amplification of 16S rRNA and for this purpose the amplified product was electrophoresed in agarose gel against a known species of Bacillus licheniformis. The 16S rRNA sequencing confirmed the Bacillus status of the strain NS1 and the nucleotide sequence BLAST results showed 98% similarity of strain NS1 having Accession No. KX765286 with few species of Bacillus licheniformis. The growth conditions of the newly isolated Bacillus licheniformis strain were investigated using submerged fermentation to understand the fermentation behaviour of the microorganism and the pattern of pectinase production by it. The growth of the organism and enzyme production by it was investigated using some local agrowastes such as wheat bran, gram bran, citrus peel, apple pomace, carrot pomace and peanut shells as carbon sources. Among these agrowastes citrus peel powder at 2.5% concentration proved as best substrate for pectinase production followed by wheat bran. Among various nitrogen sources investigated for their role in pectinase production, organic sources such as tryptone and yeast extract gave better results than inorganic nitrogen sources. Among inorganic sources Diammonium hydrogen phosphate gave more pectinase units than other inorganic nitrogen sources. Physical parameters like pH, temperature, inocula size and incubation period for high yield of pectinase in submerged fermentation were optimized by using Response Surface Methodology. Which is an efficient tool for increasing product yield many folds in short time due to limited number of experiments and lab work. In the present study, the yield of pectinase was increased 5.6 fold that optimization produced 219U/mL as compared to one variable at a time method which produced only 38.86U/mL. Several purification methods were evaluated to observe that which one is more advantageous and cost effective in the present study for pectinase purification. Three methods of protein purification (aqueous two phase purification system, macro-affinity ligand facilitated three phase partitioning and gel filtration chromatography) were used in the present study and macro-affinity ligand facilitated three phase partitioning were found to give high purification of pectinase with purification fold of 13.05. The pectinase from newly isolated Bacillus licheniformis showed some novelty in characteristics as compared to most of the pectinases produced by other species. Although its optimum activity was achieved at a temperature of 70ºC in glycine buffer pH 8 but it also showed considerable activity (26.75U/mL) even at 100ºC in phosphate buffer pH 7. Addition of 15mM CaCO3 to the enzyme assay mixture increased the pectinase activity by 3.1 fold and addition of chloroform to enzyme assay mixture increased the pectinase activity by 7.45 fold. Surfactants (CTAB, SDS and Triton X-100) increased the pectinase activity many fold as compared to control. Among various sugars investigated for their effect on pectinase activity, sorbitol was found as a stimulator of pectinase activity by increasing its activity by 1.8 fold while glucose, lactose and sucrose inhibited its activity. The pectinase produced in this study was investigated for applications such as oil extraction from sunflower seeds, apple juice extraction and clarification and starch extraction from potatoes. In all of the above applications, the locally produced pectinase enhanced the yield of apple juice, oil and starch several fold as compared to control without the application of pectinase enzymes. Apple juice yield was increased by 2.06 fold due to pectinase treatment while the juice clarification was increased by 1.62 fold. Pectinase application also increased the oil yield some 3.15 fold as compared to oil extraction by water without the addition of pectinase. Its effect on potato starch extraction was also appreciable and 3.95 fold increase in starch yield was observed due to pectinase treatment of potato slurry. All these investigations showed that the low cost pectinase produced by locally isolated microorganisms using low cost agrowastes as nutrient supplements are able to compete with costly commercial enzymes and can bring a revolution in product quality and yield if used by local industries.
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۔۔۔کا سراپا

۔۔۔۔۔۔کا سراپا

چاند سا چہرا ، بال تھے بادل
دانت تھے اُس کے موتی جیسے
ناک تھی ستواں ، پیاری آنکھیں
پتلی لمبی روشن انگلی
انگلی میں اک زرد انگوٹھی

ہاتھ تھے اُس کے چاندی جیسے
نیلے رنگ کی چادر ہوتی
کالا رنگ سکارف کا ہوتا
بہت وہ اُس کو اچھا لگتا
لان میں بیٹھے دیکھا کرتا
باتیں کرنے کو جی کرتا
لیکن اُس سے ڈر بھی لگتا

بیگ گلابی رنگ کا ہوتا
جوتا بھی تھا سفید ہی اُس کا
جیسے سفید تھے پائوں اُس کے
روز اُسی جا کرسی ہوتی
یعنی میرے عین برابر

آنکھ نہ بھر کر دیکھا ہم نے
میں نے اُس کو ، اُس نے مجھ کو
رنگ یہ سارے نقش ہیں کیسے
اُس کا میرا کیا رشتہ تھا

بشارة رسول اللہ بفتح السند والہند

The use of similes, metaphors, proverbs and idioms is given its due prestige in the annals of literature. To mention their use in the Islamic literature especially in the sayings of our Holy Prophet (PBUH) renders an ease and comfort in order to elaborate upon the Islamic teachings. It not only enhances interest but renders an inexplicable charm to explicate Islamic teachings so as to make them easy to comprehend. The use of figurative language enhances the rhythmic flow and charm of language and thus brings in a huge impact on literature. It is in this way that the proselytization task be done in an easy effective manners.

Dual Career Couples With Impact on Marital Quality and Career Satisfaction in Peshawar, Pakistan

Since fifty years or so, many studies had examined the interplay between occupational role and familial responsibilities in dual career couples, however the phenomena is yet to be explored in the context of Pakistan. This study was planned with the aim to explore the impact of role conflict (work to family and family to work conflict) on marital quality and career satisfaction of duel career couples. The study also aimed to explore that to what extent coping mechanisms are associated with role conflict, marital quality, career satisfaction and how these are experienced by respondents having various demographic features. Data was collected from 388 respondents who were selected through purposive sampling while using snowball technique in the targeted 6 public sector universities and 3 hospitals in the city of Peshawar, Khyber Pakhtunkhwa of Pakistan. A group of theories was reviewed including role strain, theory of role differentiation, theory of asymmetric permeability and expansion/scarcity hypotheses to guide this study.A sequential explanatory design of Mixed Method Research (MMR) was used to collect quantitative data by using a questionnaire and qualitative data through in-depth interviews. Statistical analysis was performed through Chi-square test, Regression analysis, T-test, ANOVA and correlation for exploring association and relation between the selected variables. A significant and negative association was found between family to work conflict (FWC) and marital quality while highly significant and negative associations were obtained with career satisfaction. Similarly, work to family conflict (WFC) was found highly significant and negatively associated with all the items loaded on the components of marital quality while found in less significant association with items loaded on the components of career satisfaction. It was also found through regression analysis that family to work and work to family conflict was significantly while negatively directed with marital satisfaction, communication, togetherness and positively related with marital disagreements. Furthermore, family to work conflict was significantly and negatively associated with career happiness, motivation and job involvement. Similarly, role conflict was found in significant and negative association with coping mechanisms while a significant positive association was obtained between coping mechanisms, marital quality and career satisfaction. It was concluded from the data obtained through T-test and ANOVA that role conflict, marital quality and career satisfaction was experienced differently by respondents having various demographic characteristics. Additionally, the data collected from the study participants through indepth interviews in the form of narratives were found in consonance with our statistical findings. The themes of the study were including 1) Role balancing is difficult; 2) Family stresses have negative implications on the job; 3) We have a happy marriage, but role overload is problematic; 4) Child care is difficult to manage with occupational role; 5) We have little but quality time together: 6) Job schedule is difficult while family responsibilities are flexible; 7) Disagreements erupts but we respond with coping; 8) Friendly communication is helpful: 9) Coping mechanisms are very useful. Considering the findings obtained from all the statistical models and in-depth interviews that family to work and work to family conflict was significantly reducing the level of marital satisfaction, marital togetherness and marital communication while causing marital disagreements in dual career couples. Likewise, family related problems and work induced stresses had certain negative outcomes for career happiness, motivation and job involvement while coping mechanisms positively negotiate the carryover of role permeability in dual career couples. Our findings support the major assumptions of the role strain theory and scarcity hypotheses by suggesting that role conflict significantly mitigate the marital quality and career satisfaction of dual career couples. Likewise, our findings supported the theory of asymmetric permeability by outlining that work related stresses were more likely permeating to cause unrest in the family because the family domain was more flexible to absorb stresses than work domain. It is recommended that medical and academic institutions should specifically formulate policies to address issues and problems of dual career couples so that their potential may be utilized for the betterment of organisations, families and society. These include flexible and favorable recruitment policies, provision of baby care and counseling centers within these settings.